THE DEVELOPMENT OF NEURODEGENERATION AND … · THE DEVELOPMENT OF NEURODEGENERATION AND BEHAVIOURAL ALTERATIONS AFTER LITHIUM/PILOCARPINE-INDUCED STATUS EPILEPTICUS IN RATS Crystal

THE DEVELOPMENT OF NEURODEGENERATION AND BEHAVIOURAL ALTERATIONS FOLLOWING LITHIUM/PILOCARPINE-INDUCED STATUS

EPILEPTICUS IN RATS

by

Crystal Maureen Dykstra

A thesis submitted in conformity with the requirements for the degree of doctor of philosophy

Institute of Medical Science University of Toronto

© Copyright by Crystal Maureen Dykstra (2011)

ii

THE DEVELOPMENT OF NEURODEGENERATION AND

BEHAVIOURAL ALTERATIONS AFTER

LITHIUM/PILOCARPINE-INDUCED STATUS EPILEPTICUS IN

RATS

Crystal Maureen Dykstra

Doctor of Philosophy

Institute of Medical Science

University of Toronto

2011

The lithium/pilocarpine model of epilepsy mimics mesial temporal lobe epilepsy with

hippocampal sclerosis (MTLE-HS) in humans. Systemic injection of pilocarpine in lithium

chloride (LiCL) pretreated adult rats results in an acute episode of severe continuous seizure

activity (status epilepticus, SE). SE causes a latent period, whereby the animal appears

neurologically normal, with subsequent development of spontaneous recurrent seizures (SRSs).

Neuropathological changes that occur during the latent period are believed to contribute to the

epileptic condition. The present thesis characterized the development of neuronal death and

behavioural alterations in rats after SE induced by the repeated low-dose pilocarpine procedure

(RLDP), and investigated the causal relationship between these two processes.

Our data demonstrated that the RLDP procedure for the induction of SE results in widespread

neurodegeneration and behavioural alterations comparable to the pilocarpine and low-dose

pilocarpine (LDP) procedures. However, the advantage to using this protocol was strain-

dependent as it reduced mortality in Wistar, but not in Long Evans Hooded (LEH), rats.

Stereological analysis of neurons (stained for the neuronal specific marker [NeuN]) at various

times (1 hr to 3 months) following SE showed that different brain regions within the

iii

hippocampus, amygdala, thalamus and piriform cortex exhibited differential rates of neuronal

loss, with the majority of SE-induced neuronal death present by 24 hours. SE resulted in

decreased exploratory behavior as assessed in the open field test, increased aggression to

handling, increased hyperreactivity as assessed in the touch-response test, and anxiolytic effects

as measured in the elevated-plus maze. Furthermore, deficits in search strategies used, as well as

impaired spatial learning and memory, contributed to poor Morris water maze (MWM)

performance. Partial neuroprotection within the hippocampus (by tat-NR2B9c) had no effect on

the number of rats developing SRSs or on behavioural alterations; this argues against a causal

relationship between neurodegeneration within this region, genesis of SRSs, and behavioural

morbidity.

iv

Acknowledgments

This thesis would not have been possible without the support of many people. First and

foremost, I offer my sincerest gratitude to Dr. James Gurd, whose encouragement, guidance and

support from the initial to the final level enabled me to complete this thesis. I greatly appreciate

his patience and knowledge whilst allowing me the room to work in my own way. I would like

to extend my gratitude to Dr. Bill Milgram, who was abundantly helpful and offered invaluable

assistance, support and guidance. I would also like to thank my program advisory committee

members, Drs. Mac Burnham and Mike Tymianski, for their time, guidance and constructive

criticism.

There are many individuals who have offered their time and expertise in training me in specific

laboratory techniques. I would like to thank Dr. Gwen Ivy for teaching me histological

techniques that were invaluable for this work and for sharing her lab space. I would like to

express my gratitude to Raymond Or for training on the microscopes, and to Candace Ikeda-

Douglas for introducing me to the lithium/pilocarpine seizure model. I am grateful to Dr.

Stephen Reid for sharing his time and expertise in animal surgeries. I am extremely grateful to

Nankie Bissoon for not only sharing her vast knowledge of experimental techniques, but also for

her guidance and friendship in the lab. I would like to extend my gratitude to Dr. Janelle

Leboutillier for her knowledge, guidance and support.

Finally, I owe my deepest gratitude to my parents for their love and support and to the rest of my

family for their encouragement.

v

List of Publications

Some of the material presented in this thesis has been published. This is to certify that I, Crystal

Dykstra, carried out the research documented in the following publications:

Dykstra CM, Ratnam M, Gurd JW (2009) Neuroprotection after status epilepticus by targeting

protein interactions with postsynaptic density protein 95. J Neuropathol Exp Neurol 68:823-831.

vi

Table of Contents

Acknowledgments ........................................................................................................................ iv

List of Publications ....................................................................................................................... v

Table of Contents ......................................................................................................................... vi

List of Tables ............................................................................................................................... xv

List of Figures ............................................................................................................................. xvi

List of Appendices ...................................................................................................................... xix

List of Abbreviations .................................................................................................................. xx

Chapter 1 General Introduction .................................................................................................. 1

1.1Main features of mesial temporal lobe epilepsy with hippocampal sclerosis ..................... 1

1.2 Animal models of seizure development and epilepsy ........................................................... 6

1.2.1 Kindling............................................................................................................................. 6

1.2.2 Post-status epilepticus models ........................................................................................... 7

1.3 Background information on the pilocarpine and lithium/pilocarpine models .................. 8

1.3.1 The pilocarpine model ....................................................................................................... 8

1.3.2 Convulsive effects of pilocarpine are mediated by activation of M1 receptors ................ 9

1.3.3 The cholinergic system is involved in the initiation but not the maintenance of SE ...... 10

1.3.4 The lithium/pilocarpine model ........................................................................................ 10

1.3.5 The proconvulsive mechanisms of lithium ..................................................................... 11

1.3.6 Use of diazepam to control SE duration and reduce mortality ....................................... 12

1.4 Behavioural and clinical features of seizure development in the lithium/pilocarpine

model ....................................................................................................................................... 12

1.4.1 The acute phase ............................................................................................................... 13

1.4.1.1 Behavioural seizures during the acute phase ........................................................ 13

1.4.1.2 Electroencephalographic patterns during motor limbic seizures and SE .............. 14

1.4.1.3 Scoring of pilocarpine-induced seizures ............................................................... 14

vii

1.4.1.4 Effect of SE duration on mortality and neuropathology ....................................... 16

1.4.2 Epileptogenesis ............................................................................................................... 17

1.4.2.1 Duration of the latent phase .................................................................................. 17

1.4.3 The chronic phase ........................................................................................................... 19

1.4.3.1 Behaviour during the chronic phase ..................................................................... 19

1.4.3.2 Electroencephalographic patterns observed during SRSs ..................................... 19

1.5 Neuropathology ..................................................................................................................... 20

1.5.1 Neurodegeneration .......................................................................................................... 20

1.5.1.1 SE-induced neurodegeneration ............................................................................. 20

1.5.1.2 Progression and severity of neuronal loss following SE ...................................... 21

1.5.1.3 Mechanisms underlying SE-induced neuronal death ............................................ 21

1.5.1.4 Types of cell death mechanisms initiated by SE .................................................. 22

1.5.1.5 Factors determining the extent and phenotype of SE-induced neuronal death ..... 24

1.5.2 Synaptic reorganization ................................................................................................... 27

1.5.3 Reactive gliosis ............................................................................................................... 28

1.5.4 Neurogenesis ................................................................................................................... 29

1.6 Co-morbid interictal disorders in mesial temporal lobe epilepsy .................................... 30

1.6.1 The relationship between epilepsy and cognitive and interictal behavioural

alterations .......................................................................................................................... 30

1.6.2 Shared neurodevelopmental, genetic or environmental causes predispose subjects to

develop both epilepsy and co-morbid behavioural and cognitive disturbances ............... 31

1.6.3 Neuropathological changes underlying the genesis of interictal behavioural

disturbances are closely related to those mediating epileptogenesis itself ....................... 32

1.6.4 Spontaneous recurrent seizures contribute to interictal behavioural and cognitive

impairment in post-SE models .......................................................................................... 33

1.6.5 Other factors affecting severity of interictal behavioural and cognitive impairment ..... 33

1.6.6 Interictal behavioural disturbances following SE ........................................................... 34

1.6.6.1 Anxiety .................................................................................................................. 34

viii

1.6.6.2 Exploration ............................................................................................................ 36

1.6.6.3 Aggression ............................................................................................................ 38

1.6.7 The effect of SE on spatial learning and memory ........................................................... 38

1.6.7.1 The Morris water maze as a test of visual-spatial learning and memory .............. 39

1.6.7.2 The effect of SE on performance in the MWM task ............................................. 40

1.7 The goals of the thesis ........................................................................................................... 42

Chapter 2 Hypotheses and Specific Objectives ........................................................................ 43

2.1 Comparison of procedures for the induction of SE ............................................................ 44

2.2 The effect of recovery time on SE-induced neurodegeneration ......................................... 44

2.3 The effect of tat-NR2B9c on SE-induced neuropathology and cognitive impairment ...... 45

2.4 The effect of SE on behavioural performance in tasks assessing anxiety, exploration

and aggression ................................................................................................................... 45

2.5 The effect of SE on search strategy use in the Morris water maze .................................... 46

Chapter 3 ..................................................................................................................................... 47

A comparison between Long-Evans hooded and Wistar rats related to the induction

and severity of status epilepticus in the low-dose and repeated low-dose

lithium/pilocarpine procedures ............................................................................................. 47

3.1 Introduction ........................................................................................................................... 47

3.2 Methods .................................................................................................................................. 48

3.2.1 Animals ........................................................................................................................... 48

3.2.2 Induction of status epilepticus ......................................................................................... 48

3.2.3 Monitoring of seizure activity ......................................................................................... 49

3.2.4 Post-seizure animal care: ................................................................................................. 49

3.2.5 Histology and Stereological analysis: ............................................................................. 50

3.2.6 Drugs: .............................................................................................................................. 54

3.2.7 Statistical Analysis: ......................................................................................................... 54

3.3 Results .................................................................................................................................... 54

ix

3.3.1 Induction of SE in Wistars and Long Evans Hooded rats ............................................... 54

3.3.2 The effect of SE on mortality .......................................................................................... 54

3.3.3 Severity of seizures in LEH and Wistar rats ................................................................... 55

3.3.4 SE-induced neuropathology in LEH and Wistar rats following SE induced with the

RLDP procedure ............................................................................................................... 59

3.3.5 Comparison of SE-induced neuropathology resulting from the LDP and RLDP

protocols in Wistar rats ..................................................................................................... 59

3.4 Discussion ............................................................................................................................... 64

3.4.1 Differential effects of induction procedure in LEH and Wistar rats ............................... 64

3.4.2 Comparison of SE-induced neuropathology in LEH and Wistar rats following SE

induction with the RLDP procedure ................................................................................. 65

3.4.3 Comparison of the effect of the LDP and RLDP protocols on SE-induced

neurodegeneration in Wistar rats ...................................................................................... 66

3.4.4 Conclusion....................................................................................................................... 67

Chapter 4 ..................................................................................................................................... 68

Temporal profile of neuronal death following lithium/pilocarpine-induced status

epilepticus................................................................................................................................ 68

4.1 Introduction ........................................................................................................................... 68

4.2 Methods .................................................................................................................................. 70

4.2.1 Animals ........................................................................................................................... 70

4.2.2 Induction of status epilepticus ......................................................................................... 70

4.2.3 Post-seizure care .............................................................................................................. 70

4.2.4 Detection of SRSs ........................................................................................................... 70

4.2.5 Histology and Stereological analysis .............................................................................. 71

4.2.6 Fluoro-Jade B staining .................................................................................................... 74

4.2.7 Statistical Analysis: ......................................................................................................... 75

4.3 Results .................................................................................................................................... 80

4.3.1 SE induction and survival rates: ...................................................................................... 80

x

4.3.2 Spontaneous seizures after lithium/pilocarpine induced SE ........................................... 80

4.3.2 Neuropathology following status epilepticus: Overview ................................................ 80

4.3.3 SE-induced neurodegeneration in the hippocampus: ...................................................... 82

4.3.5 SE-induced neurodegeneration in amygdaloid nuclei: ................................................... 98

4.3.6 SE-induced neurodegeneration in the piriform cortex: ................................................... 98

4.3.7 Detection of Fluoro-jade B stained neurons .................................................................... 98

4.4 Discussion ............................................................................................................................. 107

4.4.1 The neuropathological effect of increasing survival time following status epilepticus 107

4.4.2 The relationship between SE, SRSs and delayed neuronal death ................................. 110

4.4.3 Differences in the severity and spatial pattern of neuronal death following SE ........... 111

4.4.4 The type of cell death produced by SE ......................................................................... 112

4.4.5 Conclusion..................................................................................................................... 113

Chapter 5 ................................................................................................................................... 118

Neuroprotection following status epilepticus by targeting protein interactions with

PSD-95 ................................................................................................................................... 118

5.1 Introduction .................................................................................................................... 118

5.2 Methods ........................................................................................................................... 119

5.2.1 Induction of status epilepticus ............................................................................... 119

5.2.2 Administration of peptides ..................................................................................... 120

5.2.3 NeuN Immunohistochemistry ................................................................................ 121

5.2.4 Statistical Analysis ................................................................................................. 122

5.3 Results ............................................................................................................................. 122

5.3.1 Induction of status epilepticus ............................................................................... 122

5.3.2 SE induced by repeated low doses of pilocarpine results in neurodegeneration in

the hippocampus and piriform cortex ................................................................. 125

5.3.3 Tat-NR2B9c reduces SE-induced neurodegeneration in the hippocampus ........... 129

xi

5.3.4 Preferential neuroprotection of Tat-NR2B9c is found within specific regions of

the CA1 and CA3 ................................................................................................ 132

5.3.5 Tat-NR2B9c did not provide neuroprotection in CA1 when administered during

SE ........................................................................................................................ 135

5.4 Discussion ........................................................................................................................ 138

5.4.1 Tat-NR2B9c provided significant neuroprotection in the hippocampus ............... 138

5.4.2 Regional specificity of neuroprotection by Tat-NR2B9c within CA1 and CA3 ... 139

5.4.3 Neuroprotective effect of tat-NR2B9c is dependent on time of administration .... 140

5.4.4 Conclusion ............................................................................................................. 141

Chapter 6 ................................................................................................................................... 142

Long-lasting behavioural and anxiolytic changes in rats following status epilepticus ....... 142

6.1 Introduction .................................................................................................................... 142

6.2 Methods ........................................................................................................................... 144

6.2.1 Animals .................................................................................................................. 144

6.2.2 Induction of status epilepticus and administration of peptides .............................. 144

6.2.3 Behavioural tests .................................................................................................... 148

6.3 Results ............................................................................................................................. 151

6.3.1 SE induction ........................................................................................................... 151

6.3.2 Open field test ........................................................................................................ 152

6.3.3 Hyperexcitability tests ........................................................................................... 155

6.3.4 Elevated-plus maze: ............................................................................................... 158

6.3.5 The effect of Tat-NR2B9c on behaviour following SE ......................................... 161

6.4 Discussion ........................................................................................................................ 168

6.4.1 SE causes anxiolytic changes in behaviour and increased hyperexcitability ........ 168

6.4.2 Behavioural changes in rats following SE are long-lasting ................................... 170

6.4.3 Treatment with tat-NR2B9c did not have neuroprotective effects as assessed

behaviourally. ...................................................................................................... 171

xii

6.4.4 Conclusion ............................................................................................................. 172

Chapter 7 ................................................................................................................................... 174

The effect of SE on performance in the Morris water maze and use of exploratory

strategies ................................................................................................................................ 174

7.1 Introduction .................................................................................................................... 174

7.2 Methods ........................................................................................................................... 176

7.2.1 Animals .................................................................................................................. 176

7.2.2 Induction of status epilepticus and administration of peptides .............................. 176

7.2.3 Morris water maze testing ...................................................................................... 177

7.3 Results ............................................................................................................................. 183

7.3.1 SE induction ........................................................................................................... 183

7.3.2 Visible platform testing .......................................................................................... 183

7.3.3 The effect of SE on spatial acquisition .................................................................. 186

7.3.4 The effect of SE on spatial reversal ....................................................................... 186

7.3.5 Acquisition and reversal probe tests ...................................................................... 189

7.3.6 Effect of SE on search strategy use during spatial acquisition .............................. 192

7.3.7 Effect of SE on search strategy use during spatial reversal ................................... 192

7.3.8 Quantitative assessment of the contribution of search strategy to overall

performance ........................................................................................................ 198

7.3.9 SE results in differential impairment in Morris water maze performance and

search strategy use .............................................................................................. 202

7.3.10 The effect of Tat-NR2B9c on visual-spatial learning and use of search

strategies following SE ....................................................................................... 208

7.4 Discussion ........................................................................................................................ 213

7.4.1 SE rats exhibit impaired performance in the MWM and improve during

prolonged training ............................................................................................... 213

7.4.2 SE rats use less efficient strategies in the MWM .................................................. 214

7.4.3 Improvement in search strategy selection contributed to improved performance

epileptic rats ........................................................................................................ 215

xiii

7.4.4 The pathological effects of SE may interfere with the selection of more efficient

search strategies .................................................................................................. 216

7.4.5 Rats following SE exhibited variability in behaviour during MWM testing ......... 217

7.4.6 Neuroprotection of the dorsal hippocampus by tat-NR2B9c did not modify

performance in the MWM .................................................................................. 218

7.4.7 Conclusion ............................................................................................................. 219

Chapter 8 ................................................................................................................................... 220

General Discussion .................................................................................................................... 220

8.1 The lithium/pilocarpine model of mesial temporal lobe epilepsy ............................. 220

8.2 Comparison of the low-dose (LDP) and repeated low dose lithium/pilocarpine

(RLDP) procedures ....................................................................................................... 221

8.3 The severity and pattern of neuronal death in the lithium/pilocarpine model ........ 222

8.3.1 Pattern of neuronal death in the hippocampus ....................................................... 222

8.3.2 Pattern of neuronal death in extrahippocampal structures ..................................... 223

8.3.3 Differences in the pattern of neuronal loss between human MTLE and rats after

SE ........................................................................................................................ 225

8.4 The effect of increasing survival time on SE-induced neuronal death ...................... 226

8.4.1 Majority of neuronal death occurs early in rats following SE ............................... 226

8.4.2 Majority of neuronal death is the consequence of SE and not of spontaneous

recurrent seizures (SRSs) .................................................................................... 227

8.5 Cognitive and behavioural alterations following lithium/pilocarpine-induced SE .. 229

8.5.1 The effect of SE on spatial learning and memory .................................................. 229

8.5.2 The effect of SE on use of behavioural search strategies ...................................... 231

8.5.3 Impaired use of behavioural strategies in human MTLE-HS ................................ 233

8.6 The temporal relationship between neuronal death, behavioural alterations and

cognitive impairment following status epilepticus ..................................................... 235

8.7 The effect of neuroprotection on epileptogenesis, behavioural alterations, and

cognitive impairment .................................................................................................... 237

8.7.1 Neuroprotection within the hippocampus .............................................................. 237

xiv

8.7.2 Effect of neuroprotection in extrahippocampal regions ......................................... 240

8.8 Conclusion ........................................................................................................................... 248

Chapter 9 ................................................................................................................................... 250

Future directions ....................................................................................................................... 250

9.1 Cell death mechanisms contributing to differential rates of neuronal loss

following SE ................................................................................................................... 250

9.1.1 Previous literature .................................................................................................. 250

9.1.2 Summary of our findings ....................................................................................... 250

9.1.3 Proposed studies ..................................................................................................... 251

9.2 Specific cognitive alterations in rats following SE ...................................................... 252




9.3 The causal relationship between neurodegeneration, genesis of SRSs and

behavioural alterations ................................................................................................. 257




References .................................................................................................................................. 261

Appendices ................................................................................................................................. 308

Appendix I: Literature comparison .................................................................................. 308

Appendix II: Temporal reduction in neuron densities within regions of the

hippocampus, thalamus, amygdala and piriform cortex .................................................. 322

Appendix III: Convolution analyses .................................................................................. 326

3.1 Assessment of performance based on shift in strategy use ....................................... 326

3.2 Assessment of performance based on improved efficacy within each strategy ........ 327

xv

List of Tables

Table 1.1 Scoring system for pilocarpine-induced seizures ......................................................... 16

Table 3.1 Comparison of rat strain and SE-inducing protocols between SE induction and

mortality rates at 3 days following SE ..................................................................................... 55

Table 4.1 The effect of SE on the area (mm2) of the hippocampus, thalamus and amygdala ...... 81

Table 4.2 Temporal progression of brain regions exhibiting initial neuronal loss significantly

different from corresponding shams. ..................................................................................... 116

Table 4.3 Temporal progression of brain regions exhibiting maximal neuronal death

following ................................................................................................................................ 117

Table 5.1 Comparison of the effect of treatment on mortality, seizure severity and weight

gain following SE .................................................................................................................. 124

Table 5.2 Comparison of SE-induced pyramidal cell loss in individual counting frames ......... 126

Table 6.1 Comparison of experimental groups in seizure susceptibility and mortality at 3

months following SE .............................................................................................................. 151

Table 7.1 Performance as a function of strategy use in hidden platform Morris water maze

testing ..................................................................................................................................... 199

Table 7.2 Comparison of RLDP SE rats that exhibit differences in Morris water maze

performance ............................................................................................................................ 203

Table 8.1 Consequences of neuroprotective drug treatment ....................................................... 242

xvi

List of Figures

Figure 1.1 Epileptogenesis is caused by an initial precipitating injury .......................................... 4

Figure 3.1 Placement of counting frames within the hippocampus, hilus and piriform cortex .... 52

Figure 3.2 Comparison of behavioural seizure activity between rat strain and SE-inducing

protocol .................................................................................................................................... 57

Figure 3.3 Comparison of neuron cell densities in the hippocampus and piriform cortex of

LEH and Wistar rats following SE........................................................................................... 60

Figure 3.4 Comparison of neuronal cell densities in the hippocampus and piriform cortex of

Wistar rats following SE .......................................................................................................... 62

Figure 4.1 Placement of counting frames .................................................................................... 76

Figure 4.2 The area size of different brain regions assessed ........................................................ 78

Figure 4.3 Anti-NeuN immunohistochemical staining decreases following SE in the dorsal

and ventral hippocampus .......................................................................................................... 83

Figure 4.4 Confocal micrographs (400X) of NeuN stained cells in hippocampal subfields ........ 85

Figure 4.5 Total length of dentate gyrus remains constant following lithium/pilocarpine

induced SE ............................................................................................................................... 87

Figure 4.6 Temporal profiles of neuronal loss in hippocampal subfields following

lithium/pilocarpine induced SE ................................................................................................ 89

Figure 4.7 Anti-NeuN immunohistochemical staining decreases following SE in several

thalamic nuclei ........................................................................................................................ 92

Figure 4.8 Confocal micrographs (400X) of NeuN stained cells in several thalamic nuclei ....... 94

Figure 4.9 Temporal profiles of neuronal loss in several thalamic nuclei following

lithium/pilocarpine induced status epilepticus (SE) ................................................................. 96

Figure 4.10 Anti-NeuN immunohistochemistry staining decreases following SE in several

amygdaloid nuclei and in the posterior piriform cortex ........................................................... 99

Figure 4.11 Confocal micrographs (400X) of NeuN stained cells in several amygdaloid

nuclei and in the piriform cortex ............................................................................................ 101

Figure 4.12 Temporal profiles of neuronal loss in several amygdaloid nuclei and in the

posterior piriform cortex ........................................................................................................ 103

Figure 4.13 Confocal micrographs (400X) of Fluoro-jade B (FJB) stained cells present at 24

hours but not at 3 months after SE in the hippocampus, thalamus and amygdala ................. 105

xvii

Figure 4.14 The number of damaged brain regions as a function of increasing recovery time

after 60-min of SE .................................................................................................................. 114

Figure 5.1 Neurodegeneration depicted in NeuN-stained coronal sections of the rat dorsal

hippocampus and posterior piriform cortex (PPC) 14 days following SE ............................. 127

Figure 5.2 Tat-NR2B9c reduces pyramidal cell loss in the dorsal hippocampus when

administered 3 hours after SE ................................................................................................ 130

Figure 5.3 Tat-NR2B9c exhibits differential neuroprotection within different regions of the

CA1 and CA3 subfields of the hippocampus ......................................................................... 133

Figure 5.4 Tat-NR2B9c is not neuroprotective when administered 10 minutes following the

onset of SE ............................................................................................................................. 136

Figure 6.1 Schematic of SE-induction protocols and treatments in SE and non-SE groups ...... 146

Figure 6.2 The effect of SE on behaviour in the open field ........................................................ 153

Figure 6.3 The effect of SE on behaviour in the four hyperexcitability tests ............................. 156

Figure 6.4 The effect of SE on behaviour in the elevated-plus maze ......................................... 159

Figure 6.5 Tat-NR2B9c had no effect on behaviour in the open field ....................................... 162

Figure 6.6 Tat-NR2B9c had no effect on behaviour in the four hyperexcitability ..................... 164

Figure 6.7 Tat-NR2B9c had no effect on behaviour in the elevated-plus maze ......................... 166

Figure 7.1 Behavioural categories .............................................................................................. 181

Figure 7.2 Number of trials performed to reach criterion during visible platform

testing .......................................................................................................................................... 184

Figure 7.3 The effect of SE on hidden platform testing in the Morris water maze .................... 187

Figure 7.4 Number of platform crossings in spatial acquisition and spatial reversal probe

trials and swim speed ............................................................................................................. 190

Figure 7.5 The effect of SE on the distribution of search strategies used during spatial

acquisition and spatial reversal testing ................................................................................... 194

Figure 7.6 Summary of search strategy use between groups during Morris water maze testing 196

Figure 7.7 SE results in differential impairment in Morris water maze performance ................ 204

Figure 7.8 SE results in differential use of search strategies during Morris water maze testing 206

Figure 7.9 Tat-NR2B9c did not improve performance in SE rats during hidden platform

learning ................................................................................................................................... 209

xviii

Figure 7.10 Tat-NR2B9c has no effect on the distribution of search strategies used during

spatial acquisition in rats following SE .................................................................................. 211

xix

List of Appendices

Appendix I: Literature comparison ....................................................................................... 308

Table A1-1 Summary of studies assessing severity and progression of neuronal loss after

pilocarpine-induced SE in the hippocampus, thalamus, amygdala and

piriform cortex ........................................................................................................... 309

Table A1-2 Summary of studies investigating the effect of status epilepticus on behaviours of

anxiety, aggression and exploration in rodents .......................................................... 310

Table A1-3 Summary of studies investigating the effect of status epilepticus on visual-spatial

learning and memory ................................................................................................. 314

Appendix II: Temporal reduction in neuron densities within regions of the

hippocampus, thalamus, amygdala and piriform cortex .................................................. 322

Table A2-1 Reduction in neuronal densities within hippocampal regions following 60 min of

SE ............................................................................................................................... 323

Table A2-2 Reduction in neuronal densities within several thalamic nuclei following 60 min of

SE ............................................................................................................................... 324

Table A2-3 Reduction in neuronal densities within several amygdaloid nuclei and posterior

piriform cortex following 60 min of SE .................................................................... 325

Appendix III: Convolution analyses ....................................................................................... 326

3.1 Assessment of performance based on shift in strategy use ................................................... 326

3.2 Assessment of performance based on improved efficacy within each strategy .................... 327

xx

List of Abbreviations AD afterdischarges

Ave average

ºC degree Celsius

ACh acetylcholine

AMPA α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid

ANOVA one-way analysis of variance

BDZ benzodiazepine

CNS central nervous system

Cy5 cyanine dye 5

DAG diacylglycerol

DGCs dentate granule cells

EEG electroencephalogram

EPM elevated plus maze

FJ fluoro-jade

FJB fluoro-Jade B

g gram

GABA gamma-aminobutyric acid

GAERS genetic absence epilepsy rats from Strasbourg

GCL granule cell layer

GFAP glial fibrillary acidic protein

hr hour

HS hippocampal sclerosis

ILAE The international league against epilepsy

IMPase inositol monophosphatase

i.p. intraperitoneal injection

IPI initial precipitating injury

KA kainic acid

kg kilogram

LTP long-term potentiation

LiCl lithium chloride

LDP low-dose lithium/pilocarpine

LEH Long Evans hooded

M molar (mole/liter)

mEq milliequivalent

MPEP 2-methyl-6(pehnylethynyl)pyridine

MTLE mesial temporal lobe epilepsy

MTLE-HS mesial temporal lobe epilepsy with hippocampal sclerosis

mg milligram

mGluR5 metabotropic glutamate receptor 5 antagonist

M molarity

min minutes

ml milliliter

mm millimeter

mM millimolar

MWM Morris water maze

NeuN neuronal nuclear protein

xxi

nNOS neuronal nitric oxide synthase

NSC neuronal stem cells

NMDA N-methyl-D-aspartate

NMDAR N-methyl-D-aspartate receptor

PBS phosphate buffered saline

PTLE paradoxical temporal lobe epilepsy

IP3 inositial triphosphate

PI phosphatidylinositol cycle

PIP2 phosphatidylinositol 4,5-bisphosphate

PLCβ phospholipase C-beta

PSD-95 postsynaptic density protein-95

RLDP Repeated low-dose lithium/pilocarpine

s.c. subcutaneous

SE status epilepticus

SRS spontaneous recurrent seizure

TLE temporal lobe epilepsy

SD standard deviation

SEM standard error of means

SE status epilepticus

μg microgram

μm micrometer

w/v weight/volume

(v/v) volume/volume

For brain regions:

BLP basolateral amygdaloid nucleus, posterior part

BMP basomedial amygdaloid nucleus, posterior part

CM central medial thalamic nucleus

DG dentate gyrus

DMD dorsomedial hypothalamic nucleus, dorsal part

DMV dorsomedial hypothalamic nucleus, ventral part

d3V dorsal 3rd

ventricle

3V 3rd

ventricle

LaDL lateral amygdaloid nucleus, dorsolateral part

LaVM lateral amygdaloid nucleus, ventrolateral part

LDDM laterodorsal thalamic nucleus, dorsomedial part

LDVL laterodorsal thalamic nucleus, ventrolateral part

LV lateral ventricle

MD mediodorsal thalamic nucleus

MePV medial amygdaloid nucleus, posteroventral part

MePD medial amygdaloid nucleus, posterodorsal part

MTu medial tuberal nucleus

Rt reticular thalamus nucleus

Pir Pirform cortex

PMCo posteromedial cortical amygdaloid nucleus

xxii

Po posterior thalamic nuclei

PoDG` polymorph layer of the dentate gyrus

PPC posterior piriform cortex

pyr pyramidal cell layer

STh subthalamic nucleus

STIA the bed nucleus of the stria terminalis, intraamygdaloid division

VPL ventral posterolateral thalamic nucleus

VPM ventral posteromedial thalamic nucleus

1

Chapter 1

General Introduction

1.1 Main features of mesial temporal lobe epilepsy with hippocampal sclerosis

Epilepsy is the most frequent neurodegenerative disease after stroke (Acharya et al., 2008), and

accounts for a significant portion of the disease burden worldwide (de Boer et al., 2008). It

afflicts more than 170,000 Canadians (5.2 – 5.6 per 1000 population) (Tellez-Zenteno et al.,

2004) and at least 50 million people globally (de Boer et al., 2008). The International League

against Epilepsy (ILAE) proposed the definitions of epileptic seizures and epilepsy, stating that

―an epileptic seizure is a transient occurrence of signs and/or symptoms due to abnormal

excessive or synchronous neuronal activity in the brain. Epilepsy is a disorder of the brain

characterized by an enduring predisposition to generate epileptic seizures and by the

neurobiological, cognitive, psychological, and social consequences of this condition. The

definition of epilepsy requires the occurrence of at least one epileptic seizure (Fisher et al.,

2005).‖ The current ILAE classification of epileptic seizures was approved in 1981, and later

extended for the classification of epilepsies and epilepsy syndromes in 1989 (Commission on

classification and terminology of the international league against epilepsy. 1989). Revisions

have since been proposed to update the nomenclature to reflect modern neuroimaging, genomic

technologies and basic sciences (reviewed in: Engel, 2001; Seino, 2006; Berg et al., 2010).

The 1989 classification divides epilepsies into generalized and focal epileptic seizures,

depending on whether the characteristic seizures begin simultaneously on both sides of the brain

or are confined to one hemisphere (Commission on classification and terminology of the

international league against epilepsy, 1989). Focal epileptic seizures may spread to both

hemispheres and become secondarily generalized. Epilepsy syndromes are further classified into

idiopathic, symptomatic and cryptogenic categories based on their underlying etiologies; these

terms were recently changed to genetic, structural/metabolic and unknown cause, respectively

(Berg et al., 2010). More than half of the epilepsies are classified as structural/metabolic

disturbances (Engel and Schwartzkroin, 2006); the most common of these, and also the most

common form of human epilepsy is mesial temporal lobe epilepsy with hippocampal sclerosis

2

(MTLE-HS) (Wieser, 2004). MTLE-HS is characterized by focal epileptic seizures with or

without secondary generalization originating from the mesial temporal lobe (Margerison and

Corsellis, 1966; King and Spencer, 1995; Bartolomei et al., 2005; Bertram, 2009). While this

review focuses on the characteristics of MTLE-HS, an extensive list of other epilepsies, epileptic

syndromes and related seizure disorders are extensively reviewed elsewhere (Engel, 2001;

Chabolla, 2002; Engel and Schwartzkroin, 2006; Seino, 2006; Berg et al., 2010).

The main features of MTLE-HS are: (i) the localization of seizure foci in the limbic system,

particularly in the hippocampus, entorhinal cortex, and amygdala (Margerison and Corsellis,

1966; King and Spencer, 1995; Bartolomei et al., 2005; Bertram, 2009); (ii) the frequent finding

of an initial precipitating injury (IPI) that precedes the appearance of mesial temporal lobe

epilepsy (MTLE) (Mathern et al., 2002; Mathern et al., 1996); (iii) a seizure-free time interval

following the IPI known as epileptogenesis or latent phase ( Engel, 1993; Wieser, 2004); (iv) a

high incidence of hippocampal sclerosis (HS) (also referred to as mesial temporal sclerosis or

Ammon‘s horn sclerosis) (Babb, 1986; Babb and Brown, 1986; Babb et al., 1991; Sharma et al.,

2007) and; (v) a high prevalence of interictal behavioural disturbances and cognitive impairment

(Boro and Haut, 2003; Devinsky, 2004a; Gaitatzis et al., 2004; Swinkels et al., 2005; Cornaggia

et al., 2006; Marcangelo and Ovsiew, 2007; Garcia-Morales et al., 2008). As illustrated in

Figure 1.1, neuronal loss and synaptic reorganization are proposed to be the cause of chronic

epileptic seizures and interictal behavioural and cognitive morbidity (Wieser, 2004; Sharma et

al., 2007; Acharya et al., 2008). Even though these conditions exist prior to development of

MTLE-HS as a consequence of the IPI (Mathern et al., 1996; Wieser, 2004), several studies have

suggested that repeated ictal events may contribute to further deterioration (Mathern et al., 1996;

Wieser, 2004; Bernasconi et al., 2005).

The main features of MTLE-HS are recapitulated in chronic animal models of temporal lobe

epilepsy (TLE), particularly in the kindling and status epilepticus (SE) models. The subject of

this thesis, the lithium/pilocarpine model, belongs to SE models. The lithium/pilocarpine model

has been used in many laboratories to investigate the pathogenesis of MTLE-HS and to evaluate

the efficacy of anti-epileptogenic drugs (reviewed in: Leite et al., 2002; Curia et al., 2008;

Scorza et al., 2009). This introduction begins with a brief overview of the commonly used

animal models of TLE. The subsequent sections provide an overview on background

information and important features of the lithium/pilocarpine model, including: (i) the

3

occurrence of SE as the IPI, (ii) the presence of a latent period followed by the appearance of

spontaneous recurrent seizures (SRSs), (iii) the occurrence of widespread neurodegeneration

(including hippocampal sclerosis) and synaptic reorganization, and (iv) the development of

interictal behavioural disturbances and cognitive impairment.

4

Figure 1.1: Epileptogenesis is caused by an initial precipitating injury. During this period,

neurodegeneration and synaptic reorganization occur and contribute to the development of

recurrent seizures and behavioural and cognitive dysfunction. Recurrent seizures may also

contribute to additional morbidity and pathophysiological changes. Different factors can affect

seizure development and cognitive and behavioural outcomes.

5

6

1.2 Animal models of seizure development and epilepsy

The two most commonly used animal models to investigate epileptogenesis and MTLE-HS are

kindling and SE. Although major procedural differences exist between the two models, and each

has its own characteristics, both are capable of inducing a persistent, epileptic-like condition

(reviewed in: Leite et al., 2002; Loscher, 2002; McIntyre et al., 2002; Morimoto et al., 2004;

Martin and Pozo, 2006; Sharma et al., 2007). The major emphasis of this thesis is SE. The

ILAE defined SE in 1964 as ‗a seizure that persists for a sufficient length of time or is repeated

frequently enough to produce a fixed and enduring epileptic condition‘ (Arnautova and

Nesmeianova, 1964). Although a specific length of time is not specified in the definition, many

investigators have traditionally identified SE as prolonged seizures lasting 30 minutes or longer,

the time necessary to produce neuronal death in animal models of SE (DeLorenzo et al., 1999;

Fujikawa, 2005; Knake et al., 2009); others have argued, however, that SE should be defined as

seizures lasting 5 minutes or longer for several reasons: (1) the relationship between seizure

activity and neuronal loss in humans is not well understood, (2) self-terminating seizures rarely

last longer than 2 minutes, and (3) earlier medical treatment of SE is likely to reduce the

mortality and morbidity associated with this condition (Lowenstein et al., 1999; Meldrum, 1999).

Despite this ambiguity, SE rodent models have significantly contributed to our understanding of

the pathophysiological mechanisms underlying SE, and this is discussed in detail in the

subsequent sections. Several extensive reviews related to the kindling model are available

(McIntyre et al., 2002; Morimoto et al., 2004; Sutula and Ockuly, 2006), and this model is only

briefly described here.

1.2.1 Kindling

Induction of epilepsy by kindling involves periodic application of a brief stimulus that evokes

repetitive epileptic spikes (an afterdischarge, or AD) (reviewed in: McIntyre et al., 2002;

Morimoto et al., 2004; Sutula and Ockuly, 2006). Over repeated stimulations, the duration of

the evoked ADs and the intensity of behavioural seizures increase, while the stimulus threshold

to evoke epileptiform activity decreases. This process results in an overall increase and long-

lasting susceptibility of the animal to additional seizures. Most studies terminate kindling once

an animal exhibits a few secondary generalized (stage v) seizures (kindling scale described by

Racine, 1972). The gradual development and progression of epileptogenesis by kindling allows

7

investigators to reliably quantify both electrographic measures (AD number and duration) and

behavioural responses (seizure stages and number of class v seizures). Because this process

occurs in the absence of overt brain damage, it may be an ideal model for studying paradoxical

TLE (defined in section 1.5). A limitation of the kindling model may be that the standard stage v

criterion results in an incomplete epileptogenesis in which SRSs do not occur. A minimum of 90

to 100 kindled seizures beyond the stage v criterion is required in rats for development of SRSs

(Sutula and Ockuly, 2006). This process, referred to as over-kindling, is rarely used because of

the intensive work and time involved. Of particular interest, over-kindled rats exhibit similar

neuropathological changes to those found in SE models (Sutula and Ockuly, 2006).

1.2.2 Post-status epilepticus models

In contrast to kindling, SE is easier to produce and reliably generates epileptic animals. SE can

be induced by electrical stimulation of limbic brain structures (e.g., amygdala and hippocampus),

or by systemic administration of chemoconvulsants (e.g., kainate or pilocarpine) (reviewed in:

Leite et al., 2002; Loscher, 2002; Morimoto et al., 2004; Martin and Pozo, 2006; Sharma et al.,

2007). Loscher (2002) referred to models that involve SE as post status epilpeticus models,

since the latent phase that follows SE and precedes SRSs is of most interest to many researchers.

Pathological changes that occur during this period are proposed to contribute to the development

of epilepsy; therefore, therapeutic treatments that affect these changes may also be anti-

epileptogenic, and prevent ‗at risk‘ individuals from developing epilepsy (Loscher, 2002). The

morphological damage that occurs in animal models of SE is very similar to that seen in human

MTLE-HS, although the damage in SE models can be more severe and widespread (Sharma et

al., 2007). The use of electrical stimulation to induce SE is described as being more labour-

intensive and thus less desirable as a model as compared to the use of chemoconvulsants

(Sharma et al., 2007). However, high mortality rates are a disadvantage in some kainate and

pilocarpine models, but can be minimized by administering lower doses of the chemoconvulsant

(Hellier et al., 1998; Glien et al., 2001). In the present thesis we used pilocarpine in conjunction

with lithium chloride (LiCl) to induce SE.

8

1.3 Background information on the pilocarpine and lithium/pilocarpine models

The pilocarpine model, especially in combination with LiCl, reproduces many clinical and

morphological aspects of MTLE-HS in rodents. These features are outlined in section 1.1 and

are further described in the subsequent sections. A major drawback to the pilocarpine and

lithium/pilocarpine models is high mortality. However, mortality can be limited by modification

of the SE-inducing procedure and by controlling SE duration. In this section, background

information and cellular mechanisms underlying SE induction in the pilocarpine and

lithium/pilocarpine models are reviewed.

1.3.1 The pilocarpine model

The pilocarpine model was first used in rats (Turski et al., 1983a; Turski et al., 1983b) and then

in mice (Turski et al., 1984) to produce limbic seizures. Pilocarpine is a muscarinic

acetylcholine receptor agonist. Several extensive reviews describe a variety of protocols

available for administering pilocarpine to induce SE, and these are briefly described here

(Cavalheiro et al., 2006; Curia et al., 2008; Scorza et al., 2009). With systemic administration,

the pilocarpine dose necessary to induce SE ranges from 300 to 400 mg/kg in adult rats (Clifford

et al., 1987; Liu et al., 1994; Melloa and Mendez-Oterobs, 1996). Lower pilocarpine doses (100

- 200 mg/kg) occasionally produce brief limbic seizures, but do not result in SE (Turski et al.,

1983b). The dose of pilocarpine administered to induce SE significantly affects seizure

development, survival rates, and neuropathology. When compared to lower doses of pilocarpine

(350 mg/kg, i.p.), higher doses (380 – 400 mg/kg, i.p.) have resulted in a reduced latency to SE

onset and a greater percentage of rats developing SRSs (Clifford et al., 1987; Liu et al., 1994).

However, higher doses of pilocarpine have also resulted in greater mortality rates, and this may

be caused by the greater convulsive seizure severity and brain damage observed in these animals

(Clifford et al., 1987; Liu et al., 1994). Pilocarpine can also induce SE in rats after

intracerebroventricular (Croiset and De Wied, 1992) and intrahippocampal (Furtado et al., 2002)

administration.

9

1.3.2 Convulsive effects of pilocarpine are mediated by activation of M1 receptors

The cholinergic system is involved in the initiation of SE following pilocarpine treatment, as

pretreatment of the animals with scopolamine (a muscarinic antagonist) prevents the

development of convulsive seizures (Turski et al., 1983a). Other cholinomimetics, such as

carbachol and oxotremorine, are also able to induce seizures and seizure-induced brain damage

(Turski et al., 1983a; Olney et al., 1986). Subsequent studies have showed that the ability of

pilocarpine to induce SE is dependent on activation of the M1 muscarinic receptor subtype, since

M1 receptor knockout mice do not develop seizures in response to pilocarpine (Hamilton et al.,

1997), and epileptic activity is blocked by pirenzepine, an M1-specific antagonist (Maslansky et

al., 1994). Various mechanisms downstream of M1 receptor activation has been proposed to

account for the effect of pilocarpine on neuronal excitability, including activation of

phospholipase C-beta (PLCβ) (Scarr, 2009) and/or Src kinase (Rosenblum et al., 2000; Murthy,

2008), both of which activate subsequent downstream events resulting in enhanced excitability.

Each of these signaling mechanisms is briefly described.

1. M1 receptors are coupled to Gq/G11-type G proteins, which are in turn coupled to the

activation of PLCβ (reviewed in: Wess et al., 2007; Scarr, 2009). PLCβ cleavage of

phosphatidylinositol 4,5-bisphosphate (PIP2) generates inositial triphosphate (IP3) and

diacylglycerol (DAG) (Berridge, 2009), resulting in an alteration in a Ca2+

and K+ current

and increasing the excitability of the brain (Segal, 1988). A well-known mechanism of

cholinergic excitation involves the M1-mediated depletion of PIP2, which results in

closure of voltage-gated K+ channels (termed M-channels), since the presence of PIP2 is

necessary for the open state of these channels to remain stabilized (reviewed in: Brown,

2010).

2. An alternative mechanism by which M1 receptors may generate excitatory responses

independent of PLCβ signaling involves Src kinase activation (Rosenblum et al., 2000).

Activation of M1 receptors can induce elevation of intracellular Ca2+

, which stimulates

Src kinase activation (Felder, 1995). Src kinase is able to subsequently phosphorylate

other signaling molecules, including soluble guanylyl cyclase (Murthy, 2008) and

extracellular signal-regulated kinase (ERK) (Rosenblum et al., 2000), both of which have

10

been implicated in cholinergic excitation (Rosenblum et al., 2000; Kuzmiski and

MacVicar, 2001).

1.3.3 The cholinergic system is involved in the initiation but not the maintenance of SE

The development of SE requires a pool of neurons capable of initiating and sustaining abnormal

firing (Noe and Manno, 2005). The generation of synchronized neuronal activity is facilitated by

the loss of inhibitory synaptic transmission mediated by GABA and sustained by the excitatory

transmission mediated by glutamate (Smolders et al., 1997; Priel and Albuquerque, 2002; Noe

and Manno, 2005; Meurs et al., 2008). Experiments in cultured hippocampal neurons have

demonstrated that pilocarpine acting through muscarinic receptors causes an imbalance between

excitatory and inhibitory transmission resulting in the generation of SE (Priel and Albuquerque,

2002). Furthermore, in-vivo microdialysis has showed that pilocarpine induces an elevation in

glutamate levels in the hippocampus following the appearance of seizures (Smolders et al., 1997;

Meurs et al., 2008). The increase in neuronal activity leads to a loss of inhibition by accelerated

internalization of the GABAA receptors (reviewed in: Goodkin et al., 2005). Once seizures are

initiated, their maintenance depends on mechanisms distinct from muscarinic receptors, since

atropine becomes ineffective in controlling established seizures (Clifford et al., 1987; Curia et

al., 2008). Substantial evidence now supports the idea that following the activation of

muscarinic receptors, SE is maintained by activation of the N-methyl-D-aspartate (NMDA)

receptors (Nagao et al., 1996; Smolders et al., 1997; Deshpande et al., 2008).

1.3.4 The lithium/pilocarpine model

Honchar et al., (1983) reported that pretreatment of rats with LiCl (3 mEq/kg) permits the dose

of pilocarpine to be decreased approximately 10-fold (30 mg/kg, i.p.; referred to as the low-dose

lithium/pilocarpine (LDP) protocol in this thesis). This procedure resulted in lower mortality

rates and produced SE more reliably when compared to systematic administration of pilocarpine

alone (Clifford et al., 1987; Sharma et al., 2007). The effects of lithium are specific to the

cholinergic system since SE induced by the nerve agent soman, a cholinesterase inhibitor, is also

potentiated by lithium; however, SE induced by glutamate agonists NMDA and kainic acid, or

the gamma-aminobutyric acid (GABA) antagonists bicuculline and pentylenetetrazole are not

(McDonough et al., 1987; Ormandy et al., 1991). Neither lithium (3 mEq/kg) nor pilocarpine

11

(30 mg/kg) causes abnormal electrographic responses when administered alone (Clifford et al.,

1987; Jope and Gu, 1991). Lithium pretreatment is only effective if pilocarpine is administered

within 24 hours; the proconvulsive effects of lithium becomes less effective if pilocarpine is

administered after 24 hours, and no seizures develop after 48 hours (Clifford et al., 1987). The

lithium/pilocarpine and pilocarpine models produce similar behavioural, electrographical and

neuropathological alterations in rats following SE (Clifford et al., 1987).

Glien et al., (2001) further modified the lithium/pilocarpine model. If pilocarpine was

administered as a single dose of 30 mg/kg in lithium-pretreated rats, and SE duration was limited

to 90 min, mortality was 45% and 80% of survivors developed SRSs. However, if pilocarpine

was administered in divided doses of 10 mg/kg at 30-min interval until SE ensued (referred to as

the repeated low dose lithium/pilocarpine (RLDP) protocol in the present thesis), the mortality

rate was reduced to 7% and 85% of animals that survived SE developed SRSs. The reduced

mortality is attributed to the titrated administration of pilocarpine that accommodates for

intrastrain differences in sensitivities of individual rats to the chemoconvulsive properties of

pilocarpine (Glien et al., 2001). There was no significant difference in the induction rates of SE

between rats treated with the LDP (total of 73%) and the RLDP (total of 61%) procedures.

1.3.5 The proconvulsive mechanisms of lithium

Recent interest in determining lithium‘s physiological role in potentiating the convulsive effects

of pilocarpine stems from its role in the treatment of manic-depressive illness (Belmaker and

Bersudskya, 2007; Agam et al., 2009). Here, the main hypothesis regarding lithium‘s therapeutic

and prophylactic effect in affective disorder is that inhibition of inositol monophosphatase

(IMPase) by lithium impairs the operation of the phosphatidylinositol cycle (PI cycle) (see

reviews: Osborne et al., 1988; Berridge and Irvine, 1989; Jope and Williams, 1994; Haim and

Belmaker, 2001; Belmaker and Bersudskya, 2007). Lithium at therapeutic doses (3 mEq/Kg)

inhibits rat brain IMPase, thereby resulting in a reduction in inositol and accumulation of inositol

monophosphate (Belmaker and Bersudskya, 2007). In the absence of IMPase, lithium no longer

potentiates the effect of pilocarpine. For instance, Impa1 (encoding IMPase) knock-out mice

without lithium pre-treatment behaved similarly to lithium pre-treated rats receiving a single i.p.

subthreshold dose of pilocarpine (30 mg/kg, i.p.) (Agam et al., 2009). Because lithium inhibits

IMPase, inositol becomes less available for re-synthesis of PIP2 and the consequently reduces

12

PIP2 levels (Belmaker and Bersudskya, 2007; Brown, 2010); this increases membrane

excitability of the neuron in response to pilocarpine as described in section 1.3.2.

1.3.6 Use of diazepam to control SE duration and reduce mortality

In the pilocarpine model, SE spontaneously remits within 5 to 6 hours after initiation (Turski et

al., 1989; Lemos and Cavalheiro, 1995); however, this is often associated with exceedingly high

mortality rates (Curia et al., 2008). Limiting SE duration can improve long-term survival in rats

following SE; this is often accomplished by administrating diazepam, a GABAA agonist (Glien

et al., 2001). Curia et al., (2008) reported that, following induction of pilocarpine-induced SE,

diazepam effectively decreased mortality when administered 30, 60, 120 or 180 min after SE

induction. In addition, diazepam terminated behavioural and electrographic seizures when

administered up to 4 hrs following the initiation of SE in several seizure models (Brandt et al.,

2003a; Brandt et al., 2006; Goffin et al., 2007). The anticonvulsant action of diazepam against

pilocarpine-induced seizures was associated with prompt attenuation of extracellular glutamate

concentrations in the hippocampus (Khan et al., 1999).

1.4 Behavioural and clinical features of seizure development in the lithium/pilocarpine model

Retrospective studies show that a large proportion of patients with mesial temporal lobe epilepsy

(MTLE) undergo an IPI, including febrile convulsions, SE, encephalitis, stroke or traumatic

brain injury (Mathern et al., 1996; Mathern et al., 2002). Up to 80% of adults with MTLE are

reported to have presented childhood SE or prolonged febrile seizures (French et al., 1993;

Cendes and Andermann, 2002). In the majority of cases, SRSs appeared after a 5- to 10-year

latent period (Engel, 1993). Similarly, an epidemiologic study reported that up to 42% of

individuals with SE as their first seizure (mean age 39.7) developed epilepsy over the next 10

years (Hesdorffer et al., 1998). The pilocarpine model closely mimics the clinical manifestations

of MTLE in humans, in which an acute triggering process is frequently followed by a latent

phase and subsequent development of recurrent seizures (see Figure 1.1). An acute episode of

SE serves as the IPI (Cavalheiro et al., 2006; Curia et al., 2008; Scorza et al., 2009). Three

stages of seizure development following SE have been described below. These are the acute

phase, epileptogenesis, and the chronic phase.

13

1.4.1 The acute phase

The acute phase includes the initial occurrence of repetitive seizures, including SE, and is

typically considered to involve the initial 24-hours following seizure induction (Scorza et al.,

2009).

1.4.1.1 Behavioural seizures during the acute phase

The progression of behavioural changes following pilocarpine injection is very similar in the

pilocarpine (Turski et al., 1983b) and lithium/pilocarpine models (Clifford et al., 1987; Glien et

al., 2001). Behavioural manifestations increase with time following pilocarpine and may be

divided into 4 phases (Turski et al., 1983b; Clifford et al., 1987):

1. The first phase of behaviour changes occurs from peripheral cholinergic stimulation

within the first several minutes after pilocarpine injection. These include piloerection,

salivation, tremor, chromadacryorrhea and diarrhea.

2. In the second phase, a series of stereotyped behaviours including oro-facial movements,

salivation, eye-blinking, twitching of vibrissae and yawning subsequently develops and

persists for up to 45 min following pilocarpine injection. Animals also show a

predilection for remaining in one corner of the cage with upward extension of the nose

and neck. During the first and second phases of behavioural changes, animals are able to

be distracted by tactile or sound stimulation (Clifford et al., 1990).

3. Behavioural alterations in phase 1 and 2 subside with development of limbic motor

seizures in phase 3. These seizures are characterized by intense salivation, rearing, upper

extremity clonus, and falling, and reoccur every 3 to 15 min.

4. During phase 4 of behavioural changes, SE typically develops soon following the initial

limbic seizures (Turski et al., 1983b; Clifford et al., 1987). During SE, rats are

unresponsive to external touch and sound stimulation (Clifford et al., 1987).

In the absence of specific measures to terminate seizures (see section 1.3.6) SE spontaneously

remits within 5 to 6 hours (Turski et al., 1983b; Clifford et al., 1987). Animals are typically

found to be in a comatose state for the 24 hour period following cessation of SE.

14

1.4.1.2 Electroencephalographic patterns during motor limbic seizures and SE

Pilocarpine produces both ictal and interictal epileptiform activity in the EEG recordings, and

changes in electrographic patterns correlate well with the sequence of behavioural alterations

described in section 1.4.1.1 (Clifford et al., 1987; Turski et al., 1989; Leite et al., 1990).

Immediately following pilocarpine injection, low-voltage, fast activity with spikes appears in the

neocortex and amygdala, while a clear theta rhythm appears in the hippocampus. This EEG

pattern is correlated with phase 1 of behavioural changes (see 1.4.1.1). When behavioural

manifestations become more severe (phase 2), high voltage, fast EEG activity replaces the

hippocampal theta rhythm. Electrographic seizures characterized by high voltage, fast activity

and prominent spiking precede limbic motor seizures (phase 3), and are proposed to result from

muscarinic system activation (van Der Linden et al., 1999). This activity originates in the

hippocampus and propagates to the neocortex and amygdala (Turski et al., 1983b; Turski et al.,

1989; Leite et al., 1990). Sustained electrographic discharges occur during SE (phase 4), and

after several hours, evolve to a pattern of periodic discharges on a relatively flat background.

Studies using 14

C-2-deoxyglucose functional mapping show that specific brain structures are

associated with the different behavioural changes (Lothman and Collins, 1981; Handforth and

Ackermann, 1988; Lothman et al., 1991; Handforth and Ackermann, 1995; Handforth and

Treiman, 1995). Table 1.1 summarizes the relationship between seizure severity and the

activation of specific brain regions (see review: Veliskova, 2006). Of particular interest, brain

regions that show the highest metabolic activation during SE also exhibit the most severe

neuronal loss in the latent phase (Ingvar, 1986; Ingvar et al., 1987; Handforth and Ackermann,

1992; Handforth and Ackermann, 1995; Fernandes et al., 1999; Bouilleret et al., 2000).

1.4.1.3 Scoring of pilocarpine-induced seizures

In this thesis, the severity of seizures was assessed using a modified version of the Racine‘s scale

(1973), which at first was developed to score kindled seizures in adult rats. As summarized in

Table 1.1, stages I to V are the classical stages described by Racine (1972). Levels VI and VII

have been additionally added to the modified scale. Level VI is achieved with the occurrence of

two or more level V seizures (rearing and multiple falls) (Cammisuli et al., 1997). Level VII

consists of tonic-clonic seizures (Veliskova, 2006). The scale is based on specific characteristics

15

of epileptic seizures in the pilocarpine model, and the progression of seizure activity resulting

from gradual involvement of distinct neural networks (Veliskova, 2006).

Stages I and II involve the spread of paroxysmal activity from the original structure (e.g.,

hippocampus) to other limbic regions (Lothman and Collins, 1981; Handforth and Ackermann,

1988; Lothman et al., 1991; Handforth and Ackermann, 1995; Handforth and Treiman, 1995). In

stage III, the occurrence of clonic seizures indicates activation of structures beyond the limbic

system, namely the neocortex, thalamus and basal ganglia (Engel et al., 1978; Lothman and

Collins, 1981; Browning and Nelson, 1986; Handforth and Ackermann, 1995; Handforth and

Treiman, 1995; Veliskova et al., 2005). Clonic seizures consist of rhythmic movement of

forelimbs that are often accompanied by facial clonus. The forelimb clonus can be either

unilateral (stage III) or coordinated bilateral clonus (stage IV) with or without rearing or tail

erection (Straub tail). In stage IV, the hind limbs are usually spaced apart and the animal appears

as if in a kangaroo position (Ono et al., 1990). During seizure stages V and VI, the hind limbs

can also be involved and the rat may lose balance temporarily; however, the animal will

immediately make an effort to get back to the upright position. The tonic-clonic seizures (stage

VII) represent a spread of paroxysmal activity from the forebrain to the brainstem (Browning and

Nelson, 1986). During this type of seizures, the righting reflex is lost. The tonic phase typically

involves tonic flexion and then extension of the forelimb, hindlimb or both with variable

duration. The tonic extension of hindlimbs represents the most severe seizure phase (Swinyard,

1973). After the tonic phase, long-lasting clonus of all limbs may develop. Wild running and

jumping in some rats may also be observed. Scoring systems specific to the type of seizures

generated by other models exist and are reviewed by others (Veliskova, 2006).

16

Table 1.1: Scoring system for pilocarpine-induced seizures

Seizure

stage 1

Behavioural expression Righting

reflex

Structures involved2

I Staring with mouth clonus Preserved Limbic structures

II Head nodding, automatisms (i.e.,

scratching, sniffing orientation)

III Unilateral forelimb clonus Preserved Other forebrain regions,

specifically neocortex,

thalamus and basal ganglia

IV Bilateral forelimb clonus (rearing)

V Forelimb clonus with rearing and

one fall

Brief loss of

postural

control

VI Forelimb clonus with rearing and

multiple falls

VII Tonic/clonic seizures. Infrequent

observation of wild running and

jumping with vocalization

Lost Brainstem

1. Seizure stages as described by Cammisuli et al., 1997 and by Veliskova, 2006. 2. Structures

mapped by 2-deoxyglucose studies and EEG patterns of specific brain regions (see section

1.4.2.2)

1.4.1.4 Effect of SE duration on mortality and neuropathology

SE duration is considered a critical factor in predicting outcome of SE in humans ( DeLorenzo et

al., 1992; Scholtes et al., 1994; Towne et al., 1994; DeLorenzo et al., 1999; Drislane et al.,

2009;). Towne et al., (1994) highlighted the prognostic significance of SE duration in reporting

a 2.7% mortality for episodes lasting less than an hour, but 32% for those lasting more than an

hour. DeLorenzo et al., (1999) reported that seizures lasting 10 to 29 min often stop on their

own, but very few seizures lasting over 30 min do so; mortality is substantially higher in the

latter group (DeLorenzo et al., 1999). Thirty to 60 min appears to be a critical time frame after

which the brain loses its ability to autonomously terminate seizure activity; beyond this time,

neurodegeneration and SE-related morbidity ensue (Scholtes et al., 1994; DeGiorgio et al., 1995;

DeGiorgio et al., 1999; DeLorenzo et al., 1999; Fujikawa, 2005; Nandhagopal, 2006; Knake et

al., 2009).

In accord with human data, a higher mortality rate is correlated with longer SE duration in the

pilocarpine and lithium/pilocarpine models (reviewed in section 1.3.6). Prolonged seizures are

17

also associated with increased refractoriness to benzodiazepine (BDZ) treatment, and may

account for the higher mortality since SE activity becomes more difficult to control with passing

time. Several extensive reviews related to pharmacoresistance to BDZ in several SE animal

models and in patients with MTLE or SE are available (Macdonald and Kapur, 1999; Avoli et

al., 2005; Loscher, 2007; Goodkin and Kapur, 2009).

A minimum of 40 to 60 minutes of continuous electrographic seizure discharges is required for

neuronal death to occur in rats (Nevander et al., 1985; Fujikawa, 1996). Fujikawa et al., (1996)

showed that no neuronal injury was present in rats after 20 min of SE, but increased substantially

between 40 and 60 min, and only slightly between 1 and 3 hrs. The proposed mechanisms of

neuronal death include excitotoxicity, energy impairment, free radical generation and apoptosis

(Fujikawa, 2005; Niquet et al., 2005); these processes are discussed in section 1.5. The duration

of SE is also critical to the development of SRSs and is discussed in section 1.4.2.1.

1.4.2 Epileptogenesis

Epileptogenesis, also referred to as the latent phase, is the period between the acute phase and the

occurrence of SRSs. This stage is characterized by a progressive normalization of EEG and an

absence of seizure activity. The animals are often in poor physical shape following cessation of

SE, and post-seizure care is critical to increase survival rates (Glien et al., 2001). Body weight

typically decreases after SE (10-20%), but recovers to pre-treatment values during the early

latent phase (Turski et al., 1989).

1.4.2.1 Duration of the latent phase

Several studies investigating the latency to development of SRSs have used a single high dose of

pilocarpine (320 – 400 mg/kg) to induce SE, and allowed SE to spontaneously remit (Cavalheiro

et al., 1991; Liu et al., 1994; Arida et al., 1999; Goffin et al., 2007). In these studies, continuous

video-EEG recordings were used to detect for the onset and progression of SRSs. The end of the

latent period was defined as the first occurrence of a stage IV or greater epileptic seizure. Latent

periods ranging between 3 days and 44 days were reported (Cavalheiro et al., 1991; Liu et al.,

1994; Arida et al., 1999). Arida et al., (1999) additionally showed a relationship between the

frequency of SRSs and the duration of the latent period. Shorter latent periods (3-5 days) were

18

associated with a greater number of SRSs (range 124-727 seizures in 135 days) than longer (28 –

30 days) latent periods (range 45 – 584 seizures in 135 days) (Arida et al., 1999).

Two lines of evidence indicate that SE duration affects the epileptogenic process. Unless

otherwise indicated, the following studies investigating this relationship have used the high-dose

pilocarpine model.

1. First, a minimum duration of SE is required for the development of chronic epilepsy.

Using diazepam to terminate SE, Klitgaard et al., (2002) reported that SRSs developed in

rats with a SE duration of 30 min, but not in rats with a SE duration of 7.5 and 15 min

(Klitgaard et al., 2002). On the other hand, Lemos and Cavalheiro (1995) reported that

SRSs developed in rats with a SE duration of 60 min, but not in rats with only 30 min of

SE; in this study, SE was terminated with combined treatment of diazepam (10 mg/kg,

i.p.) and pentobarbital (30 mg/kg, i.p.). A possible rationale for the difference in results

between studies is the difference in procedures used to terminate SE.

2. Second, studies have shown that SE duration can affect the duration of the latency period

and frequency of SRSs. Longer durations of SE were correlated with increased latencies

to the development of SRSs, and this was suggested to be related to the increased

production of neurosteroids from activated glial cells (Biagini et al., 2006; Biagini et al.,

2009). Because neurosteroids, such as allopregnanolone, act as positive GABAA receptor

modulators, an increase in their induction was proposed to retard epileptogenesis (Biagini

et al., 2006; Biagini et al., 2009). Using the lithium/pilocarpine model, Glien et al.,

(2001) also showed that a longer SE duration was associated with increased latency to the

occurrence of SRSs. In both of these studies, SE duration was controlled with diazepam

(10 – 20 mg/kg, i.p.). In contrast, Lemos and Cavalheiro (1995) obtained different results

by terminating SE in rats with combined treatment of diazepam (10 mg/kg, i.p.) and

pentobarbital (30 mg/kg, i.p.). This study reported a progressive increase in the latent

period and a decrease in seizure frequency in animals with shorter SE. In addition,

animals with shorter SE (1 and 2 hrs) exhibited less neuronal loss and axonal sprouting in

the hippocampal formation. Consequently, the severity of neuropathology was suggested

to affect the length of the latent period. The combined treatment of diazepam (10 mg/kg)

and pentobarbital (30 mg/kg) has been reported to reduce mossy fiber sprouting and

neuronal loss after SE (Luongo et al., 2009). It is possible that this interference with

19

histopathological changes contributes to the difference in results between this study and

others that used only diazepam to control SE duration (Glien et al., 2001; Klitgaard et al.,

2002; Biagini et al., 2006; Goffin et al., 2007; Biagini et al., 2009).

1.4.3 The chronic phase

The chronic phase is characterized by the appearance of SRSs, which persist for the life of the

animal.

1.4.3.1 Behaviour during the chronic phase

As proposed by Goffin et al., (2007), the classification of pilocarpine-induced seizures (see

Table 1.1) can be simplified by referring to stages I - III as partial seizures, and stages IV – VII

as secondarily generalized seizures. According to this classification, recurrent seizures start

appearing approximately 7 days after SE as partial seizures, and develop into generalized

seizures in the following days (Goffin et al., 2007). The evolution of SRSs in the pilocarpine and

lithium/pilocarpine models mimic the behavioural and electrographic stages of kindling (see

section 1.4.3.2) (Leite et al., 1990; Cavalheiro et al., 2006; Goffin et al., 2007). Once the SRSs

resemble a stage V seizure of amygdala kindling (Racine, 1972), the majority of subsequent

seizures are also generalized (Cavalheiro et al., 2006). At this point, SRSs appear to recur in

clusters with cyclicity, peaking every 5 to 8 days (Goffin et al., 2007) or more (Arida et al.,

1999). The frequency of SRSs increases and then remains constant 2 months after SE, and

persists throughout the lifetime of the animal (Priel et al., 1996; Arida et al., 1999; Goffin et al.,

2007).

1.4.3.2 Electroencephalographic patterns observed during SRSs

The first spontaneous seizures are partial seizures characterized by paroxysmal activity in the

hippocampus without changes in cortical recordings (Leite and Cavalheiro, 1995). Subsequent

seizures show a gradual spreading of paroxysmal activity from the hippocampus to cortical

recordings and longer duration of ictal events. The fully developed generalized seizures are

characterized by bursts of spiking activity in the hippocampus that spread to the neocortex (Leite

and Cavalheiro, 1995; Goffin et al., 2007). Electrographic seizures rarely last more than 60 sec

and are followed by depressed background activity with frequent EEG interictal spikes. The

interictal spikes are more intense when animals are seizure-free and in the period of slow-wave

20

sleep and are nearly non-existent during motor activity and paradoxical sleep (Arida et al., 1999).

Consequently, a higher seizure frequency (i.e., lower seizure threshold) tends to occur during

daylight hours (Arida et al., 1999; Goffin et al., 2007).

1.5 Neuropathology

Several pathophysiological phenomena that occur during the latency phase are suggested to

contribute to epileptogenesis. These include neurodegeneration, synaptic reorganization, glial

cell activation, and ectopic cell proliferation (see reviews: Fujikawa, 2005; Dalby and Mody,

2001; McNamara et al., 2006; Pitkänen and Lukasiuk, 2009). In the present thesis, we have

specifically focused on neurodegeneration following SE, and discussed this process in the

subsequent sections. The other histopathological changes have been briefly described here but

extensive reviews on each of these are available.

1.5.1 Neurodegeneration

1.5.1.1 SE-induced neurodegeneration

In most patients, MTLE is believed to be initiated by brain damage and synaptic reorganization

secondary to an IPI (Babb, 1986; Babb and Brown, 1986; Babb et al., 1991; Sharma et al., 2007).

MTLE can be subclassified histopathologically as paradoxical temporal lobe epilepsy (PTLE) or

hippocampal sclerosis (HS) (Sharma et al., 2007). No lesions are observed in PTLE, whereas in

70% of MTLE patients with HS, lesions include neuronal degeneration, gliosis and aberrant

mossy fiber sprouting in the inner molecular layer of the dentate gyrus. Neuronal loss is also

frequently observed in the amygdala and the surrounding entorhinal, perirhinal and para-

hippocampal cortices, and in extra-temporal areas, including the thalamus and cerebellum (Jutila

et al., 2002; Bernasconi et al., 2003; Bonilha et al., 2010). In the absence of systematic

complications or pre-existing epilepsy, SE in humans produces similar widespread neuronal loss

and reactive gliosis in the hippocampus, amygdala, thalamus, piriform and entorhinal cortices

and Purkinje cell layer of the cerebellum (Fujikawa et al., 2000). This pattern of neuronal loss is

replicated in the pilocarpine and lithium/pilocarpine models of SE (Honchar et al., 1983; Turski

et al., 1983a; Turski et al., 1983b; Fujikawa, 1996; Covolan and Mello, 2000), and is generalized

in other chemoconvulsant models of epilepsy including kainic acid, bicuculline, picrotoxin and

21

pentetrazole (Lothman and Collins, 1981; Ben-Ari et al., 1981; Ben-Ari, 1985; Turski et al.,

1985).

1.5.1.2 Progression and severity of neuronal loss following SE

Previous studies assessing the progression of neuronal death in rats after SE have been limited by

a semi-quantitative assessment of tissue damage and/or by the incomplete nature of the time

frame examined (Fujikawa, 1996; Motte et al., 1998; Covolan and Mello, 2000; Peredery et al.,

2000; Poirier et al., 2000). For example, timeframes are restricted to less than 3 to 5 days

(Fujikawa, 1996; Covolan and Mello, 2000) or timeframes examined are spaced far apart (Motte

et al., 1998; Peredery et al., 2000; Poirier et al., 2000b). A summary of these studies is provided

in appendix I, Table A1-1. SE has been shown to primarily contribute to neuronal death

(Peredery et al., 2000; Pitkänen et al., 2002; Gorter et al., 2004; Deshpande et al., 2007), with the

majority of neuronal injury/death occurring in the several days following SE (Fujikawa, 1996;

Motte et al., 1998; Covolan and Mello, 2000; Peredery et al., 2000; Poirier et al., 2000). The

effect of SRSs on neurodegeneration, however, remains unclear. While some studies report no

correlation of neuronal death and the frequency of SRSs (Pitkänen et al., 2002; Gorter et al.,

2004), others have demonstrated progressive neuronal loss during the chronic stage of seizure

development (Roch et al., 2002). In the kindling model, brief recurrent seizures can cause

neuronal loss and hippocampal sclerosis (Cavazos et al., 1991; Cavazos et al., 1994), supporting

a potential role of SRSs in neuronal damage. The difficulty in verifying whether the chronic

epileptic seizures that follow SE lead to brain damage is delineating what extent of neuronal loss

is attributed to SRSs, and what occurs because of delayed damage from the IPI (reviewed in:

Dudek et al., 2002). In the present thesis, a detailed quantitative time course of

neurodegeneration between 1 hr and 3 months following SE was completed: (1) to improve our

understanding and to compare the evolution of neuronal death in different brain structures, (2) to

determine which of these structures exhibit delayed neuronal loss extending past epileptogenesis,

and (3) to establish a critical timeframe for the feasibility of neuroprotective strategies.

1.5.1.3 Mechanisms underlying SE-induced neuronal death

Early studies demonstrated that electrographic seizures can cause neuronal death in artificially

ventilated animals, even in the absence of underlying systematic physiological complications

(Meldrum and Brierley, 1973; Meldrum et al., 1974; Nevander et al., 1985). Prolonged seizures

22

initiate neuronal death by excitotoxicity (reviewed in: Fujikawa, 2005). This occurs when

glutamate receptors are excessively stimulated. The specific role of NMDA receptors in SE-

induced neurodegeneration is strongly supported by two main findings.

(1) NMDA receptor antagonists administered in rats before or after SE provide significant

neuroprotection (Fariello et al., 1989; Clifford et al., 1990; Fujikawa et al., 1994;

Fujikawa, 1995).

(2) In an in-vitro model of SE, specific entry of Ca2+

through NMDA receptors results in

more cell death as opposed to Ca2+

entering through non-NMDA glutamate receptors or

voltage-gated calcium channels (Deshpande et al., 2008). Similar findings have been

demonstrated in other models of glutamate neurotoxicity (Tymianski et al., 1993; Sattler

et al., 1998).

During SE, excessive synaptic release of glutamate can cause excitotoxicity by binding to

NMDA receptors and allowing high levels of Ca2+

to enter the cells. Ca2+

influx into the cells

activates a number of enzymes, including neuronal nitric synthase, phospholipases,

endonucleases, and cysteine proteases (i.e., calpains and caspases). These enzymes subsequently

damage cell structures such as components of the cytoskeleton, membrane and DNA (reviewed

in: Fujikawa, 2005; Forder and Tymianski, 2009; Hardingham, 2009; Lau and Tymianski, 2010;

Wang and Qun, 2010), leading to different forms of cell death (see section 1.5.1.4).

1.5.1.4 Types of cell death mechanisms initiated by SE

Even though it is generally accepted that neuronal death following SE is excitotoxic in nature

(Fountain, 2000; Fujikawa, 2005; Deshpande et al., 2008), it is not clear if the cell death

phenotype is primarily necrotic or apoptotic (reviewed in: Fujikawa, 2005; Lau and Tymianski,

2010; Wang and Qun, 2010). A detailed discussion of necrosis and apoptotsis is beyond the

scope of this thesis, but numerous reviews of this topic are available (Saraste and Pulkki, 2000;

Manning and Zuzel, 2003; Goldstein and Kroemer, 2007; Doonan and Cotter, 2008;

Vanlangenakker et al., 2008). Briefly, necrosis results from loss of cellular homeostasis and

membrane integrity and acute mitochondrial dysfunction, all leading to massive energy failure

(reviewed in: Goldstein and Kroemer, 2007; Vanlangenakker et al., 2008). Apoptosis is

characterized by cell shrinkage, membrane blebbing, DNA internucleosomal degradation,

chromosome condensation and formation of membrane-bound apoptotic bodies, and relies on

23

preserved mitochondrial functioning and integrity and ATP synthesis (reviewed in: Saraste and

Pulkki, 2000; Doonan and Cotter, 2008).

A myriad of neurotoxic signaling cascades are capable of initiating apoptosis and/or necrosis,

and several extensive reviews of these pathways are available (Yakovlev and Faden, 2004;

Harwood et al., 2005; Henshall, 2007; Henshall and Murphy, 2008; Vosler et al., 2008). This

section briefly describes the roles of caspases and calpains in apoptotic and necrotic cell death,

respectively. Specific caspases are suggested to mediate apoptosis and can be activated by either

intrinsic (death receptor-mediated) or extrinsic (mitochondria-mediated) pathways (Harwood et

al., 2005; Henshall, 2007). In response to a stimulus, the initiator caspases (e.g., caspase-8 and

caspase-9) activate executioner (or effector) caspases (e.g., caspase-3), which subsequently

mediate the biochemical and morphological features of apoptosis (Harwood et al., 2005;

Henshall, 2007). On the other hand, calpain is activated by an increase in intracellular Ca2+

and

cleaves multiple substrates including cytoskeletal and associated proteins, kinases, phosphatases,

membrane receptors and transporters; excessive calpain activity leads to cytoskeletal protein

breakdown and subsequent loss of structural integrity and disturbances of axonal transport,

accelerating cell death and contributing to necrotic morphology (Pang et al., 2003 and reviewed

in: Fujikawa, 2005; Vosler et al., 2008). Still, generalizing the involvement of caspases and

calpains to specific cell death morphologies is not straightforward. Extensive cross-talk between

the cysteine proteases occurs, and this causes the boundary between necrotic and apoptotic cell

death phenotypes to become blurred (Harwood et al., 2005). Instead, a widely accepted view is

that excitotoxic cell death occurs along a continuum of necrotic and apoptotic morphologies

(Fujikawa et al., 2000b; Doonan and Cotter, 2008; Kotariya et al., 2010; Wang and Qun, 2010).

Necrosis is generally considered the principal morphological phenotype of dying cells after SE

(Fujikawa, 2005; Kotariya et al., 2010). Despite the predominance of necrotic morphology in

degenerated neurons, others suggest that apoptosis may play a critical role in seizure-induced

brain damage (reviewed in: Bengzon et al., 2002; Henshall, 2007; Henshalla and Murphy,

2008). Often, a mixed form of neuronal death with apoptotic and necrotic features following SE

is reported (Sloviter et al., 1996; Becker et al., 1999; Fujikawa et al., 1999; Covolan et al., 2000;

Fujikawa et al., 2000b; Weise et al., 2005). Autophagy is also up-regulated and contributes to

neuronal death following prolonged seizures (Cao et al., 2009a; Cao et al., 2009b); this process

can induce cell death through excessive self-digestion and degradation of essential cellular

24

constituents (reviewed in: Levine and Klionsky, 2004; Codogno and Meijer, 2005), and is

initiated with increased oxidative stress (Chu, 2006). A heterogeneous population of cell death

morphologies is similarly reported in animal models of stroke (Snider et al., 1999) and traumatic

brain injury (Singleton and Povlishock, 2004).

1.5.1.5 Factors determining the extent and phenotype of SE-induced neuronal death

Several factors have been shown to affect the extent and phenotype of neuronal death following

prolonged seizures. These include severity of SE, intracellular ATP levels, and regional and

neuronal specific cell-death signaling pathways. Each of these factors is briefly described.

(1) The intensity of continuous convulsive seizures in Wistar rats was shown to affect cell

death morphology of hippocampal pyramidal cells; more severe SE (induced by 12

mg/kg of KA, i.p.) led to an increase of necrotic cell death, whereas milder SE (induced

by 9 mg/kg of KA, i.p.) increased the presence of dying neurons exhibiting apoptotic

features (Tokuhara et al., 2007 and reviewed in: Meldrum, 2002). In contrast, Fujikawa

et al., (2010) only detected necrotic cell death in Wistar rats subjected to different

durations of pilocarpine-induced SE (1 hr or 3 hrs), failing to establish a relationship

between SE duration and cell death morphology.

(2) A critical determinant of the cell death mechanism is intracellular ATP concentration, the

production of which depends on the structural and functional integrity of the

mitochondria. Whereas ATP depletion is associated with necrosis, ATP is required for

the development of apoptosis. Cheung et al., (2009) demonstrated apoptotic cell death in

the CA1 and CA3 pyramidal cell layer of Sprague Dawley rats following 40 min of SE

(induced by unilateral microinjection of KA into CA3). Of particular interest, these

regions had maintained ATP levels, and the majority of degenerating pyramidal cells

exhibited intact structural integrity of the mitochondria. In the pilocarpine and

lithium/pilocarpine models, variability in the severity of mitochondrial damage following

SE is reported; the ultrastructural damage of the mitochondria in hippocampal pyramidal

cells ranges from no visible changes, to mitochondrial swelling, which is most frequently

detected, and/or severe damage characterized by rupture of the inner and outer

mitochondrial membranes ( Fujikawa et al., 1999; Jing et al., 2007).

25

(3) Repetitive or prolonged seizures can lead to region-specific cell death pathways. For

example, Sloviter et al., (1996) demonstrated that repetitive seizures caused apoptotic

cell death in dentate granule cells (DGCs) and necrotic cell death in the CA1 and CA3

pyramidal cells. Of particular interest, Lopez-Meraz et al., (2010) showed that in

immature rats following KA-induced SE, caspase-9 was up-regulated in DGCs exhibiting

apoptotic cell death, while caspase-8 was up-regulated in pyramidal CA1 cells exhibiting

necrotic cell death (caspases described in section 1.5.1.4). Differences in post-synaptic

cell-death pathways can occur within the same neuronal population. KA or pilocarpine-

induced SE in adult rats resulted in two types of pyramidal cell death: early necrosis (1

day after SE) and delayed cell death with apoptotic features (3 to 7 days after SE). One

possibility is that the morphological differences in neuronal death may be attributed to

differential activation of cystein proteases (see section 1.5.1.4). For example, the

expression and activation of calpain was detected in early necrotic cell death following

SE (Araújo et al., 2008; Wang et al., 2008), whereas caspase-3 was detected in delayed

neuronal death exhibiting apoptotic features (Narkilahti et al., 2003; Weise et al., 2005;

Wang et al., 2008).

1.5.1.6 The role of neurodegeneration in epileptogenesis

Although SE results in significant neurodegeneration and in the development of epilepsy, the

contribution of neuronal death in the genesis of SRSs is unclear. Two lines of evidence suggest

neuronal loss is not required for the genesis of chronic epilepsy. First, several studies have

showed that nearly complete protection of limbic structures, including the hippocampus and

amygdala, did not prevent the development of epilepsy (Ebert et al., 2002; Loscher, 2002; Brandt

et al., 2006). Second, several animal models of epilepsy can lead to development of SRSs in the

absence of overt brain damage. For example, seizures produced by electroconvulsive shock

repeated for several days initiate mossy fiber sprouting and development of SRSs without

neuronal loss. Similarly, in immature rats, febrile seizures can lead to SRSs without neuronal

death (Baram et al., 2002; Kapur, 2006).

Still, others have reported that SE-induced neurodegeneration can exacerbate the epileptogenic

process and functional outcome. For instance, Zhang et al., (2002) compared two groups of rats

treated differently with kainic acid. Rats in one group experienced two short sessions of priming

26

seizures and one sustained episode of SE; this priming effect resulted in no obvious neuronal loss

or mossy fiber sprouting. The second group experienced only a single episode of SE. Though

both groups developed epilepsy, the massive neuronal damage and mossy fiber sprouting in the

latter group resulted in more frequent and intensified SRSs (Zhang et al., 2002). Similarly,

Persinger and Dupont (2004) showed that the extent of neuronal loss in the temporal cortex,

dentate gyrus, hilus and CA3 region correlated with the frequency of SRSs (Persinger and

Dupont, 2004). Others showed that neuroprotective agents improved behavioural and cognitive

functioning in rats following SE (Rice et al., 1998; dos Santos et al., 2005; Brandt et al., 2006;

Cunha et al., 2009; Jun et al., 2009). Thus, although it seems that neurodegeneration is not a

prerequisite for the development of epilepsy, neuroprotective strategies may have a role in

modifying the disease outcome (Loscher, 2002; Pitkanen and Kubova, 2004; Naegele, 2007;

Acharya et al., 2008), and in mitigating interictal behavioural and cognitive morbidity (see

section 1.6).

In the present thesis, we investigated the effectiveness of a neuroprotective strategy in rats

following lithium/pilocarpine-induced SE. As previously described in section 1.5.1.3,

excitotoxicity mediated via the NMDAR is recognized as a major mechanism in

neurodegeneration resulting from SE. Consistent with a critical role of NMDARs in SE-induced

neurodegeneration, previous studies showed that systematic administration of NMDAR

antagonists are neuroprotective in rodent models of epilepsy, even when given after the onset of

SE (Fariello et al., 1989; Clifford et al., 1990; Fujikawa et al., 1994; Fujikawa, 1995). The

clinical efficacy of NMDAR antagonists is limited, however, due to psychomimetic side-effects

in humans (Krystal et al., 1994; Lahti et al., 1995; Malhotra et al., 1996; Rowland et al., 2005).

An alternative approach to preventing NMDAR-mediated excitotoxicity would be to disrupt the

interaction of the receptor with downstream signaling molecules (see section 5.1). PSD-95 is a

critical scaffolding protein that links the NMDAR to signaling enzymes within the postsynaptic

density, and suppression of the expression of PSD-95 selectively attenuated excitotoxicity

triggered via NMDARs (Sattler et al., 1999). Tat-NR2B9c is a synthetic peptide, consisting of

the C-terminal 9 amino acids of the NR2B subunit of NMDARs fused to the membrane

transduction domain of the HIV-1-Tat protein, that was designed to disrupt excitotoxic signaling

from the NMDAR by interfering with protein interactions involving the PDZ1 and PDZ2

domains of PSD-95(see section 5.2.2). Tat-NR2B9c was previously shown to provide

27

significant neuroprotection and preserve cognitive function after transient stroke in rats (Aarts et

al., 2002; Sun et al., 2008). Here, we assessed the ability of tat-NR2B9c to provide

neuroprotection (see chapter 5) and mitigate behavioural morbidity (see chapters 6 and 7)

following lithium/pilocarpine-induced SE in rats. The effectiveness of other neuroprotective

strategies assessed in post-status epilpeticus rodent models is further discussed in section 8.7 and

compared in Table 8.1.

1.5.2 Synaptic reorganization

Mossy fiber sprouting is observed in the human epileptic temporal lobe (Sutula et al., 1989;

Houser et al., 1990; Franck et al., 1995), and has been extensively studied in animal models of

TLE (reviewed in: Dalby and Mody, 2001; Sutula, 2002; Nadler, 2003; Cavazos and Cross,

2006; Pitkänen and Lukasiuk, 2009). Axons of granule cells (mossy fibers) are most often

described as sprouting aberrantly into the dentate supragranular layer; new synapses on granule

cell dendrites increase the overall excitatory connection between granule cells (Okazaki et al.,

1995; Buckmaster et al., 2002). Additional mossy fiber reorganization includes axonal growth in

the hilus, development of infrapyramidal and suprapyramidal (interblade) connectivity, and

expansion of the terminal field of the mossy fiber pathway in CA3 and along the septotemporal

axis of the hippocampus over distances as long as 700 to 800 microns (Sutula et al., 1998;

Buckmaster and Dudek, 1999; Holmes et al., 1999). Seizure-induced reorganization along the

septotemporal axis provides a mechanism for translamellar synchronization of cellular

hyperexcitability within the hippocampus to occur (reviewed in: Cavazos and Cross, 2006).

Although mossy fiber sprouting can occur in the absence of neurodegeneration caused by

repeated seizures (e.g., in the kindling model; reviewed in Morimoto et al., 2004), neuronal death

(e.g., in the hilus and CA3 pyramidal cell layer) can also initiate mossy fiber reorganization

following an IPI (reviewed in: Sutula, 2002; Nadler, 2003). Mossy fiber sprouting appears prior

to the occurrence of SRSs and persists for the lifetime of the animal (Cavazos et al., 1991;

Nissinen et al., 2001; Wuarin and Dudek, 2001). Several studies have demonstrated a direct

correlation between the degree of neuronal loss, mossy fiber sprouting into the dentate

supragranular layer and granule cell hyperexcitability (Sutula, 2002; Cavazos and Cross, 2006;

Sutula and Dudek, 2007). Recent studies indicate that the functional effects of the recurrent

excitatory circuits formed by mossy fiber reorganization may only be apparent under conditions

28

of reduced inhibition, or alterations in the extracellular ionic environment (e.g., elevation of

[K+]o) (reviewed in: Sutula, 2002; Sutula and Dudek, 2007).

Similar to SE-induced neurodegeneration, the development of mossy fiber sprouting following

prolonged seizures can exacerbate the epileptogenic process and functional outcome (see figure

1.1). Jing et al., (2009) showed that suppression of mossy fiber sprouting, by combined

treatment consisting of intrahippocampal transplantation of adult neural stem cells and

intraventricular erythropoietin- infusion, reduced hippocampal excitability and prevented

development of SRSs in kainate-treated rats (Jing et al., 2009). In other studies, pre-treatment

with cycloheximide in rats has been shown to reduce mossy fiber reorganization (Longo and

Mello, 1997; dos Santos et al., 2005). It also concomitantly reduced behavioural alterations (Lee

et al., 2003; dos Santos et al., 2005), indicating a connection between mossy fiber sprouting and

SE-induced behavioural morbidity. Synaptic reorganization has been detected elsewhere,

including the CA1, entorhinal cortex and neocortex (Esclapez et al., 1999; Wuarin and Dudek,

2001; Smith and Dudek, 2002; Cavazos et al., 2004).

1.5.3 Reactive gliosis

Reactive gliosis is a prominent characteristic in the epileptic brains of humans (Spencer et al.,

1999; D'Ambrosio, 2004) and is recapitulated in rodent models of TLE. It is characterized by

the hypertrophy (intensive outgrowth of cellular processes) of astroctyes as well as by the

proliferation of microglial cells and astrocytes (Ridet et al., 1997). Glial cells perform many

functions, including structural support, water and ionic homeostasis, regulation of

neurotransmission, inflammatory responses and neurogenic potential (Ridet et al., 1997);

reactive gliosis results in acute and long-term alterations of these functions, which in turn

contribute to the epileptogenic process. Several extensive reviews on the role of glial cells in

epilepsy are available (D'Ambrosio, 2004; Eid et al., 2008; Jabs et al., 2008; Binder and

Steinhäuser, 2006; Seifert et al., 2010). Of particular interest, reactive astrocytes were shown to

contribute to delayed neuronal death of cortical and hippocampal pyramidal cells following

pilocarpine-induced SE (Ding et al., 2007). In this study, prolonged elevation of astrocytic Ca2+

signaling correlated with the temporal profile of neuronal death, which was maximal at 3 days

following SE. Astrocytic Ca2+

signaling leads to gliotransmission of glutamate and D-serine;

administration of MPEP (2-methyl-6(pehnylethynyl)pyridine), a metabotropic glutamate receptor

29

5 antagonist (mGluR5), blocked astrocytic Ca2+

signaling and provided significant

neuroprotection, linking astrocytic gliotransmission to delayed neuronal death. The

neuroprotective effect of ifenprodil, an antagonist specific for NR2B-containing NMDA

receptors, indicates that gliotransmission induces neuronal excitotoxicity by activation of these

receptors (Ding et al., 2007).

1.5.4 Neurogenesis

Adult neurogenesis is the generation of new neurons in the central nervous system through

division of neural stem cells (NSCs). These stem cells divide to form neuroblasts, which

eventually differentiate into neuronal or glial phenotypes and are integrated into functional

networks. The majority of NSCs are located in two neurogenic regions: (1) Neuroblasts from

the subventricular zone lining the lateral ventricles migrate along the rostral migratory stream

into the olfactory bulb, where the majority differentiate into interneurons (Whitman and Greer,

2009). (2) Neuroblasts in the subgranular zone of the DG, a lamina between the granule cell

layer (GCL) and the hilus, migrate into the GCL and mainly mature into functional granule cells

(Parent, 2007).

In the pilocarpine model, SE increases the rate of neurogenesis in the DG for a 2-week period

(Parent et al., 1997). While the majority of cells migrate appropriately into the DG following

SE, approximately 20% migrate aberrantly to ectopic locations, most notably the dentate hilus

(Walter et al., 2007). Hilar ectopic granule cells are invariably bipolar, in contrast to normally

situated granule cells, which have only an apical dendrite. Even though their membrane

properties closely resemble normally positioned granule cells, they are notable for bursting in

synchrony with CA3 pyramidal cells (Scharfman et al., 2000). The increased excitability of

these cells is attributed to their abnormal innervations; hilar ectopic granule cells receive mossy

fiber input from other granule cells and innervate other granule cells, in addition to the typical

targets of the mossy fiber pathway (CA3 pyramidal cells and inhibitory interneurons, hilar mossy

cells and inhibitory interneurons) (Parent et al., 1997; Scharfman et al., 2000; Dashtipour et al.,

2001). These cells also receive few inhibitory synapses (Dashtipour et al., 2001). Of particular

interest, Gong et al., (2007) demonstrated that loss of a subset of interneurons expressing reelin,

a migration guidance cue that influences neuronal migration, contributes to the ectopic location

of granule cells following SE.

30

The altered innervations of hilar ectopic granule cells and the recurrent synapses between CA3

pyramidal neurons combine to form a reverberatory loop, which is proposed to participate in

synchronization and propagation of seizure activity (Nadler, 2003; Parent and Murphy, 2008).

During the chronic phase of seizure development a decrease in neurogenesis occurs (Hattiangady

et al., 2004). The increase in neurogenesis following SE and the decrease in neurogenesis during

chronic epilepsy both contribute to the epileptogenic process and to the development cognitive

and behavioural impairments (reviewed in: Hattiangady and Shetty, 2008; Kuruba et al., 2009).

Several extensive reviews on the role of neurogenesis in epilepsy are available (Parent, 2007;

Danzer, 2008; Hattiangady and Shetty, 2008; Parent and Murphy, 2008; Kuruba et al., 2009).

1.6 Co-morbid interictal disorders in mesial temporal lobe epilepsy

MTLE is often associated with interictal behavioural disturbances, including anxiety, depression,

and psychosis, as well as learning and memory impairments ( Boro and Haut, 2003; Devinsky,

2004; Gaitatzis et al., 2004; Swinkels et al., 2005; Cornaggia et al., 2006; Marcangelo and

Ovsiew, 2007; Garcia-Morales et al., 2008). Because of several confounding variables in the

clinical setting, however, the causal relationships between epilepsy, affective disturbances and

cognitive impairments are poorly understood. These confounders preclude direct demonstration

that seizures themselves are responsible for behavioural and cognitive deficits, and include the

underlying etiology and brain pathology, present and past anticonvulsant treatments, degree of

seizure control, and developmental status of the patient (Heinrichs and Seyfried, 2006). Many of

these problems are circumvented by the use of animal models of epilepsy, which allow for a

more direct correlation of behavioural outcomes with physiological and histological data

(reviewed in: Majak and Pitkanen, 2004; Post, 2004; Heinrichs and Seyfried, 2006). The present

section reviews current hypotheses regarding the relationship between epilepsy and interictal

behavioural and cognitive disturbances, and specifically focuses on data derived from post-status

epilepticus animal models.

1.6.1 The relationship between epilepsy and cognitive and interictal behavioural alterations

Based on animal models of epilepsy, three hypotheses regarding the causal relationship between

seizures and behavioural morbidity have been proposed:

31

1. Shared neurodevelopmental, genetic or environmental causes predispose subjects to

develop both epilepsy and co-morbid behavioural and cognitive disturbances

2. Neuropathological changes underlying the genesis of interictal behavioural disturbances

are closely related to those mediating epileptogenesis itself

3. SRSs contribute to cognitive and interictal behavioural impairment

1.6.2 Shared neurodevelopmental, genetic or environmental causes predispose subjects to develop both epilepsy and co-morbid behavioural and cognitive disturbances

Evidence for this hypothesis is supported by studies showing cognitive and interictal behavioural

disturbances prior to seizure development in genetically-predisposed epileptic rats (Kelly et al.,

2003; Sarkisova et al., 2003; Midzyanovskaya et al., 2005; Jones et al., 2008; Runke and

McIntyre, 2008). For instance, Jones et al., (2008) illustrated that genetic absence epilepsy rats

from Strasbourg (GAERS, a genetic model of absence epilepsy in rats; epilepsy model reviewed

in: Marescaux et al., 1992; Danober et al., 1998) exhibit an increase in anxiety- and depressive-

like behaviours compared to their non-epileptic controls. This behavioural phenotype of GAERS

is present prior to the occurrence of spontaneous spike-and-wave discharges (non-convulsive

absence seizures), which develop between postnatal days 30 to 60, and persists throughout the

life of the animal. Similarly, FAST kindling rats that have been bred to exhibit enhanced rates of

amygdala kindling, a model of temporal lobe epileptogenesis (model reviewed in: McIntyre et

al., 1999; Racine et al., 1999; McIntyre et al., 2002), display different behaviours of stress

responsivity and anxiety prior to kindling compared to SLOW rats (McIntyre et al., 2002; Kelly

et al., 2003; McIntyre and Gilby, 2007; Runke and McIntyre, 2008). For example, FAST-

kindling rats exhibit less anxiety in the elevated-plus maze, and increased exploratory behaviour

without habituation over repeated trials in the open field (McIntyre et al., 2002). Additionally,

FAST-kindling rats display inferior performance in the Morris water maze prior to kindling,

suggesting both working and reference memory impairments (Anisman and McIntyre, 2002).

32

1.6.3 Neuropathological changes underlying the genesis of interictal behavioural disturbances are closely related to those mediating epileptogenesis itself

The second hypothesis states that neuropathological changes underlying the genesis of interictal

behavioural and cognitive disturbances are closely related to those mediating epileptogenesis

itself. This is strongly supported by studies of post-status epilepticus animal models. As

described previously, epilepsy can be seen as a process whereby an IPI (like SE) results in a

latent phase (epileptogenesis) and, eventually, the occurrence of SRSs (see section 1.4). As

described in section 1.5, neuronal death and synaptic reorganization caused by an initial brain

injury contribute to the genesis of recurrent seizures (Fujikawa, 2005; Acharya et al., 2008;

Sharma et al., 2008), and have also been proposed to underlie interictal behavioural and

cognitive morbidity (Majak and Pitkanen, 2004; Sayin et al., 2004; de Oliveira et al., 2008). For

example, experimental febrile seizures cause long-lasting behavioural and cognitive disturbances

and epileptogenesis, and are accompanied by neuropathological changes including neuronal loss,

mossy fiber sprouting, and gliosis (Kubova et al., 2004; Sayin et al., 2004; de Oliveira et al.,

2008; and reviewed in Stafstrom, 2002). Similar results are obtained in adult rats following SE

induced by epileptogenic agents (Milgram et al., 1988; Detour et al., 2005; dos Santos et al.,

2006; Cardoso et al., 2008; de Oliveira et al., 2008; Cardoso et al., 2009; Cardoso et al., 2009).

The severity of behavioural and cognitive deficits is correlated with the extent of neuronal death

(Milgram et al., 1988; Niessen et al., 2005; Cilio et al., 2003) and mossy fiber sprouting (de

Oliveira et al., 2008; de Rogalski et al., 2001; Sogawa et al., 2001). Neuroprotection within

selective brain regions or a reduction in mossy fiber sprouting has been demonstrated to mitigate

SE-induced morbidity(Bolanos et al., 1998; Rice et al., 1998; Cilio et al., 2001; dos Santos et al.,

2005; Brandt et al., 2006; Frisch et al., 2007; Cunha et al., 2009; Jun et al., 2009), providing

direct support for the hypothesis that neuropathological changes during epileptogenesis cause

interictal behavioural and cognitive impairments in rats after SE.

33

1.6.4 Spontaneous recurrent seizures contribute to interictal behavioural and cognitive impairment in post-SE models

The temporal onset of behavioural and cognitive impairments has been shown to occur as early

as 24 hours following SE induced by kainic acid or pilocarpine, and precedes development of

SRSs (Milgram et al., 1988; Rice et al., 1998; Mikati et al., 2001; Sogawa et al., 2001; Chauviere

et al., 2009). These data are consistent with neuropathological changes contributing to

epileptogenesis and early cognitive and behavioural disturbances. As illustrated in Figure 1.1,

however, an alternative hypothesis suggests that SRSs contribute to neuronal death, synaptic

reorganization, and interictal behavioural and cognitive decline. SE-induced epileptogenesis

results in the appearance of SRSs within a few weeks (discussed in sections 1.4.2.1 and 1.4.3).

Hort et al., (1999) and Sun et al., (2009) demonstrated that although MWM performance is

impaired after SE, learning and memory are further deteriorated with development of SRSs (see

appendix I, Table A1-2 for details). In another study, the impaired performance in the MWM

was less severe in rats with sporadic seizures (<1/day) than in frequently seizing animals

(≥1/day) (Nissinen et al., 2000). In spontaneous seizing animals, however, it is difficult to

differentiate the contribution of SE-induced damage to cognitive and behavioural impairment

from the damage caused by SRSs, particularly because the rats with frequent seizures begin with

greater SE-induced damage (Roch et al., 2002; Majak and Pitkanen, 2004). Additionally, brain

damage in specific brain regions can progress weeks to months after SE, complicating the

assessment of neuronal loss caused by SRSs in post-status epilepticus models (Fujikawa, 1996;

Pitkänen et al., 2002).

1.6.5 Other factors affecting severity of interictal behavioural and cognitive impairment

In various animal models of epilepsy, additional factors are shown to affect the severity of

interictal behavioural and/or cognitive impairment. These include genetic background (Hort et

al., 2000), age at the time of the epileptogenic insult (Stafstrom et al., 1993; Cilio et al., 2003;

Kubova et al., 2004), extent of brain damage (Milgram et al., 1988; Mohajeri et al., 2003),

location of seizure focus (Becker et al., 1997), seizure frequency (Nissinen et al., 2000) and

environmental conditions (Faverjon et al., 2002; Wang et al., 2007) (also reviewed in: Post,

2004; Majak and Pitkanen, 2004). Many of these factors also influence the severity of

behavioural disturbances in human epilepsies (Cornaggia et al., 2006; Titlic et al., 2009).

34

1.6.6 Interictal behavioural disturbances following SE

The effect of SE and epilepsy on a variety of different behaviours has been assessed. Because

behavioural tasks assessing anxiety, exploration and aggression have been shown to be

particularly affected in rodents following prolonged seizures, we focused our attention on the

analyses of these behaviours.

1.6.6.1 Anxiety

Anxiety disorders are commonly reported in patients with epilepsy (Cornaggia et al., 2006; Titlic

et al., 2009). Previous researchers have proposed that the behavioural symptoms of human

anxiety disorders, which include hypervigilance, exaggerated startle, avoidance and escape

reactions (Vazquez and Devinsky, 2003), reflect the inappropriate activation or exaggeration of

normally adaptive defence responses (Rosen and Schulkin, 1998). Because the neural

mechanisms underlying anxiety are conserved across mammalians, this behaviour may be

investigated using behavioural tasks that are ethologically designed to evaluate defence

responses in rodents; the repertoire of defence reactions in rodents can differ between tasks, and

include flight/escape, freezing, startle, and defensive threat/attack (Rodgers, 1997). Defence

responses are believed to have originated as anti-predator strategies, but mammals additionally

exhibit defensive reactions to other threatening stimuli, including those associated with

aggressive conspecifics and dangerous/potentially dangerous objects or environments (Griebel,

1995; Rodgers, 1997; Rodgers et al., 1997). Over 30 different types of behavioural tasks

designed to assess anxiety have been developed (Rodgers, 1997; Rodgers and Dalvi, 1997).

Wall and Messier (2001) caution that in using behavioural tests of anxiety, fundamental

differences between anxiety in humans and animals may exist. In humans, anxiety can also be

described as an affective, emotional state that may or may not have an externally observable

correlate. In testing anxiety in rodents, we must rely on observable behaviours and assume that

these reflect the internal emotional state (Griebel, 1995). Because the elevated plus maze (EPM)

and open field are the most commonly used tests of anxiety and exploration (see section 1.6.6.2)

in animal models of epilepsy (Wall and Messier, 2001; Heinrichs and Seyfried, 2006; Ramos,

2008), we have primarily focused our discussion on the effect of SE on behaviour in these tasks.

The EPM consists of two elevated, open (brightly lit) arms perpendicular to two enclosed (dark)

arms (Rodgers and Dalvi, 1997; Stafstrom, 2006). Rodents prefer dark, enclosed spaces to

35

brightly lit, open areas, but they are also spontaneously exploratory by nature. Because rodents

have an innate fear of heights, elevating the EPM off the floor also contributes to the anxiety

level. The EPM is regarded as an unconditioned spontaneous behavioural conflict test that uses

an ethologically relevant situation to measure the conflict between the drive to explore a new

environment, and the tendency to avoid a potentially dangerous area (File, 1993; Treit et al.,

1993; Carobrez and Bertoglio, 2005). It has also been suggested that a rodent‘s aversion to open

spaces can be attributed to thigmotaxis, a reaction in which the animal remains close to the

vertical surfaces, and which is part of their natural defensive repertoire (Carobrez and Bertoglio,

2005). Similar to the EPM, the open field is another unconditioned spontaneous behavioural

conflict test (Falter et al., 1992; Treit et al., 1993). The open field test evaluates the conflict

between exploration of a novel environment and aversion to open spaces from which escape is

prevented by a surrounding wall. Typically, rodents will explore a new environment but become

less active on subsequent exposure to the environment, a process referred to as habituation

(Walsh and Cummins, 1976; Prut and Belzung, 2003).

SE induced by pilocarpine or kainic acid results in a long-lasting anxiolytic response as assessed

by the EPM test, with adult (Choleris et al., 2001; Prut and Belzung, 2003; Chauviere et al.,

2009) and immature rats (Santos et al., 2000; Sayin et al., 2004; Kubova et al., 2004; Detour et

al., 2005; dos Santos et al., 2005; Cardoso et al., 2009; Sun et al., 2009) spending a greater

amount of time in the unprotected open arm. In contrast, Groticke et al., (2008) failed to find

behavioural changes with this task in mice. Less consistent results are reported in the open field

test. Brandt et al., (2006) reported an anxiolytic response in rats following self-sustaining status

epilepticus (SSSE) induced by electrical stimulation of the basolateral amygdala, with an

increased amount of time spent in the central zone of the open field. In contrast, others found

that SE caused by pilocarpine or kainic acid resulted in an anxiogenic response, whereby rats

(Santos et al., 2000; dos Santos et al., 2000; Sayin et al., 2004) or mice (Cardoso et al., 2009)

exhibited increased thigmotaxis (e.g., increased locomotor activity and/or time spent in the

periphery of the open field). Finally, several studies have reported no differences in locomotor

activity or time spent in the central or peripheral zones of the open field (Groticke et al., 2008;

Cardoso et al., 2009; Müller et al., 2009). Because behavioural responses of rodents in these

tasks are extremely sensitive to differences in the species/strain used, the environmental

conditions (e.g., lighting) and the procedures used, these factors are likely to have contributed to

36

differences in results between the studies. Impaired anxiety in rodents following SE has been

reported in other behavioural tasks, which include the hole-board test (Kubova et al., 2004; Sayin

et al., 2004; Szyndler et al., 2005; de Oliveira et al., 2008; dos Santos et al., 2006), the light-dark

box test (Groticke et al., 2008; Müller et al., 2009), the novel-object recognition test (de Oliveira

et al., 2008; Groticke et al., 2008; Müller et al., 2009), and the elevated T-maze test (Groticke et

al., 2008; Müller et al., 2009). Results are summarized in appendix I, Table A1-2.

1.6.6.2 Exploration

Although exploratory activity is less routinely assessed in the open field and EPM tests, it can

provide critical information on different aspects of behavior (Rodgers et al., 1997; Heinrichs and

Seyfried, 2006 Milgram et al., 1988). This includes the well-being of the animal, the presence or

increase of inadaptive behaviour, and the reactivity of the animal to a novel environment. In

these tasks, exploratory activity can be analyzed by the frequency and/or duration of rearing

activity (also referred to as vertical activity), by the frequency of risk assessments, and by

locomotor activity (Rodgers and Dalvi, 1997; Carobrez and Bertoglio, 2005). Risk assessments

include stretched-attend postures, and involve the reluctance of the animals to leave the confines

of the enclosed protected arm of the EPM (Rodgers and Dalvi, 1997; Carobrez and Bertoglio,

2005).

In the EPM test, studies have demonstrated that SE causes a long-lasting increase in exploratory

behaviour (e.g., frequency of rearing and risk assessments) (Detour et al., 2005; Sun et al., 2009).

While some authors have similarly reported an increase in rearing activity in the open field test

(Kubova et al., 2004; Erdogan et al., 2005; Sun et al., 2009), others have found diminished

rearing activity (dos Santos et al., 2005; Groticke et al., 2008). An increase in locomotor activity

has also been reported in the EPM test (e.g., number of closed or total arm entries) and/or the

open field test (e.g., total distanced travelled) ( Milgram et al., 1988; Santos et al., 2000; Kubova

et al., 2004; Sayin et al., 2004; Detour et al., 2005; dos Santos et al., 2005; Szyndler et al., 2005;

Brandt et al., 2006; Cardoso et al., 2009; Müller et al., 2009; Sun et al., 2009). Impaired

exploratory activity in rodents following SE has been detected in other behavioural tests,

including the hole-board test and the novel object recognition test (Groticke et al., 2008; Müller

et al., 2009). Results are summarized in appendix I, Table A1-2.

37

A change in exploratory and/or locomotor activity in rats following SE can indicate a decrease in

the well-being of the animal (Heinrichs and Seyfried, 2006). In a series of comprehensive

articles on animal well-being, Clark and colleagues defined specific indicators of wellness in

multiple species (Clark et al., 1997a; Clark et al., 1997b; Clark et al., 1997c; Clark et al., 1997d).

For instance, wellness is determined by the presence of certain species-typical behaviour, such as

grooming and exploratory activity (e.g., rearing and risk assessments) in rats. An absence of

abnormal behaviours is also required, such as non-goal directed activities; in rodents, this can

include motor stereotypes, increased non-directed locomotion, flight and immobility, social

withdrawal and aggression (Heinrichs and Seyfried., 2006). A human analog of the well-being

assessment for epileptic animals is the Quality of Life in Epilepsy inventory (QOLIE) (Heinrichs

and Seyfried., 2006), which includes assessment of physical, affective, and social behaviours

(Devinsky et al., 1995). Not surprisingly, patients with epilepsy show impairment in these

aspects of their daily lives (Devinsky et al., 1995; Devinsky., 2000).

As outlined previously, a change in exploratory behavior can indicate the presence of an

inadaptive response. For instance, a decrease in exploratory behaviour can result from changes

of other behaviours, such as increased non-directed locomotor activity (Heinrichs and Seyfried,

2006). In the EPM, Groticke et al., (2008) and Müller et al., (2009) showed that decreased

exploratory behaviour (i.e., stretch-attend postures) was accompanied by increased locomotion

in mice following pilocarpine-induced SE. A decrease in exploratory behaviour and risk-

assessments can also indicate an animal exhibiting hyporeactivity to a stressful environment (dos

Santos et al., 2005). In support of this view, several studies showed a decrease in rearing activity

in rats and mice following SE (dos Santos et al., 2005; Groticke et al., 2008).

In several studies, animals were repeatedly assessed in the open field test (Krsek et al., 2001;

Kubova et al., 2004; Sun et al., 2009). In a control (or non-epileptic) animal, the behavioural

responses to the open field normally habituates over subsequent exposures (e.g., attenuation of

rearing activity and/or locomotion). However, pilocarpine-treated rats exhibited no signs of

habituation, suggesting impairment in a simple nonassociative form of learning and memory

(Krsek et al., 2001; Kubova et al., 2004; Sun et al., 2009).

38

1.6.6.3 Aggression

Increased aggressiveness is reported in patients with epilepsy (Geschwind, 1983; Cornaggia et

al., 2006). Psychological stress, the effects of anticonvulsant therapy and the actual occurrence

of seizures are ruled out as possible causes (Geschwind., 1983). Geschwind (1983) speculated

that interictal emotional disturbances in patients with TLE are the result of an intermittent spike

focus (i.e., increased hyperexcitability) in the temporal lobe that leads to a permanent alteration

in the responsiveness of the limbic system. In particular, the temporolimbic structures of the

brain that subserve emotional representation are highly epileptogenic, and are involved in the

hypothalamic-pituitary-adrenal axis, which modulates hormonal secretion and mediates

hormonal feedback (Herzog, 1999). These alterations in neural signaling may produce a

heightened emotional response to many stimuli as well as a decrease in sexual responsiveness

(Herzog, 1999). Of particular interest, brain damage to the temporolimbic structures is

correlated with increased aggressiveness (Hallera and Kruk, 2006).

A number of simple tests are available to assess aggressiveness and hyperexcitability in rodents

following SE (Rice et al., 1998; Heinrichs and Seyfried, 2006). Rice et al., (1998) demonstrated

that rats following pilocarpine-induced SE behaved more aggressively toward the experimenter

in the handling (or pickup) test (e.g., involves picking the rat up around its midsection and

ranking its behavioural response), and showed increased excitability in the touch-response test

(e.g., ranking the startled response when the animal is prodded from behind). Kainic acid also

resulted in increased aggression in rats when handled (Milgram et al., 1988; Stafstrom et al.,

1993). Increased hyperexcitability and aggression in rats were detected within the first day

following SE, and the behavioural alterations were long-lasting (Milgram et al., 1988; Rice et al.,

1998).

1.6.7 The effect of SE on spatial learning and memory

Impaired learning and memory has been reported in adults and children following SE

(Helmstadter et al., 2007), and in patients with TLE (Motamedi and Meador, 2003; Vingerhoets,

2006). More specifically, patients with TLE frequently showed deficits in declarative memory

(ability to acquire facts and events related to one‘s personal past, which is often compared with

visual-spatial learning in rats (Guerreiro et al., 2001; Heinrichs and Seyfried, 2006) and in the

performance of visuospatial tasks ( Hermann et al., 1997; Gleissner et al., 1998; Abrahams et al.,

39

1999). Cognitive impairment in human epilepsies is one aspect that can be modeled quite

effectively and reliably in post-status epilepticus animal models (Heinrichs and Seyfried, 2006).

The MWM and the radial arm maze are the most commonly used methods to assess spatial

learning and memory in rodents, and both have been used frequently in epilepsy research

(Stafstrom, 2006). Because the MWM was used to assess spatial learning and memory in the

present thesis, this method and the effect of SE on MWM performance were discussed in detail

in the subsequent sections.

1.6.7.1 The Morris water maze as a test of visual-spatial learning and memory

The majority of research assessing the effects of SE on cognitive functioning is based on data

from rats being tested on the hidden platform version of the MWM (Stafstrom, 2006). Briefly,

the MWM consists of a large circular pool filled with opaque water in which a small escape

platform is hidden (Morris, 1984; Brandeis et al., 1989; Vorhees and Williams, 2006). During a

number of training trials, the animals learn to navigate to the submerged platform using distal,

visual cues surrounding the pool. Rats are proficient swimmers, and the water maze utilizes

negative reinforcement (water immersion) to motivate the animal to learn and recall the platform

location. Performance is primarily assessed by the latency (or distance travelled) for the animal

to locate and escape onto the platform.

It is now recognized that acquisition of the MWM task has two main components: (1)

behavioural-strategies learning and (2) spatial learning (McDonald and White, 1994; Morris and

Frey, 1997; Inglish and Morris, 2004). The first component involves learning behavioural

strategies that allow the rat to move around in its spatial environment and to learn the most

effective strategies for locating and reaching its target (e.g., the submerged platform).

Acquisition of behavioural strategies involves selection from a variety of instinctive behaviours,

such as suppression of the rat‘s normal response of thigmotaxic swimming in close proximity to

the pool wall, in favour of searching the inner portion of the pool where the platform is located,

and recognizing and consistently using the submerged platform as an escape (McDonald and

White, 1994; Whishaw, 1989). The second component is the spatial learning component,

whereby the rat builds a spatial cognitive map correlating context information (extramaze cues)

with platform location (Morris, 1984; DiMattia and Kesner, 1988; Fenton and Bures, 1993).

Once this map is created, the rat is able to swim directly to the escape platform from any point of

40

the circumference of the tank. Success in the spatial learning component requires prior learning

of the behavioural-strategies component (Whishaw, 1989; Morris, 1989; Bannerman et al., 1995;

Saucier and Cain, 1995; Baldi et al., 2003).

Independent memory systems appear to be required for learning of behavioural-strategies and for

spatial learning (McDonald and White, 1994; McDonald and White, 1993; Miranda et al., 2006).

Evidence strongly supports the role of the hippocampus and posterior parietal cortex in spatial

mapping and memory (Cain et al., 2006). The prefrontal cortex, striatum, cerebellum and medial

thalamus appear to be involved in acquisition of behavioural strategies (Cain et al., 2006;

Packard and McGaugh, 1996; Leggio et al., 1999). For instance, rats with hippocampal damage

acquired the visible platform version of the MWM normally but were impaired on acquisition of

the hidden platform version of the task (Fenton and Bures, 1993; Broadbent et al., 2004; Leggio

et al., 2006). In the visible (or cued) platform task, animals only require acquisition of

behavioural strategies to locate the platform made visible either with a flag and/or with the

platform elevated above the water surface. On the other hand, rats with damage to the striatum

or cerebellum exhibited a characteristic behavioural pattern in which they did not swim towards

the center of the pool, but continually scratched at and/or swam around the periphery of the walls

(McDonald and White, 1994; Whishaw et al., 1987; Federico et al., 2006). This behaviour was

interpreted as the animal‘s inability to suppress their thigmotaxic response to the task. In

general, performance in the hidden platform version of the MWM task involves acquisition of

behavioural strategies and spatial learning, with each component dependent on separate memory

systems that work in concert to achieve optimal performance in the task (McDonald and White,

1994; McDonald and White, 1993; Miranda et al., 2006).

1.6.7.2 The effect of SE on performance in the MWM task

Numerous studies have demonstrated impaired MWM performance as assessed by the hidden

platform MWM task in immature (Hung et al., 2002; Stafstrom, 2002; Kubova et al., 2004; Sayin

et al., 2004) and adult (Milgram et al., 1988; Rice et al., 1998; Hort et al., 1999; Mikati et al.,

2001;; Hort et al., 2000; Nissinen et al., 2001; McKay and Persinger, 2004; dos Santos et al.,

2005; Frisch et al., 2007; Zhou et al., 2007; Cunha et al., 2009; Jun et al., 2009; Sun et al., 2009)

rats and in mice (Mohajeri et al., 2003; Groticke et al., 2008; Müller et al., 2009) following SE.

Impaired MWM performance has been detected as early as 3 to 7 days after SE ( Rice et al.,

41

1998; Hort et al., 1999; Hort et al., 2000; Cunha et al., 2009) and appeared to be long-lasting

(testing conducted 1 to 5 months after SE (Rice et al., 1998; Hort et al., 2000; Kubova et al.,

2004; McKay and Persinger, 2004; dos Santos et al., 2005; Frisch et al., 2007; Groticke et al.,

2008; Müller et al., 2009). Because animals are in poor physical condition for 3 days after SE,

testing in the MWM has never been conducted earlier than 3 days (Rice et al., 1998; Hort et al.,

1999; Hort et al., 2000; Cunha et al., 2009). Immature rats exhibited less severe cognitive

deficits when compared to adult rats, and this difference is attributed to less severe neuronal loss

occurring in immature brains (Stafstrom et al., 1993; Kubova et al., 2004; Stafstrom, 2006).

Milgram et al., (1998) reported that MWM performance is improved when the task is repeated.

However, this finding may depend on the SE-induction protocol or the rodent strain/species

used. For instance, Hort et al., (2000) found that although LEH rats showed improved

performance when the task was repeated, no improvement was detected in Wistar rats.

Although SE is often suggested as being the primary cause of impaired MWM performance

(Rice et al., 1998; Hort et al., 1999; Hort et al., 2000; dos Santos et al., 2005), two studies

showed that development of SRSs can also contribute to cognitive decline (Hort et al., 1999; Sun

et al., 2009). Of particular interest, Mohajeri et al., (2003) found that the severity of SE (i.e.,

intensity of seizures) and the extent of brain damage correlated with the severity of impairment

in MWM performance (Mohajeri et al., 2003). Cognitive deficits in rats after SE have been

reported in other behavioural tasks, including the radial arm maze (Letty et al., 1995; Wu et al.,

2001; Sayin et al., 2004; Detour et al., 2005) and the displaced object-recognition task

(Chauviere et al., 2009). Results are summarized in appendix I, Table A1-3.

Although the MWM task has revealed impaired performance in rats following SE, the exact

nature of this impairment is uncertain. This is because the commonly reported dependent

measure, escape latency to locate the submerged platform, cannot distinguish between deficits in

spatial learning and deficits in behavioural strategies. Most studies attribute poor MWM

performance to impaired spatial learning and memory (see appendix I, Table A1-3). However,

several studies have additionally reported a thigmotaxic response in rats after SE, and that this

behaviour was not accounted for by the presence of sensory or motor impairment (results

described in appendix I, Table A1-3; Milgram et al., 1988; Hort et al., 1999; Kubova et al., 2004;

McKay and Persinger, 2004; Groticke et al., 2008; Jun et al., 2009); these findings indicate

deficits in behavioural strategies. In the present thesis, a detailed analysis on search strategy use

42

during hidden platform testing in the MWM was completed to determine: (1) the effect of SE on

search strategy selection, and (2) whether impaired search strategy use, along with deficits in

spatial learning and memory, contribute to poor MWM performance in rats after SE.

1.7 The goals of the thesis

The goals of the present thesis were threefold:

1. The first aim was to optimize our use of the lithium/pilocarpine model. This was

accomplished by comparing the effects of different rat strains and procedural protocols

on SE induction and mortality rates, seizure severity, and neuropathology (see chapter 3).

2. Our second goal was to characterize neurodegeneration and behavioural alterations in rats

after SE induced by the RLDP lithium/pilocarpine procedure. Briefly, this was achieved

by (1) completing a detailed timecourse analysis of neuronal loss following SE (see

chapter 4), (2) determining the effect of SE on behavioural alterations as assessed by the

open field test, elevated plus maze test, and four hyperexcitability tests (see chapter 6),

and (3) assessing the effect of SE on MWM performance and on use of behavioural

search strategies (see chapter 7).

3. Finally, our third goal was to test the effectiveness of a neuroprotective strategy using the

RLDP lithium/pilocarpine procedure we developed and characterized. Briefly, this was

achieved by assessing the ability of tat-NR2B9c to provide neuroprotection (see chapter

5) and mitigate behavioural alterations (see chapters 6 and 7) following

lithium/pilocarpine-induced SE in rats. Tat-NR2B9c is a synthetic peptide contructed to

disrupt the NMDAR signaling complex, and has been previously described in section

1.5.1.6.

The general hypothesis, specific hypotheses and specific objectives are discussed in chapter 2.

43

Chapter 2

Hypotheses and Specific Objectives

Patients with MTLE-HS frequently have a history of an IPI such as febrile convulsions, SE,

encephalitis, stroke or traumatic brain injury (Mathern et al., 1996; Mathern et al., 2002). The

lithium/pilocarpine model reproduces most clinical and neuropathologic features of MTLE-HS

(see section 1.1). In adult rats, pilocarpine leads to an acute episode of SE which serves as the

IPI (see section 1.3). This is followed by a latent period and subsequent development of SRSs

(see section 1.4). Data from humans and post-status epilepticus rodent models suggest that both

the risk of epilepsy (Hesdorffer et al., 1998; Zhang et al., 2002; Persinger and Dupont, 2004) and

the severity of behavioural alterations (Shorvon, 1994; Loscher, 2002; Devinsky, 2004a;

Pitkanen and Kubova, 2004; Naegele, 2007; Acharya et al., 2008) are associated with

neuropathological changes caused by the IPI (see section 1.5). In both conditions, neuronal

death is observed in the hippocampus, amygdala, thalamus, and piriform and entorhinal cortices

(see section 1.5.1).

The relationship between neuronal death, epileptogenesis, and cognitive/behavioural alterations

remains unclear. While some studies report a positive relationship between neuronal death and

the severity of epileptic seizures, others have shown that epileptogenesis can occur in the

absence of neuronal loss (see section 1.5.1.6). Some (Bolanos et al., 1998; Rice et al., 1998; dos

Santos et al., 2005; Brandt et al., 2006; Cunha et al., 2009; Jun et al., 2009), but not all (Cilio et

al., 2001; Halonen et al., 2001; Narkilahti et al., 2003b; Pitkänen et al., 2004; Zhou et al., 2007),

studies have demonstrated that reducing neuronal death can also alleviate SE-induced

behavioural and cognitive morbidity in rats (see Table 8.1). Our general hypothesis is that

genesis of SRSs, cognitive impairment and behavioural alterations are associated with neuronal

death caused by SE. The causal relationship between neuronal death, epileptogenesis and

cognitive and behavioural morbidity is discussed in chapter 8. This thesis was centered on 5

specific hypotheses and associated objectives that examined SE-induced neurodegeneration and

behavioural alterations in the lithium/pilocarpine model; each of these is presented in the

subsequent chapters (3 – 8).

44

2.1 Comparison of procedures for the induction of SE

High mortality is a major drawback in using the lithium/pilocarpine model (section 1.3).

Mortality can be abated, however, by administering lower doses of pilocarpine. Glien et al.,

(2001) demonstrated that when compared to a single dose of pilocarpine (30 mg/kg, LDP

protocol), repeated administration with lower doses of pilocarpine (10 mg/kg, RLDP protocol)

every ½ hour until SE onset significantly reduced mortality rates in Wistar rats (see section

1.3.5). Whether the RLDP protocol can similarly reduce mortality in other commonly used rat

strains has not been determined. Previous studies have demonstrated interstrain differences with

respect to seizure intensity, mortality rates and SE-induced neuropathology in various models of

epilepsy, which includes kindling (Loscher, 2002; Brandt et al., 2003a), and chemically-induced

SE produced by kainic acid (Sanberg and Fibiger, 1979; Golden et al., 1995; Xu et al., 2004),

pentylenetetrazol (Becker et al., 1997a) or pilocarpine (Hort et al., 2000; Xu et al., 2004).

Because interstrain differences exist with other models of SE (section 3.1), the ability of the

RLDP procedure to reduce mortality rates in Wistar rats may not similarly reduce mortality rates

in other rat strains.

Specific hypothesis: The ability of the RLDP procedure to lower mortality and increase the

incidence of SE in Wistar rats will not generalize to other rat strains.

Specific objectives: To compare the effect of the LDP and RLDP procedures on the induction

of SE and on mortality in Wistar and LEH rats (see chapter 3).

2.2 The effect of recovery time on SE-induced neurodegeneration

Presently, it remains unclear how SE-induced neurodegeneration in different brain regions

evolves over time (section 1.5.1.2). Previous studies have described early (<24 hrs) and delayed

(>24 hrs) neuronal death following prolonged seizures, and suggested that they may be attributed

to necrotic (Fujikawa et al., 1999; Fujikawa et al., 2000b; Fujikawa, 2005; Kotariya et al., 2010)

and apoptotic (Narkilahti et al., 2003a; Weise et al., 2005) processes, respectively. Furthermore,

early necrotic and delayed neuronal death with apoptotic features may occur in different brain

regions (Sloviter et al., 1996; Lopez-Meraz et al., 2010), or even within the same neuronal

population (e.g., pyramidal cell layer of the hippocampus) (Narkilahti et al., 2003a; Weise et al.,

2005). Neurodegeneration has not been previously investigated with the RLDP procedure.

45

Specific hypothesis: SE induced by the RLDP procedure results in differential rates of neuronal

death in different brain regions.

Specific objectives: To determine in which brain regions neuronal cell death first appears, how

neuronal loss evolves over time, and the extent of neuronal death that occurs early (<24hrs) or is

delayed (>24 hrs) (see chapter 4).

2.3 The effect of tat-NR2B9c on SE-induced neuropathology and cognitive impairment

NMDA receptors mediate seizure-induced neuronal death by excitotoxicity (section 1.5.1.3).

Binding of the NMDA receptor to the postsynaptic density protein-95 (PSD95) links the receptor

to downstream signaling molecules (section 5.1). During SE, excessive synaptic release of

glutamate activates NMDA receptors resulting in the influx of Ca2+

which, in turn, can activate

signaling molecules located proximal to the NMDA receptor ion channel by virtue of their

association with PSD-95, and initiate neurotoxic downstream signaling cascades. Tat-NR2B9c

was constructed to interfere with the binding of the NR2B subunit of NMDA receptors with the

PDZ1 and PDZ2 domains of PSD-95 (described in Methods 5.2.3). Administration of tat-

NR2B9c protected cultured neurons from excitotoxicity, and markedly reduced focal ischemic

brain damage and preserved cognitive function in rats when applied before or up to 3 hours after

insult (Aarts et al., 2002; Sun et al., 2008).

Specific hypothesis: Tat-NR2B9c will provide neuroprotection and mitigate cognitive outcome

in rats following SE.

Specific objectives: To determine the effects of tat-NR2B9c on (1) the neuronal death induced

by SE and (2) the behavioural/cognitive deficits associated with SE (see chapter 5).

2.4 The effect of SE on behavioural performance in tasks assessing anxiety, exploration and aggression

SE leads to development of behavioural alterations in the EPM test, the open field test, and the

hyperexcitability tests (section 1.6.6). When these changes first occur and whether or not they

are long lasting remains unclear. Previous studies have shown that SE-induced

neurodegeneration occurs within days following SE (section 1.5.1.2). Because the majority of

46

neuronal death occurs within the first several days (section 1.5.12), and is associated with

behavioural morbidity (sections 1.5.1.6 and 1.6.3), it is likely that behavioural alterations in

anxiety, exploration and aggression similarly develop within the first several days after SE.

Furthermore, since the bulk of SE-induced neuronal death precedes the development of epilepsy,

SRSs are unlikely to contribute to these behaviours.

Specific hypothesis: SE will result in early and long-lasting behavioural changes in tasks

assessing anxiety, exploration and aggression and will not be affected by the development of

SRSs.

Specific objectives: To determine the effect of SE on the behaviour of rats in the elevated plus

maze, open field, and four hyperexcitability tests at early and late times following SE and

following the development of SRSs (see chapter 6).

2.5 The effect of SE on search strategy use in the Morris water maze

Although it has been shown that SE results in impaired performance in the hidden platform

version of the MWM task (section 1.6.7), the effect of SE on search strategy use has not been

previously assessed. SE-induced neuronal death affects the neural systems that underlie spatial

learning and behavioural strategies learning, both components which are critical in MWM

performance (section 1.6.7.1). Since behavioural strategies are acquired before spatial learning

in rats during testing (section 1.6.7.1), impaired search strategy use may underlie the impaired

MWM performance in epileptic rats.

Specific hypothesis: SE results in the impaired development of search strategies that in turn

contributes to suboptimal performance in the MWM task.

Specific objectives: To determine the effect of SE on the development of search strategies in

the MWM task, and to assess the relationship between search strategies and performance in the

MWM (see chapter 7).

47

Chapter 3

A comparison between Long-Evans hooded and Wistar rats related to the induction and severity of

status epilepticus in the low-dose and repeated low-dose lithium/pilocarpine procedures

3.1 Introduction

Treatment with pilocarpine, alone or in combination with lithium, is widely used to produce

seizures in animal models of both status epilepticus (SE) and latent seizure development (Turski

et al., 1989; Pitkänen and McIntosh, 2006). Pilocarpine when used alone requires high doses

(320-400 mg/kg i.p or s.c.), and is associated with high mortality rates and low rates of seizure

induction (Turski et al., 1983b; Turski et al., 1989; Cavalheiro et al., 2001). Pretreatment of rats

with lithium chloride permits the dose of pilocarpine to be decreased approximately 10-fold (30

mg/kg) and results in both lower mortality rates and higher rates of seizure induction (Clifford et

al., 1987). The repeated administration of pilocarpine to lithium pretreated rats allows the

dosage of pilocarpine to be even further reduced (10 mg/kg) and is effective in reducing

mortality, while producing SE and chronic epilepsy in a high proportion of animals (Glien et al.,

2001).

Interstrain difference with respect to seizure intensity, mortality rates and SE-induced

neuropathology has been reported in various models of epilepsy, including kindling (Loscher,

2002; Brandt et al., 2003a), and chemically-induced SE produced by kainic acid (Sanberg and

Fibiger, 1979; Golden et al., 1995; Xu et al., 2004), pentylenetetrazol (Becker et al., 1997a) or

pilocarpine (Hort et al., 2000; Xu et al., 2004). For instance, Wistar rats showed greater

sensitivity and exhibited more reliable convulsant responses to kainic acid than Sprague Dawley

and Long-Evans hooded (LEH) rats (Golden et al., 1991; Golden et al., 1995). Such strain

differences, however, may not be consistent across models. Hort et al., (2000) reported that

pilocarpine effects were more pronounced in LEH rats that exhibited a higher rate of SE

induction, higher mortality rate, more severe neuropathology and a behavioural outcome worse

than Wistar rats.

48

In view of the reported advantages of using repeated administration of low-doses of pilocarpine

in combination with lithium for inducing SE in Wistar rats (Glein et al., 2001), it was important

to establish whether this effect generalizes across other strains. Accordingly, our first objective

was to compare the rates of seizure induction, intensity of behavioural seizures, and mortality

rates of Wistar and LEH rats subjected to either the single-dose injection of 30 mg/kg

pilocarpine, or the repeated injections of 10 mg/kg pilocarpine, in combination with lithium. The

results identified several differences between the responses of the two strains to the SE-inducing

protocols, and demonstrated that the repeated administration of low-doses of pilocarpine

significantly reduced mortality rates in Wistar, but not in LEH, rats.

The relationship between convulsive seizure intensity and neuropathology remains unclear.

Although a few studies report a correlation between seizure severity and neuronal injury (Brandt

et al., 2003a; Tilelli et al., 2005), others have failed to detect such a relationship (McKhann et al.,

2003; Schauwecker et al., 2004; McLin and Steward, 2006; Lorenzana et al., 2007). Because

seizure severity during SE varied with rat strain and seizure-induction protocol, we also

examined the relationship between seizure intensity and neuropathology.

3.2 Methods

3.2.1 Animals

All procedures were approved by the University of Toronto Scarborough Animal Care

Committee and were in accordance with the guidelines established by the Canadian Council on

Animal Care. Male LEH and Wistar rats (Charles River, Montreal) weighing between 300 and

350 g were individually housed with free access to food and water for at least 7 days in 12 h

light/dark cycles before experimental use. A total of 89 Wistar rats and 88 LEH rats were used

in the analyses of the behavioural responses to convulsant treatment.

3.2.2 Induction of status epilepticus

Two procedures were used for the induction of SE. In the low-dose lithium/pilocarpine ( LDP)

procedure rats were pretreated with lithium chloride (3mEq/kg, i.p.) 24 hours before the injection

of pilocarpine, and received methylatropine nitrate (10 mg/kg, i.p) 30 min prior to pilocarpine.

LEH (n=42) or Wistar (n=25) rats were then administered an initial injection of pilocarpine (30

mg/kg, i.p.). If SE did not develop within 60 min, a second pilocarpine injection (15 mg/kg) was

49

administered. A total of 5 LEH and 2 Wistar rats required the second injection. Diazepam (4

mg/kg, i.p.) was administered at 1, 3 and 5 hours following the onset of SE to control the

duration of seizure activity.

For the repeated low-dose lithium/pilocarpine (RLDP) procedure, rats were pretreated with

lithium chloride (3mEq/kg, i.p.) 24 hours before and methylatropine nitrate (10 mg/kg, i.p) 30

min before the initial injection of pilocarpine. Pilocarpine (10 mg/kg, i.p.) was then administered

to LEH (n=46) or Wistar (n=64) rats every 30 min as described by Glien et al., (2001) until the

rat experienced a generalized, class IV/V seizure (see section 3.2.3), since rats generally

developed SE shortly thereafter. Animals that did not develop SE within 30 min of the first class

IV/V seizure received additional pilocarpine injections at 30-min intervals up to a maximum of 6

injections. Diazepam (4 mg/kg, i.p.) was administered at 1, 3 and 5 hours following onset of SE

to terminate seizure activity.

3.2.3 Monitoring of seizure activity

The behavioural progression of pilocarpine-induced seizures was assessed using a modified

Racine scale (1972) as described previously in Cammisuli et al., (1997) and in Veliskova (2006).

Seizure activity was recorded as: stage I, staring with mouth clonus; stage II, head nodding,

automatisms (e.g., scratching, sniffing orientation); stage III, unilateral forelimb clonus; stage

IV, bilateral forelimb clonus (rearing); stage V, forelimb clonus with rearing and one fall; stage

VI, forelimb clonus with rearing and multiple successive falls; stage VII, tonic/clonic seizures

(e.g., running and jumping). Animals were continuously monitored following the first injection

of pilocarpine, and the maximum stage of seizure activity occurring in each 5 minute interval

following SE onset was analyzed.

3.2.4 Post-seizure animal care:

Immediately following the first injection of diazepam to terminate seizures, animals received 5

ml 0.9% saline (3ml i.p. and 2ml s.c). Additional saline (5ml i.p) was given on the morning and

evening of the next day (Glien et al., 2001). Starting on the second day after SE, animals were

tube-fed with softened rat chow mixed with applesauce for 3 days on average. Softened rat

chow was also provided in dishes until the rats commenced to eat hard pellets.

50

3.2.5 Histology and Stereological analysis:

The extent of neuronal injury after SE was assessed by staining brain sections with NeuN

antibodies, a specific neuronal marker (see section 4.2.5). Because of high mortality rates in

LEH rats, the comparison of neuropathology between rat strains was conducted at 24 hours after

SE induced with the RLDP protocol. In contrast, comparison of neuropathology between the

LDP and RLDP procedures was assessed in Wistar rats at 3 months, subsequent to behavioural

tests conducted for a separate study. Animals were anaesthetized with a mixture of xyaline (26

mg/kg i.p.; CDMV, Saint-Hyacinthe, Quebec) and ketamine (174 mg/kg i.p., CDMV, Saint-

Hyacinthe, Quebec), and sequentially perfused transcardially with 90 ml of 0.1 M phosphate-

buffered saline, pH 7.4 (PBS), followed by 400 to 500 ml of 4% (w/v) paraformaldehyde, pH 7.4

in PBS. Brains were removed and left overnight at 4˚C in the paraformaldehyde solution. The

following day, brains were soaked for cryoprotection in 30% (w/v) sucrose in PBS until they

sank at room temperature. Next, brains were frozen in 2-methylbutane at -35˚C, and stored at -

80˚C until further use. Forty µm coronal sections were prepared with a freezing microtome.

Sections were placed in 24-well culture plates containing antifreeze solution (50 mM of pH 7.4

phosphate buffer, 30% (v/v) ethylene glycol, 15% (w/v) glucose), and stored at -20˚C.

For each animal, 3 coronal brain sections containing the dorsal hippocampus were selected

between Bregma -3.2 mm through -3.72 mm (Paxinos and Watsons rat brain atlas, 5th

edition),

with the first section presenting all hippocampal subfields, exhibiting only the dorsal portion of

the lateral ventricle, and containing the most ventral portions of the capsular division (CeC) and

lateral division (CeL) of the central amygdala. The subsequent sections were selected ventrally

at 240 µm intervals, and exhibited the lateral ventricle extending to the ventral portion of the

brain, replacing the structures CeC and CeL detected in the initial section. Section were rinsed in

PBS (3 x 5 min washes), reacted overnight at 4˚C with NeuN antibody (1:1000; Chemicon,

Billerica, MA, USA) in 0.2% (v/v) goat serum, 0.3% (v/v) Triton X-100 in PBS followed by 2

hours at room temperature with Cy3 conjugated secondary antibody (1:200; Chemicon, Billerica,

MA, USA) in 0.2% (v/v) goat serum, 0.3% (v/v) Triton X-100 in PBS.

Neurons were imaged using a Zeiss LSM510 Laser Scanning Confocal microscope equipped

with a 40x/1.3 oil-immersion objective lens, and neuronal density determined using the unbiased

optical dissector technique as described by West and Gunderson, 1990. Briefly, three counting

51

frames (120 X 60 µM) were positioned in the CA1, CA3, hilus, and posterior piriform cortex

(PPC), two counting frames were positioned in the CA4 region, and 1 counting frame was placed

in CA2 of the left brain hemisphere as shown in Figure 3.1, A-C. Right brain structures were

visually inspected and found to be comparable to the left side. Neurons were identified as

NeuN-positive cells that contained a relatively large (>8µm) soma (Shi et al., 2004).

Chromophilic somas contained within each counting frame, or touching the inclusion borders of

the frame (upper and right borders) were counted (West and Gunderson, 1990). The dissector

height equivalent to the known tissue height prior to staining (40 µm) was used in all

calculations (Hatton and Von Bartheld, 1999), and upper or lower exclusion borders in the z

plane were excluded as previous studies demonstrated that no significant variation in neuronal

density occur whether such borders were used or not (Harding et al., 1994; Gardella et al., 2003;

Azcoitia et al., 2005). Unless otherwise specified, cell densities for individual animals represent

the average densities of all counting frames in a particular region for 3 brain sections. All results

are expressed as neurons per mm3.

52

Figure 3.1 Placement of counting frames within the hippocampus, hilus and piriform

cortex. Representative images of NeuN stained coronal sections of a sham animal, depicting the

left dorsal hippocampus (A), hilus (B) and posterior piriform cortex (PPC) (C). Boxes represent

placement of counting frames in the pyramidal cell layer (pyr) of the CA1 (1a-c), CA2 (2a), CA3

(3a-c) and CA4 (4a-b), in the hilus (5a-c), or also referred to as the polymorph layer of the

dentate gyrus (PoDG), and in layer II of the PPC (a-c).

53

54

3.2.6 Drugs:

Pilocarpine, lithium chloride and methylatropine nitrate were purchased from Sigma (St Louis,

Missouri) and dissolved in 0.9% (w/v) saline prior to administration. Diazepam was purchased

from CDMV (Saint-Hyacinthe, Quebec) and used as the commercial solution (5 mg/ml).

3.2.7 Statistical Analysis:

Analysis of variance (ANOVA) and chi-square analysis were performed using Statistica 9.0

software. Non-parametric testing was performed using GraphPad Prism 5 software.

Significance was set at a p-value of 0.05 or less.

3.3 Results

3.3.1 Induction of SE in Wistars and Long Evans Hooded rats

We initially compared the effectiveness of the RLDP and LDP procedures in inducing SE in

Wistar and LEH rats. SE induction rates between groups were assessed using chi-square

analysis. The number of pilocarpine injections administered in the RLDP protocol and the

latency to SE onset were assessed by the Student‘s t-test. The results are summarized in Table

3.1, and indicate that the LEH rats are more sensitive to the convulsive effects of pilocarpine

compared to Wistar rats. The average number of pilocarpine injections required to induce SE

using the RLDP procedure was significantly lower for LEH than for Wistar rats. With the LDP

procedure, Wistar rats had a significantly longer latency to SE development than the LEH rats.

Although both the RLDP and LDP procedures resulted in similar rates of SE induction within

strains (LEH: RLDP - 80% induction, LDP – 88%; Wistars: RLDP – 59%, LDP – 68%), a

greater percentage of LEH rats, as compared to Wistar rats, entered SE with both procedures (see

Table 3.1).

3.3.2 The effect of SE on mortality

Mortality rates between groups were assessed using chi-square analysis. Table 3.1 summarizes

mortality data as a function of strain and protocol. LEH rats exhibited a higher rate of mortality

than Wistar rats with both the LDP and RLDP induction protocols. Wistars showed a significant

reduction in mortality from 41% with the LDP protocol to 16% with the RLDP procedure.

55

Although more Long Evans rats died following the LDP procedure than the RLDP procedure

(80% as compared to 65%), this difference was not significant (p = 0.09).

Table 3.1: Comparison of rat strain and SE-inducing protocols between SE induction

and mortality rates at 3 days following SE.

Treatment Total #

of rats

per

group

# of pilocarpine

injections for

RLDP protocol1

Latency to

SE onset for

LDP

protocol2

SE

Induction3

Survived4 Died

4

LEH

RLDP

46 2.7 ± 1.1 ---- 37 (80%) 13 24 (65%)

LEH LDP 42 ---- 36 ± 16 min 37 (88%) 7 30 (80%)

Wistar

RLDP

64 4.0 ± 1.4 * ---- 38 (59%)* 32 6

(16%)*^

Wistar

LDP

25 ---- 48 ± 16 min* 17 (68%)* 10 7 (41%)*

1. Number of repeated low-dose pilocarpine injections (10 mg/kg) administered with the RLDP

protocol. * Wistars required a greater # of pilocarpine injections compared to LEH rats (p<0.05).

2. Latency to SE onset in the LDP protocol (average ± SD). * Wistars had longer latency to SE

onset compared to LEH rats (p<0.05). 3. Number of animals that entered SE for 60 min.

Numbers in () represent % of animal that developed SE. * Strain difference in SE induction rates

detected across LDP and RLDP protocols (p<0.05). 4. Number of animals that survived or died

within 3 days following SE. Numbers in ( ) represent the % of animals that died. * Reduced

mortality rates detected in Wistar compared to LEH rats across LDP and RLDP protocols

(p<0.05). ^ Wistar RLDP has a lower mortality rate than Wistar LDP (p<0.05). Data analyzed

by Chi-square analysis or Student‘s t-test.

3.3.3 Severity of seizures in LEH and Wistar rats

We next compared the effect of SE induction protocol and rat strain on the intensity of

behavioural seizures during 60 min of SE (see Methods 3.2.3). Because behavioural seizures

was assessed using a I- to VII-grade ordinal scale, data from the initial 10 min of SE and from

the subsequent 50 min of SE was first analyzed using the non-parametric Kruskall-Wallis

56

ANOVA by ranks, followed by the Dunn‘s test to determine individual group differences. With

both SE induction protocols, the severity of seizures declined during the initial 10 min of SE,

with initial seizures scores of 4 to 5 decreasing to 2.5 to 3.5 over time for both LEH and Wistar

rats (Figure 3.2). No group differences in seizure severity were detected within the initial 10 min

of SE (Fig 3.2, black bars). During the subsequent 50 min of SE, LEH rats exhibited

significantly more severe seizure activity than Wistar rats with both procedures (Figure 3.2, grey

bars; p<0.05). Furthermore, Wistar rats treated with the RLDP protocol consistently exhibited

significantly less severe convulsive seizures than Wistar rats treated with the LDP protocol, or

LEH rats treated with either seizure-inducing protocol (Figure 3.2 B, grey bars; p<0.05).

57

Figure 3.2 Comparison of behavioural seizure activity between rat strain and SE-inducing

protocol. (A) Average seizure activity was determined for the initial 10 min (black bars) and for

the subsequent 50 min (grey bars) of SE as described in Methods (mean ± SEM). * Behavioural

seizure activity less severe than the corresponding 10 min value (p<0.05). ** Behavioural

seizure activity less severe than the values for LEH rats treated with either the LDP or RLDP

procedures, and Wistar rats treated with the LDP procedure (p<0.05). *** Behavioural seizure

activity less severe than the value for LEH rats with the LDP procedure (p<0.05). LEH RLDP

(n=37), LEH LDP (n=37), Wistar RLDP (n=38), Wistar LDP (n=17). Data analyzed using the

non-parametric Kruskall-Wallis ANOVA by ranks, followed by the Dunn‘s test to determine

individual group differences.

58

59

3.3.4 SE-induced neuropathology in LEH and Wistar rats following SE induced with the RLDP procedure

The effect of the RLDP procedure on SE-induced neuronal death was compared between LEH

and Wistar rats to determine if there were any strain differences in neuropathology. Because of

difficulty in keeping LEH rats alive for prolonged recovery times, neuron density was assessed

24 hours following termination of SE. Data were first analyzed using one-way ANOVA,

followed by the Newman-Keuls post-hoc test to detect group differences. As illustrated in

Figure 3.3 (A, B), SE induced with the RLDP procedure resulted in significant neuron loss in the

CA1, CA3, and CA4 pyramidal cell layers in both LEH and Wistar rats. Neuronal damage was

more severe in LEH rats for each of these regions. Extensive neurodegeneration was observed in

the PPC in both strains, whereas neuron loss in the hilus was only observed in Wistar rats. No

neuron loss was detected within the CA2 subfield with either rat strain.

3.3.5 Comparison of SE-induced neuropathology resulting from the LDP and RLDP protocols in Wistar rats

Since the RLDP protocol reduced the intensity of convulsive seizures in Wistar rats compared to

the LDP protocol, the extent of SE-induced neuronal loss resulting from each procedure was

examined. Brain sections were stained with anti-NeuN antibodies and quantified as in Methods

(section 3.2.5) 3 months after SE, subsequent to behavioural testing conducted as a separate

study. Data were first analyzed using one-way ANOVA, followed by the Newman-Keuls post-

hoc test to detect group differences. As illustrated in Figure 3.4 (A, B), significant neuron loss

was detected in the CA1, CA3, CA4, hilus and PPC, but not in the CA2. No difference in neuron

densities between LDP and RLDP protocols was detected.

60

Figure 3.3 Comparison of neuron cell densities in the hippocampus and piriform cortex of

LEH and Wistar rats following SE. SE was induced with the RLDP procedure, terminated

after 60 minutes, and animals sacrificed 24 hours later. (A) Confocal micrographs (400X) of

NeuN stained cells in the pyramidal cell layer of the CA1-CA4 subfields of the hippocampus,

hilus, and in layer II of the PPC. Scale bar = 20 µm. (B) Stereological analysis of NeuN-

positive cells was determined as in Methods (section 3.2.5), and neuron densities for individual

animals plotted as open circles. Mean cell density for each group is represented as a horizontal

bar. * Reduced neuron density in animals after SE compared to sham animals (p<0.05).

** Reduced neuron density in LEH SE compared to Wistar SE animals (p<0.05). Data analyzed

by on-way ANOVA followed by Newman-Keuls multiple comparison procedure.

61

62

Figure 3.4 Comparison of neuronal cell densities in the hippocampus and piriform cortex

of Wistar rats following SE. SE induced with the RLDP or LDP procedure was terminated

after 60 minutes, and animals sacrificed 3 months later. (A) Confocal micrographs (400X) of

NeuN stained cells in the pyramidal cell layer of the CA1-CA4 subfields of the hippocampus,

hilus, and in layer II of the PPC. Scale bar = 20 µm. (B) Stereological analysis of NeuN-

positive cells was determined as in Methods (section 3.2.5), and neuron densities for individual

animals plotted as open circles. Mean cell density for each group is represented by a horizontal

bar. * Reduced neuron density in animals after SE compared to sham animals (p<0.05). Data

analyzed by one-way ANOVA followed by Newman-Keuls multiple comparison procedure.

63

64

3.4 Discussion

High mortality rates have been a major drawback in chemoconvulsant models of seizure

development. Previous studies have suggested two possible means of reducing mortality:

modification of the SE induction procedure and/or selection of rat strains (Hellier et al., 1998;

Hort et al., 2000; Glien et al., 2001). In the lithium/pilocarpine model, Glien et al., (2001)

demonstrated that repeated low-doses of pilocarpine (10 mg/kg) increased survival rates in

Wistar rats. However, the effectiveness of this procedure in other rat strains was not determined.

We therefore compared the RLDP protocol with the LDP protocol in LEH and Wistar rats, and

identified several interstrain differences in response to the two seizure-inducing procedures. The

main findings were: 1) Wistar rats showed greater sensitivity to the convulsive effects of

pilocarpine and exhibited less severe status in the LDP and RLDP procedures than LEH rats, 2)

the RLDP protocol reduced mortality and severity of status in the Wistar rats when compared to

the LDP protocol, but had no significant effect in LEH rats, 3) with the RLDP procedure, LEH

rats exhibited more severe SE-induced damage in CA1, CA3 and CA4 than Wistar rats, while

hilar neuron loss was only detected in Wistar rats, and 4) the differences in seizure severity

between the LDP and RLDP procedures in Wistar rats did not result in detectable differences in

neuronal death.

3.4.1 Differential effects of induction procedure in LEH and Wistar rats

Differences in the responses of LEH and Wistar rats with respect to seizure susceptibility and

mortality were found. Wistar rats were more resistant to the convulsive effects of pilocarpine

than LEH rats, requiring a greater number of pilocarpine injections to induce SE with the RLDP

protocol, displaying a longer latency to SE development with the LDP protocol, and exhibiting a

lower rate of SE induction with both seizure-inducing procedures. Wistar rats also exhibited less

severe behavioural seizures and lower mortality rates when compared to LEH rats with the LDP

and RLDP procedures. Strain differences in response to pilocarpine have previously been

reported. Consistent with our results, Hort et al., (2000) reported that LEH rats exhibit more

severe convulsive SE and sustained higher mortality rates when compared to Wistars following a

single high-dose injection of pilocarpine (330-350 mg/kg). In contrast, Xu et al., (2004) failed to

detect any interstrain differences in status severity or mortality after the administration of high-

doses of pilocarpine (300 mg/kg). Since the length of SE affects mortality (Curia et al., 2008),

65

progression of behavioural seizures (Turski et al., 1983b) and ensuing neuropathology

(Fujikawa, 1996), the shorter duration of SE (approximately 20 min) used by Xu et al., (2004)

may not have been long enough to detect interstrain differences reported after 3 hrs of SE by

Hort et al., (2000), or after 1 hr of SE in the present study. The present findings also indicate

that the beneficial effects of the RLDP protocol are strain dependent. Compared to the LDP

procedure, the RLDP protocol reduced mortality and severity of SE in Wistar rats, but had no

significant effect in LEH rats. Presently, the molecular or genetic basis of the difference in

response to pilocarpine of LEH and Wistar rats is not known.

3.4.2 Comparison of SE-induced neuropathology in LEH and Wistar rats following SE induction with the RLDP procedure

Previous studies using electrical stimulation of the basolateral amygdala to induce SE have

demonstrated that the severity of SE affects the subsequent extent of neuronal loss (Brandt et al.,

2003a; Tilelli et al., 2005). In the present study, we investigated whether a similar relationship

can be detected in the lithium/pilocarpine model. Because LEH rats experienced more severe

seizures than Wistars following SE induced with the RLDP protocol, we compared SE-induced

neuropathology between these strains. Since LEH rats exhibited high mortality rates following

pilocarpine treatment, quantitative stereological analysis was conducted 24 hrs after SE.

Consistent with the occurrence of more severe seizures, LEH rats exhibited greater pyramidal

cell loss in the CA1, CA3 and CA4 regions of the hippocampus than Wistar rats. In contrast,

however, the loss of hilar neurons only occurred in Wistar rats. Both strains exhibited similar

and severe neurodegeneration within layer II of the piriform cortex and preservation of

pyramidal cells in CA2. As described in the subsequent chapter, maximum pyramidal cell loss

in Wistar rats occurs in the CA3, CA4 and piriform cortex within 24 hours after SE; the majority

of CA1 pyramidal cell loss, however, is delayed, with only a 29% decrease by 24 hours, and an

80% decrease within 3 days. In contrast to this delay in Wistar rats, CA1 pyramidal cells in LEH

rats had decreased by 80% within 24 hours, indicating that the initial loss of CA1 pyramidal cells

is more rapid in the LEH strain. In general agreement with our results, Hort et al., (2000) also

found less pathology in Wistar rats than in LEH rats. The different pattern of neuronal injury

between the two strains may be caused by specific factors that affect the phenotype of cell death.

For instance, the latency to onset of SE and intensity of convulsive seizures have been reported

to be correlated with the type of cell death observed; shorter latencies or more severe seizures

66

correlated with necrotic cell death, whereas longer latencies or milder seizures resulted in

delayed cell death with apoptotic features (Kondratyev and Gale, 2004; Tokuhara et al., 2007).

Although the phenotype of cell death in the present study cannot be confirmed without

ultrastructural analysis, the rapid pyramidal cell loss in the CA1 of LEH rats may be attributed to

the more severe seizure intensity and shorter latency to SE development compared to Wistar rats.

3.4.3 Comparison of the effect of the LDP and RLDP protocols on SE-induced neurodegeneration in Wistar rats

The differences in neuronal death between LEH (more severe) and Wistar (less severe) rats

correlated with the relative severity of SE exhibited by these two strains. The relationship

between seizure severity and pathology suggested by these results is, however, confounded by

possible differences in genetic influences between the strains. To remove the possible influence

of strain difference in assessing the relationship between seizure severity and neuropathology,

we exploited our finding that Wistar rats treated with the RLDP protocol experienced less severe

seizures than Wistars treated with the LDP protocol. The extent of neurodegeneration under both

conditions was examined 3 months after SE. In the subsequent chapter, we demonstrated that by

3 months, maximum cell loss occurs within the regions assessed and is unaffected by

development of SRSs. Despite the differences in seizure intensities and mortality rates between

the two procedures, the pattern of neuron loss in the hippocampus and piriform cortex was

similar in both cases.

Although some studies have demonstrated a link between seizure severity and neuronal death,

others have failed to detect this relationship. For example, in comparing five different strains of

mice, McKhann et al., (2003) failed to detect a relationship of kainic acid susceptibility,

behavioural seizure severity, and loss of hippocampal pyramidal cells. Subsequent studies

confirmed these results, indicating that seizure susceptibility, convulsive seizure intensity, and

SE-induced neuropathology are controlled independently by genetic factors (McKhann et al.,

2003; Schauwecker et al., 2004; McLin and Steward, 2006; Lorenzana et al., 2007). In contrast,

studies that involved electrical stimulation in the left basolateral amygdala of rats to induce

different types of SE supported a positive correlation between behavioural seizure intensity and

severity of neurodegeneration in the hippocampus and piriform cortex (Brandt et al., 2003;

Tilelli et al., 2005). Although it is possible that the difference in seizure severity between the

LDP and RLDP procedures in the present study was insufficient to result in detectable

67

differences in neuropathology, our findings support the conclusion that the extent of

neuropathology is not directly related to seizure severity.

3.4.4 Conclusion

In summary, the present study identifies several differences between the response of LEH and

Wistar rats to SE-inducing protocols, and demonstrates that the repeated administration of low-

doses of pilocarpine is most effective in reducing mortality in Wistar rats. Furthermore, in spite

of eliciting less severe seizures during SE, the repeated low-dose injections of pilocarpine did not

reduce the extent of SE-induced neurodegeneration in the hippocampus and piriform cortex.

Based on these findings, we investigated SE-induced neuropathology and the effects of

neuroprotective peptides in Wistar rats treated with the RLDP protocol.

68

Chapter 4

Temporal profile of neuronal death following

lithium/pilocarpine-induced status epilepticus

4.1 Introduction

Status epilepticus (SE) is a prolonged seizure condition representing a major medical and

neurological emergency (Delorenzo, 2006). The incidence rates of SE in epidemiological studies

range from 9.9 to 41 per 100,000 population annually (Chin et al., 2004; Rosenow et al., 2007).

SE is associated with substantial mortality and morbidity (Lowenstein, 1999), including

epileptogenesis (Hesdorffer et al., 1998) and cognitive decline (Shorvon, 1994; Devinsky,

2004a). Data from humans and post-status epilepticus animal models suggest that both the risk

of epilepsy and the severity of cognitive impairments are associated with brain damage caused

by prolonged seizure activity (Shorvon, 1994; Lowenstein, 1999; Fountain, 2000; Devinsky,

2004b; Delorenzo, 2006; Helmstaedter, 2007). Histological studies in experimental models

indicate that 30 to 60 minutes of SE is required to initiate neurodegeneration (Nevander et al.,

1985; Fujikawa, 1996).

In 1983, Turski and colleagues described a rodent model of seizure-induced brain damage

produced by the systemic administration of pilocarpine, a muscarinic cholinergic agonist. The

same year Honchar et al., (1983) reported that if rats were pretreated with lithium-chloride,

seizure-induced brain damage could be produced with a substantially lower dose of pilocarpine.

In humans, SE results in neurodegeneration in the hippocampus, neocortex, piriform and

entorhinal cortices, septum, amygdaloid and thalamic structures (Fujikawa et al., 2000a). This

pattern of neuronal loss is replicated in the pilocarpine and lithium/pilocarpine models of SE, and

is generalized in other post-status epilepticus rodent models including kainic acid, bicuculline,

picrotoxic and pentetrazole (Ben-Ari et al., 1981; Lothman and Collins, 1981; Ben-Ari, 1985;

Turski et al., 1985).

Whereas others have described the extent and severity of SE-induced neurodegeneration (Turski

et al., 1983b; Honchar et al., 1983; Fujikawa, 1996; Motte et al., 1998; Covolan and Mello, 2000;

69

Peredery et al., 2000; Poirier et al., 2000), a detailed analysis comparing the temporal pattern of

neuronal death in different brain regions has not been reported. Previous studies using the

lithium or lithium/pilocarpine models have been limited by the semi-quantitative assessment of

neuronal damage, and/or by the restricted recovery times examined (see appendix I, Table A1-1).

For instance, times are restricted to less than 72 hours after SE (Fujikawa, 1996; Covolan and

Mello, 2000) or times examined are spaced far apart (Motte et al., 1998; Peredery et al., 2000;

Poirier et al., 2000). SE activates different cell death mechanisms that result in early necrotic (≤

1 day after SE) (Fujikawa et al., 1999; Araújo et al., 2008; Wang et al., 2008) and delayed

apoptotic (3 to 7 days after SE) (Narkilahti et al., 2003a; Weise et al., 2005; Wang et al., 2008)

degenerative morphologies. These different cell-death morphologies can occur in different brain

regions (Sloviter et al., 1996; Lopez-Meraz et al., 2010) or within the same population of

neurons (e.g. pyramidal cell layer of the hippocampus) (Narkilahti et al., 2003a; Weise et al.,

2005). Thus, we hypothesized that regions in the hippocampus, thalamus, amygdala and

piriform cortex would exhibit different temporal profiles of neuronal death following SE.

In the present study, our objective was to provide a comparative, quantitative analysis of SE-

induced neurodegeneration in several brain structures. Specifically, we sought to determine: (1)

in which brain regions neuronal death first appeared, (2) how neuronal loss evolved over time,

and (3) the extent of brain damage attributed to early (<24hrs) and to delayed (>24 hrs) neuronal

loss. We therefore analyzed neurodegeneration in 19 brain regions following 60 minutes of SE

induced by the repeated low-dose lithium/pilocarpine (RLDP) procedure (Glien et al., 2001).

Because neurodegeneration has not been previously investigated with the RLDP procedure, we

also sought to determine the effect of SE induced with this procedure on neuronal loss and to

compare this to other models. Neuronal death was assessed by stereological analysis of neurons

(stained for the neuronal specific marker [NeuN]) at times ranging from 1 hr to 90 days after SE.

The results demonstrate that depending upon the brain region, neuronal death occurred as early

as 1 hour following SE, and that different brain regions exhibit differential rates of neuronal loss,

with the majority of SE-induced neuronal death present by 24 hours.

70

4.2 Methods

4.2.1 Animals

All procedures were approved by the University of Toronto Animal Care Committee and were in

accordance with the guidelines established by the Canadian Council on Animal Care. Male

Wistar rats (Charles River Laboratories, Sherbrooke, Quebec, Canada) weighing between 300

and 350 g were individually housed with free access to food and water for at least 7 days in 12 h

light/dark cycles (i.e., lights on between 7 a.m. and 7 p.m.) before experimental use. A total of

64 Wistar rats were used in the present study.


SE was induced using the RLDP procedure exactly as described in Methods section 3.2.2.

Duration of seizure activity was controlled by the administration of diazepam (4 mg/kg, i.p.) 1, 3

and 5 hours following the onset of SE and animals were allowed to survive for various times up

to 3 months following SE. Sham animals underwent identical procedures but received saline in

place of pilocarpine, and did not exhibit any seizures.

4.2.3 Post-seizure care

Following the termination of SE, animals were kept in a quiet room for 3 days. Immediately

following the first injection of diazepam, animals received 5 ml 0.9% saline (3ml i.p. and 2ml

s.c.), and this was repeated in the morning and evening of the following day (5ml i.p.) as

described by Glien et al., (2001). Starting on the second day after SE, animals were tube-fed

softened rat chow mixed with applesauce for 3 days on average. Softened rat chow was also

provided in dishes until the rats commenced to eat hard pellets.

4.2.4 Detection of SRSs

Glein et al., (2001) previously demonstrated that the average latency onset to SRSs in rats treated

with the RLDP protocol was 6 weeks. In the present study, two methods for recording of

spontaneous seizures were used. First, all seizures observed during handling, or by direct

observation of the rats in their home cages were noted. The rats were continuously monitored

during the first 3 days following SE in their home cages, and checked on daily thereafter.

Second, a group of 8 rats was videotaped between 6 and 8 weeks after SE to confirm that rats

71

used for histological analyses at the 3 months recovery time were epileptic. The group of 8 rats

was videotaped for 4 hours per day, 6 days each week. Because the frequency of SRSs in rats

after pilocarpine-induced SE is much higher during the light (diurnal) compared to the dark

(nocturnal) period (Arida et al., 1999; Goffin et al., 2007), all recordings for spontaneous

seizures were done during the light period (7 a.m. – 6 p.m.). All 4-hr video recordings were

analyzed for seizures by using the fast-forward speed (six times the normal speed) of the video

recorder. Once a seizure-like activity was observed, the videotape was rewound to the beginning

of the behavior and examined at real-time speed. Since most SRSs following pilocarpine-

induced SE are generalized (Cavalheiro et al., 2006), only the occurrence of class 4/5

behavioural seizures was recorded. A rat was considered epileptic after exhibiting one or more

SRSs.

4.2.5 Histology and Stereological analysis

The extent of neuronal injury at time points ranging from 1 hour to 3 months after SE was

compared by staining brain sections with NeuN antibodies, a specific neuronal marker. NeuN

antibody recognizes the DNA-binding, neuron-specific protein NeuN, which is present in most

neuronal cell types of vertebrates tested (i.e., rats, humans, chicks, salamander) (Mullen et al.,

1992; Wolf et al., 1996). Developmentally, immunoreactivity of NeuN antibodies appears

initially after neurons become post-mitotic, and remains present throughout the life of these cells

(Mullen et al., 1992; Wolf et al., 1996). The immunohistochemical staining is primarily

localized in the nucleus of the neurons with lighter staining in the cytoplasm. The strong nuclear

staining suggests a nuclear regulatory protein function; however, no evidence currently exists as

to whether the NeuN protein antigen has a function in the cytoplasm, or whether it is simply

synthesized there before being transported into the nucleus (Mullen et al., 1992). Animals were

anaesthetized with a mixture of xyline (26 mg/kg i.p., Rompun, CDMV, Saint-Hyacinthe,

Quebec, Canada) and ketamine (174 mg/kg i.p., Ketalar, CDMV, Saint-Hyacinthe, Quebec,

Canada), and perfused transcardially with 90 ml of 0.1 M phosphate-buffered saline, pH 7.4

(PBS) followed by 400-500 ml of 4% (w/v) paraformaldehyde in PBS. Brains were removed

and left overnight at 4˚C in the paraformaldehyde solution. The following day, brains were

soaked for cryoprotection in 30% (w/v) sucrose in PBS until they sank at room temperature.

Next, brains were frozen in 2-methylbutane at -35˚C and stored at -80˚C. Forty µm coronal

sections were prepared with a freezing microtome. Sections were placed in 24-well culture

72

plates containing antifreeze solution (0.05 M sodium phosphate buffer, pH 7.4, 30% (w/v)

ethylene glycol, 15% (w/v) glucose), and stored at -20˚C.



edition),





brain, replacing the structures CeC and CeL detected in the initial section. Coronal brain section

containing the ventral hippocampus were selected between Bregma -4.92 mm through 5.4, with

the first section clearly presenting a continuous pyramidal cell layer connecting the dorsal and

ventral CA1 and CA3 regions. Section were rinsed in PBS (3 x 5 min washes), reacted overnight

at 4˚C with NeuN antibody (1:1000; Chemicon, Billerica, MA, USA) in 0.2% (v/v) goat serum,

0.3% (v/v) Triton X-100 in PBS followed by 2 hours at room temperature with Cy3 conjugated

secondary antibody (1:200; Chemicon, Billerica, MA, USA) in 0.2% (v/v) goat serum, 0.3%

(v/v) Triton X-100 in PBS.

Neurons were imaged using a Zeiss LSM510 Laser Scanning Confocal microscope (Carl Zeiss)

equipped with a 40x/1.3 oil-immersion objective lens. Digital images were acquired with the

Zeiss LSM 510 software. Neuronal density was determined using the unbiased optical dissector

technique as described by West and Gundersen (1990). As illustrated in Figure 4.1, images

were captured in the left brain hemisphere of specific regions within the hippocampus, thalamus,

amygdala and piriform cortex. In the dorsal hippocampus (Fig 4.1 A, B), images were taken in

the pyramidal cell layer of CA1 (boxes 1a-c), CA2 (2a), CA3 (3a-c), and CA4 (4a-b) and in the

polymorphic layer of the hilus (5a-f). In the ventral hippocampus (Fig 4.1 C), images were taken

in the pyramidal cell layer of CA1 (6a-c) and CA3 (7a-c). For the thalamus, images were

captured in the following regions (Figure 4.1 D): laterodorsal thalamic nucleus, dorsomedial

part (LDDM, 8a-b); laterodorsal thalamic nucleus, ventrolateral part (LDVL, 9a-b); posterior

thalamic nuclei (Po, 10a-b); ventral posteromedial thalamic nucleus (VPM, 11a-b); ventral

posterolateral thalamic nucleus (VPL, 12a-b); reticular thalamus nucleus (Rt, 13a-b). Likewise,

images were captured in the following regions of the amygdala (Figure 4.1 E): lateral

amygdaloid nucleus, dorsolateral part (LaDL, 14a-b); lateral amygdaloid nucleus, ventrolateral

73

part (LaVM, 15a-b); basolateral amygdaloid nucleus, posterior part (BLP, 16a-b); basomedial

amygdaloid nucleus, posterior part (BMP, 17a-b); posteromedial cortical amygdaloid nucleus

(PMCo, 18a-b). Finally, images were also taken within layer II of the posterior piriform cortex

(Figure 4 E, 19a-c). Since the pyramidal cell layer of the CA1, CA2, CA3, and CA4 and layer II

of the piriform cortex formed a thick row of densely packed neurons, a single counting frame

(120 X 60 µM) was positioned within the images captured (depicted as green boxes in Figure

4.1). Two counting frames (120 X 60 µM) were positioned within the images captured for the

thalamus, amygdala and hilus, since neurons were less numerous and uniformly scattered within

these structures (depicted as small white boxes in Figure 4.1). Right brain structures were


NeuN-positive cells that contained a relatively large (≥ 8µm) soma (Shi et al., 2004).






density occur whether such borders were used or not (Harding et al., 1994; Gardella et al., 2003;

Azcoitia et al., 2005). Unless otherwise specified, cell densities for individual animals represent



To assess whether tissue shrinkage occurred at specific times after SE, the area size (expressed as

mm2) of the dorsal and ventral hippocampus, thalamus and amygdala in the left brain hemisphere

was determined. Images were captured by the fluorescent microscope equipped with a 2.5x

objective lens. The AxioVision LE 4.0 analysis software was used to measure area size by

drawing an outline around the region of interest. As illustrated in Figure 4.2, the brain areas

assessed were based on regional boundaries defined in the Paxinos and Watson‘s rat brain atlas,

5th

edition. For the dorsal (Figure 4.2A, grey region) and ventral (Figure 4.2B, grey region)

hippocampus, an outline was drawn around the hippocampal pyramidal cell layer (pyr) and

included the dentate gyrus (DG). The area size of the thalamus and nearby structures (Figure

4.2A, green region) included an outline encircling the perimeter of the lateral posterior thalamic

nuclei (LPMR, LPLR), the reticular thalamic nucleus (Rt), the subthalamic nucleus (STh), the

74

medial tuberal nucleus (MTu), the dorsomedial hypothalamic nucleus (DMV, DMD), the central

medial thalamic nucleus (CM), and the mediodorsal thalamic nucleus (MD). The area excluded

the dorsal and ventral 3rd

ventricular spaces (d3V, 3V). Finally, for the area size of the amygdala

and adjoining structures (Figure 4.2, blue region), an outline was traced starting at the rhinal

fissure, and continued along the perimeter of the piriform cortex (Pir), the posteromedial cortical

amygdaloid nucleus (PMCo), the medial amygdaloid nuclei (MePV, MePD), the bed nucleus of

the stria terminalis, intraamygdaloid division (STIA), and the lateral amygdaloid nucleus

(LaVM, LaDL). The area excluded the lateral ventricular space (LV). The area sizes of the

brain structures assessed for individual animals represent the average area sizes of the

corresponding structure for 3 brain sections (the same sections used for stereological analysis of

neurons described above).

4.2.6 Fluoro-Jade B staining

For each animal, coronal brain sections were selected as described in section 4.2.5, and stained

with Fluoro-Jade B (FJB). FJB is an anionic fluorochrome reported to selectively stain

degenerating neurons (Poirier et al., 2000; Schmued and Hopkins, 2000). Briefly, brain sections

were immersed in 100% ethanol for 5 min, followed by 2 min in 70% ethanol and two 1-min-

long rinses in distilled water. Slides were then transferred to a solution of 0.06% potassium

permanganate for 10 min, gently shaken, to minimize background staining. After two more

rinses, slides were placed in FJB staining solution for 30 min at room temperature. The staining

solution was prepared from a 0.01% stock solution of FJB (Histo-Chem Inc., Jefferson AR,

USA) that was made by adding 10 mg of the dye powder to 100 ml of distilled water. To make

up 100 ml of staining solution, 4 ml of stock solution was added to 96 ml of 0.1% acetic acid

vehicle. This results in a final dye concentration of 0.0004%. Following staining, the sections

were rinsed three times with distilled water. The slides were dried, immersed in xylene, and

mounted. Slides were examined on a Zeiss LSM510 Laser Scanning Confocal microscope (Carl

Zeiss) equipped with an Argon laser. Digital images were acquired using the Zeiss LSM 510

software.

75

4.2.7 Statistical Analysis:

Statistical analysis was performed using Statistica 6.0 software. Significant differences were

determined using one-way analysis of variance (ANOVA). The Newman-Keuls post-hoc test

was used to determine differences between treatment groups. Significance was set at a p-value

of 0.05 or less. Moving average trendlines were drawn for the temporal profiles of neuronal

densities of each brain region (shown in Figures 4.6, 4.9, and 4.12) using the Excel Microsoft

2007 software program. Moving average trendlines are used to smooth out fluctuations in data

to show a trend (i.e., reduction in neuronal densities over a period of time) more clearly. In the

treadlines, two data points were averaged, and the average value was used as a point in the line.

76

Figure 4.1: Placement of counting frames in the dorsal hippocampus (A): pyramidal cell layer

of CA1 (boxes 1a-c), CA2 (2a), CA3 (3a-c), and CA4 (4a-b). Hilus (B): polymorphic layer of the

hilus (5a-f). Ventral hippocampus (C): pyramidal cell layer of CA1 (6a-c) and CA3 (7a-c).

Thalamus (D): laterodorsal thalamic nucleus, dorsomedial part (LDDM, 8a-b); laterodorsal

thalamic nucleus, ventrolateral part (LDVL, 9a-b); posterior thalamic nuclei (Po, 10a-b); ventral

posteromedial thalamic nucleus (VPM, 11a-b); ventral posterolateral thalamic nucleus (VPL,

12a-b); reticular thalamus nucleus (Rt, 13a-b). Amygdala (E): lateral amygdaloid nucleus,

dorsolateral part (LaDL, 14a-b); lateral amygdaloid nucleus, ventrolateral part (LaVM, 15a-b);

basolateral amygdaloid nucleus, posterior part (BLP, 16a-b); basomedial amygdaloid nucleus,

posterior part (BMP, 17a-b); posteromedial cortical amygdaloid nucleus (PMCo, 18a-b).

Piriform cortex (E): layer II of posterior piriform cortex (PPC, 19a-c). See Methods section

4.2.5 for details on stereological analysis of neurons.

77

78

Figure 4.2: The area size of different brain regions assessed. A: Dorsal hippocampus

(indicated by grey region), thalamus and adjoining structures (green region), amygdala and

adjoining structures (blue region). Image depicted: Bregma -3.24 mm (Paxinos and Watson,

2005). B: Ventral hippocampus (indicated by grey region). Image depicted: Bregma -5.28 mm

(Paxinos and Watson, 2005). Area size determined as described in Methods section 4.2.5.

79

80

4.3 Results

4.3.1 SE induction and survival rates:

Sixty-four lithium-pretreated rats were administered repeated doses of pilocarpine (10 mg/kg), of

which 44 (69%) developed SE. Eight rats died between recovery time points and were not used

in the present study. Four rats were analyzed for each of the 9 recovery times. An additional 4

rats were used as sham animals.

4.3.2 Spontaneous seizures after lithium/pilocarpine induced SE

A group of eight rats were videotaped between 6 and 8 weeks after SE to determine whether they

were epileptic. All 8 of the rats exhibited one or more SRSs within this timeframe. No

difference in the frequency of SRSs between the weeks assessed were found. The average

frequency in the eight rats was 0.75 ± 0.3 seizures per 24 hours of recording during week 6, 1.5 ±

0.5 during week 7, and 0.9 ± 0.3 during week 8. Four of these animals randomly selected were

later used for histological analyses at the 3 month recovery time. Although we cannot rule out

the presence of SRSs at earlier times, no SRSs were noted during handling of the animals, or by

direct observation of the animals in their home cages, during the first 2 weeks following SE.

4.3.2 Neuropathology following status epilepticus: Overview

Because the hippocampal formation, thalamus, amygdala and piriform cortex are highly

susceptible to SE-induced neurodegeneration (Honchar et al., 1983; Clifford et al., 1987), we

concentrated our analyses on 19 regions within these structures. With the exception of

hippocampal subfield CA2, significant neuronal loss was observed in all other regions assessed.

The severity and temporal profile of neuronal loss for regions within each brain structure are

described in detail below. Results for each brain structure assessed are presented as follows: (1)

low-powered images of NeuN immunohistochemical staining providing an overall view of

damage in each structure, (2) representative high-powered images depicting NeuN positive cells

(neurons) that were quantified as in Methods (section 4.2.5), and (3) graphs displaying changes

in neuron density, expressed as percent of controls, over time. With the exception of the

amygdala at 90 days following SE (see section 4.3.5), no significant tissue shrinkage at times

after SE occurred in the dorsal and ventral hippocampus, thalamus, or amygdala (Table 4.1),

81

permitting the direct comparison of neuron densities in SE and sham animals. Actual neuron

densities are given in appendix II (Tables AII-1, AII-2 and AII-3).

Table 4.1: The effect of SE on the area size (mm2) of the hippocampus, thalamus and

amygdala

Recovery after

SE1

Dorsal

hippocampus

Ventral

hippocampus

Thalamus Amygdala

Sham 3.55 ± 0.19 9.71 ± 0.17 14.95 ± 0.28 3.31 ± 0.14

12 hours 3.53 ± 0.18 10.40 ± 0.20 15.78 ± 0.33 3.45 ± 0.17

3 days 3.52 ± 0.37 10.01 ± 0.19 14.53 ± 0.28 3.27 ± 0.18

14 days 3.62 ± 0.14 9.48 ± 0.14 14.27 ± 0.20 3.00 ± 0.23

3 months 3.54 ± 0.10 9.37 ± 0.30 15.28 ± 0.26 1.66 ± 0.15 *

Area size of the left brain hemisphere in various brain regions determined at specific times (1)

following SE (see Methods 4.2.5). Data expressed as mm2 ± SEM. * Area size of the amygdala

decreased between 14 days and 3 months following SE. No change in area size of the dorsal and

ventral hippocampus and thalamus was detected. A total of 4 animals were analyzed at each

recovery time.

82

4.3.3 SE-induced neurodegeneration in the hippocampus:

The effect of SE on the hippocampus is shown in Figures 4.3 - 4.6. Neuronal edema was

detected by 3 hours in CA1, CA3 and CA4 (Fig 4.4, indicated by solid arrows). Enlargement of

the 3rd

and lateral ventricles was visible by 3 months after SE (Fig 4.3B and D, indicated by φ).

SE resulted in the loss of neurons in all hippocampal regions except CA2 (Fig 4.4). Likewise,

with the exception of 3 animals that exhibited partial or complete destruction of the

suprapyramidal (or enclosed) blade of the granule cell layer (<8% of SE animals), no loss of

dentate granule cells, defined indirectly by a shortening of the total length of the dentate gyrus

(Bertram et al., 1990), was detected (Fig 4.5). The three animals that exhibited damage within

the dentate gyrus were assessed at the 24-hours, 7 days, and 3 months recovery times,

respectively.

Quantification of NeuN-stained neurons showed that the temporal profile of neuronal density

varied between hippocampal regions (Fig 4.6). SE-induced loss of pyramidal cells in the dorsal

CA1 occurred within two distinct times following SE: a 28% decrease in cell density occurred

within 1 hour, followed by a further 53% decrease in cell density between 1 and 3 days (Fig

4.6A). By 3 days after SE, 27 ± 8% (avg ± SEM) of pyramidal cells remained in dorsal CA1,

with no further decrease after this time. In contrast to the rapid initial loss of pyramidal cells in

the dorsal CA1, neuronal loss in ventral CA1 was delayed for at least 12 hrs, after which time the

density of neurons decreased to a minimum of 33 ± 4% of shams by 7 days (Fig 4.6A). Dorsal

and ventral CA1 were the most severely damaged hippocampal regions, while less extensive

damage occurred in CA3, CA4 and hilus. Although neuronal loss in dorsal CA3 was initially

slower than in ventral CA3, the ultimate severity of damage in these regions was not

significantly different, with 57 ± 10% and 42 ± 2% of pyramidal cells remaining after 3 months,

respectively (Fig 4.6B). Significant neuronal loss was detected in the CA4 and hilus within 3-6

hrs and 1-3 hrs after SE, respectively. No further damage evolved after the initial loss in the

CA4 and hilus, with 43 ± 14% and 57 ± 7% of neurons remaining at 3 months, respectively (Fig

4.6A, B).

83

Figure 4.3: Anti-NeuN immunohistochemical staining decreases following SE in the dorsal

and ventral hippocampus. Representative images of the left dorsal (A, B) and ventral (C, D)

hippocampus from a sham rat (A, C), and from a rat 3 months after SE (B, D). Dotted line

indicates the border between dorsal CA3 and CA4 (A). Arrows depicts decreased NeuN staining

in the pyramidal cell layer: more severe damage is detected in CA1 (unfilled arrows) compared

to CA3 and CA4 (solid arrows). Φ Ventricular enlargement detected within 3 months after SE.

Scale bar = 500 µm. 25X magnification. Abbreviations: dorsal 3rd

ventricle (DV3); lateral

ventricle (LV); polymorphic layer of the hilus (PoDG); pyramidal cell layer (pyr).

84

85

Figure 4.4: Confocal micrographs (400X) of NeuN stained cells in hippocampal subfields:

dorsal CA1 (A), CA2 (B), CA3 (C) and CA4 (D) in a sham rat, and in rats at different recovery

times after SE. Neuronal edema detected in CA1, CA3, and CA4 by 3 hrs after SE (solid

arrows). No damage detected in CA2 (B). Scale bar = 50 µm.

86

87

Figure 4.5 Total length of dentate gyrus remains constant following lithium/pilocarpine

induced SE. The length of the dentate gryus in the left (black bars) and right (grey bars) brain

hemispheres were determined at different time points (1 hr – 3 months) after SE. Values

represent length of dentate gyrus (µm ± SEM). Four animals were analyzed at each time point.

88

89

Figure 4.6: Temporal profiles of neuronal loss in hippocampal subfields following

lithium/pilocarpine induced SE. Coronal brain sections prepared at various times following

SE were stained with anti-NeuN antibodies and neurons counted as in Methods (section 4.2.5).

Neuron density was graphed as percentage of control (± SEM) values. A: * Dorsal CA1 at ≥ 1 hr

different from shams (p<0.05), ** hilus at ≥ 3 hrs different from shams (p<0.05), *** ventral

CA1 at ≥ 1 day different from shams (p<0.05), ^ dorsal CA1 at ≥ 3 days different from 1 day

after SE (p<0.05), ^^ ventral CA1 at ≥ 7days different from 1 day after SE, # neuronal loss more

severe in CA1 compared to all other regions with combined analyses at 14 and 90 days (p<0.05).

B: * Ventral CA3 at ≥ 1 hour different from shams (p<0.05), ** CA4 at ≥ 6 hours different from

shams (p<0.05), *** dorsal CA3 at ≥ 12 hours different from shams (p<0.05).

90

91

4.3.4 SE-induced neurodegeneration in thalamic nuclei:

All thalamic regions exhibited SE-induced neurodegeneration as shown in Figures 4.7-4.9.

However, there were differences between the thalamic nuclei in the temporal profiles and

severity of neuronal loss. Neuronal death in the VPM and VPL occurred within two separate

times following SE: an initial phase within the first 12 hours after SE, and a second phase

between 2 weeks and 3 months. While the initial loss of neurons was similar in both regions, by

3 months damage to the VPL was more severe than to the VPM, with 24± 3% and 40 ± 1% of

neurons remaining, respectively (Fig 4.9A). Neuronal loss occurred more rapidly in RT than in

either VPM or VPL, with only 57 ± 3% of RT neurons remaining 6 hours after SE (Fig 4.9A).

Compared to RT and VPM at 3 months, more severe damage occurred in the LDDM, LDVL and

Po. In the LDDM, neuron density was reduced by 25 ± 3.7% of shams within the first hour after

SE, with 26 ± 3% of neurons remaining after 24 hours (Fig 4.9B). Although the onset of damage

was slightly delayed in the LDVL, the progression of neuronal loss was similar to LDDM

thereafter, with 24 ± 7% of neurons remaining 24 hours after SE. In Po, a 45 ± 5.9% reduction

in neuron density occurred between 1 - 3 hours after SE, with 33 ± 6% neurons remaining by 6

hours.

92

Figure 4.7: Anti-NeuN immunohistochemical staining decreases following SE in several

thalamic nuclei. Representative images of the thalamus in a sham rat (A), and in rats at

recovery time points 24 hours (B) and 3 months (C). Arrows depict decreased NeuN staining in

the LDVL, LDDM, Po and Rt by 24 hours after SE. * Additional damage detected in the VPM

and VPL by 3 months after SE. Φ Ventricular enlargement detected within 3 months after SE.

Scale bar = 500 µm. 25X magnification. Abbreviations: laterodorsal thalamic nucleus,

dorsomedial part (LDDM); laterodorsal thalamic nucleus, ventrolateral part (LDVL); posterior

thalamic nuclei (Po); ventral posteromedial thalamic nucleus (VPM); ventral posterolateral

thalamic nucleus (VPL); reticular thalamus nucleus (Rt); 3rd

ventricle (3V).

93

94

FIGURE 4.8: Confocal micrographs (400X) of NeuN stained cells in several thalamic

nuclei: (A) Laterodorsal thalamic nucleus, dorsomedial part (LDDM), (B) posterior thalamic

nuclei (Po), (C) ventral posterolateral thalamic nucleus (VPL), and (D) reticular thalamus

nucleus (Rt) in a sham rat, and in rats at different recovery time points after SE. Scale bar = 50

µm.

95

96

FIGURE 4.9: Temporal profiles of neuronal loss in several thalamic nuclei following

lithium/pilocarpine induced status epilepticus (SE). Coronal brain sections prepared at

various time points following SE were stained with anti-NeuN antibodies and neurons counted as

in Methods (section 4.2.5). Neuron density graphed as percentage of control (± SEM) values.

A: * Rt at ≥ 3 hrs different from shams (p<0.05), ** VPM and VPL at ≥ 6 hrs different from

shams (p<0.05), ^ VPM and VPL at 3 months different from 14 days (p<0.05), # VPL more

severely damaged compared to VPM (p<0.05). B: * LDDM at ≥ 1 hr different from shams

(p<0.05), **LDVL and Po at ≥ 3 hrs different from shams (p<0.05), ^ LDDM and LDVL at ≥ 1

day different from 12 hrs (p<0.05). Abbreviations: laterodorsal thalamic nucleus, dorsomedial

part (LDDM), laterodorsal thalamic nucleus, ventrolateral part (LDVL), posterior thalamic

nuclei (Po), ventral posteromedial thalamic nucleus (VPM), ventral posterolateral thalamic

nucleus (VPL), reticular thalamus nucleus (Rt).

97

98

4.3.5 SE-induced neurodegeneration in amygdaloid nuclei:

Extensive damage was observed in the amygdala, with necrotic lesions (Fig 4.10, indicated by *)

and enlargement of the lateral ventricles (Fig 4.10, indicated by φ) present by 3 months after SE.

A significant decrease in the area size of the amygdala was also detected between 2 weeks and 3

months following SE (Table 4.1). As illustrated in Figures 4.11 and 4.12, differences in the

temporal profiles and severity of neuronal loss were observed between amygdaloid nuclei.

Neuronal loss in the LaDL and LaVM was similar, and occurred within two separate times:

following an initial decrease within 12 hours after SE, neuron densities remained constant until

additional losses occurred between 1 and 2 weeks, with 33 ± 5% and 24 ± 6% of neurons

remaining, respectively (Fig 4.12A). In contrast to the pattern of neuronal loss observed in the

lateral nucleus, neuronal loss in the BLP and BMP occurred during the first week after SE,

reaching minimum neuron densities of 9 ± 4% and 7 ± 6% of shams, respectively (Fig 4.12B).

Although severity of damage by 3 months in the BLP and BMP was similar, the progression of

neuronal loss in the first 24-hours after SE was different between these regions. While a

decrease of 53 ± 3.1% occurred between 3 and 6 hours in the BMP, neuronal loss was delayed in

BLP, with only an initial 13 ± 4.7% reduction occurring within 12 hours. The PMCo was the

least severely damaged region within the amygdala, with neuron density decreasing to a

minimum of 72 ± 5% of shams between 1 and 3 hours after SE (Fig 4.12A).

4.3.6 SE-induced neurodegeneration in the piriform cortex:

Severe damage was detected in layer II of the posterior piriform cortex (PPC), with necrotic

lesions visible within this region by 3 months after SE (Fig 4.10C). In PPC, initial neuronal loss

occurred between 3 and 6 hours after SE, with a minimum of 35 ± 18% of pyramidal cells

remaining by 12 hours (Fig 4.11D and Fig 4.12B).

4.3.7 Detection of Fluoro-jade B stained neurons

As shown in Figure 4.13, FJB stained neurons were present by 24 hours following SE in the

hippocampus, thalamus, and amygdala. In contrast, no FJB stained neurons were observed in

epileptic rats at 3 months following SE, or in shams.

99

Figure 4.10: Anti-NeuN immunohistochemistry staining decreases following SE in several

amygdaloid nuclei and in the posterior piriform cortex. Representative images of the

amygdala and PPC from a sham rat (A), and from rats at recovery time points 24 hours (B) and 3

months (C) after SE. Arrows illustrate decreased NeuN staining in the lateral and basolateral

amygdaloid nuclei and piriform cortex 24 hours after SE. * depicts necrotic lesion in amygdala

by 3 months. Φ Ventricular enlargement detected at 3 months. Scale bar = 500 µm. 25X

magnification. Lateral amygdaloid nucleus, dorsolateral part (LaDL); lateral amygdaloid

nucleus, ventrolateral part (LaVM); basolateral amygdaloid nucleus, posterior part (BLP);

basomedial amygdaloid nucleus, posterior part (BMP); posteromedial cortical amygdaloid

nucleus (PMCo); posterior piriform cortex (Pir); lateral ventricle (LV).

100

101

FIGURE 4.11: Confocal micrographs (400X) of NeuN stained cells in several amygdaloid

nuclei and in the piriform cortex: (A) Lateral amygdala nucleus, ventrolateral part (LaVM),

(B) basolateral amygdaloid nucleus, posterior part (BLP), (C) posteromedial cortical amygdaloid

nucleus (PMCo), and (D) posterior piriform cortex (PPC) in a sham rat, and in rats at different

recovery times following SE. Scale bar = 50 µm.

102

103

FIGURE 4.12: Temporal profiles of neuronal loss in several amygdaloid nuclei and in the

posterior piriform cortex. Coronal brain sections prepared at various time points following SE

were stained with anti-NeuN antibodies and neurons counted as in Methods (Section 4.2.5).

Neuron density graphed as percentage of control (± SEM) values. A: * LaDL at ≥ 1 hr different

from shams (p<0.05), ** PMCo at ≥ 3 hrs different from shams (p<0.05), *** LaVM at ≥ 6 hrs

different from shams (p<0.05), ^ LaDL and LaVM at ≥ 14 days different from 7 days (p<0.05).

B: * BMP at ≥ 3 hrs different from shams (p<0.05), ** PPC at ≥ 6 hrs different from shams

(p<0.05), *** BLP at ≥ 12 hrs different from shams (p<0.05), ^ PPC at ≥ 12 hrs different from 6

hrs (p<0.05), ^^ BLP and BMP at ≥ 7 days different from 3 days (p<0.05). Abbreviations:

lateral amygdaloid nucleus, dorsolateral part (LaDL), lateral amygdaloid nucleus, ventrolateral

part (LaVM), basolateral amygdaloid nucleus, posterior part (BLP), basomedial amygdaloid

nucleus, posterior part (BMP), posteromedial cortical amygdaloid nucleus (PMCo), Posterior

piriform cortex (PPC).

104

105

FIGURE 4.13: Confocal micrographs (400X) of Fluoro-jade B (FJB) stained cells present

at 24 hours but not at 3 months after SE in the hippocampus, thalamus and amygdala.

Coronal brain sections were stained with FJB, a marker for degenerationing neurons. (A) Dorsal

CA1 subfield of the hippocampus, (B) ventral posteromedial thalamic nucleus (VPM), (C)

basolateral amygdaloid nucleus, posterior part (BLP) in a sham rat, and in rats at 24 hours and 3

months recovery times following SE. Scale bar = 50 µm.

106

107

4.4 Discussion

In the present study, we performed a detailed quantitative analysis of neuronal death in 19 brain

regions at times ranging from 1 hr to 3 months following 60 minutes of SE. The major findings

of this study were that: (1) the RLDP procedure resulted in widespread neuronal death that was

generally similar to that found with the low-dose lithium/pilocarpine and the high-dose

pilocarpine procedures, (2) in some brain regions, neuronal death appeared as early as 1 hr

following SE, with the majority of neuronal death in all brain structures present by 24 hrs after

SE, (3) while specific regions within the hippocampus (dorsal and ventral CA1) and amygdala

(LaDL, LaVM, BLP, BMP) showed additional neuronal loss between 1 and 14 days after

recovery, the somatosensory thalamic nuclei (VPM, VPL) were the only areas with additional

neuronal death between 2 weeks and 3 months of recovery, and finally, (4) different regions

within the hippocampus, thalamus, amygdala and piriform cortex exhibited differential rates of

neuronal loss. These results are further discussed in the subsequent sections.

4.4.1 The neuropathological effect of increasing survival time following status epilepticus

In the present study, we provided the first analysis of neuronal loss with the RLDP protocol for

the induction of SE, and found that the extent of brain damage was similar to that previously

described with both the low-dose lithium/pilocarpine (LDP) and the high-dose pilocarpine

procedures (Honchar et al., 1983; Turski et al., 1983a; Turski et al., 1983b; Clifford et al., 1987;

Fujikawa, 1996; Motte et al., 1998; Covolan and Mello, 2000; Peredery et al., 2000; Poirier et

al., 2000). In chapter 3, we showed that the severity of neuronal death between rats treated with

the LDP and the RLDP protocols was similar at 3 months, despite procedural differences in the

mortality rates and in the severity of seizures. Because we did not directly compare the 2

methods at all the different times analyzed here, however, we cannot determine whether the rates

of neuronal death between brain regions also occurred similarly. In apparent contrast, it was

previously reported that in rats without lithium-pretreatment, seizure severity, mortality rates and

neuropathology was affected by different doses of pilocarpine administered (Clifford et al., 1987;

Liu et al., 1994; Curia et al., 2008). With sub-convulsive doses of pilocarpine alone (100

mg/kg, i.p.), neuronal damage was confined to the piriform cortex and anterior olfactory nuclei

(Clifford et al., 1987). Damage was extended to include the amygdala, cortical and basal nuclei

108

if the dosage was increased to 200 mg/kg. Rats exhibiting convulsive seizures after an injection

of 200 mg/kg showed additional damage in the medio-thalamic nuclei and neocortex. Injection

of pilocarpine at the highest dose of 400 mg/kg resulted in rats exhibiting the highest mortality

rates, the most severe convulsive seizures, and extensive neuronal damage that were the most

comparable to that observed in the lithum/pilocapine model (Clifford et al., 1987). Therefore,

the RLDP protocol has the advantage of reducing mortality rates in rats while producing a

similar pattern of neurodegeneration to both the low-dose lithium/pilocarpine and high-dose

pilocarpine procedures.

Because several recovery times ranging between 1 hr and 3 months were analyzed in the present

study, we were able to provide a detailed description of the progression of neuronal death

between brain regions following lithium/pilocarpine-induced SE. As illustrated in Figure 4.13,

the number of brain regions first exhibiting SE-induced neuronal loss (Fig 4.14, grey bars), and

brain regions sustaining maximum neuronal death (Fig 4.14, black bars), increased quickly with

time following cessation of SE; a list of these brain regions can be found in Tables 4.2 and 4.3,

respectively. A decrease in the density of neurons was apparent as early as 1 hr in 3 brain

regions (dorsal CA1, dorsomedial part of the laterodorsal thalamic nucleus (LDDM) and

basolateral amygdaloid nucleus (BLP)), and the number of brain regions exhibiting significant

neuronal death increased to 10 by 3 hrs, 15 by 6 hrs, and all brain regions, with the exception of

CA2, by 24 hrs (see Table 4.2). Eleven out of 19 brain regions exhibited no further neuronal loss

after 24 hrs of recovery (see Table 4.3). Of the 8 brain regions exhibiting the loss of neurons

after 24 hrs of recovery, 6 (dorsal and ventral CA1, dorsolateral (LaDL) and ventrolateral

(LaVM) parts of the lateral amygdaloid nucleus, basolateral amygdaloid nucleus (BLP), and

basomedial amygdaloid nucleus (BMP)) sustained additional neuronal loss between 1 and 14

days after SE. The thalamic somatosensory nuclei (ventral posteromedial (VPM) and ventral

posterolateral (VPL) thalamic nuclei) were the only areas for which additional neuronal death

occurred after 2 weeks. Although there was a delay between the times at which neuronal loss

first became apparent (Table 4.2), and when maximal neurodegeneration occurred for most brain

regions examined (Table 4.3), this period was brief since most of the damage was present by 24

hrs (depicted in Fig 4.14).

Our findings demonstrated that with the RLDP model of SE, the majority of neuronal death in

the hippocampus, thalamus, amygdala and piriform cortex occurred by 24 hrs after SE. Previous

109

studies using the LDP or the high-dose pilocarpine procedures and different staining procedures

analyzed the same brain regions, and similarly showed that most of the neuronal damage

appeared within the initial 24 hrs of recovery (Fujikawa, 1996; Motte et al., 1998; Covolan and

Mello, 2000; Poirier et al., 2000) (see table A1-1 in appendix I). For example, Fujikawa et al.,

(1996) used hematoyxlin and eosin (H&E) staining to qualitatively assess for the presence of

acidophilic neurons, which was used as a criterion of irreversible neuronal injury; the majority of

neuronal damage was observed between the 0-4 hrs and 24 hrs time points following SE in the

dorsal and ventral CA1 and CA3 subfields of the hippocampus, hilus, amygdala, thalamus, and

piriform cortex. The mediodorsal and lateroposterior thalamic nuclei were the only regions to

exhibit additional damage between the 24 hrs and 72 hrs recovery times (Fujikawa, 1996).

Because injured neurons are also agryophillic (Horvath et al., 1997), Covolan and Mello (2000)

used the Gallyas‘s silver impregnation method to qualitatively assess neuronal damage, and

showed that the most intense staining appeared in regions within the hippocampus, amygdala and

thalamus between the 8 hrs and 24 hrs recovery times. Finally, Poirier, Capek and Koninck

(2000) showed that the most intense Dark Nuclear (silver) stain occurred at 3 hrs following SE in

the hippocampus, and preceded the presence of Flouro-jade (FJ) staining (a marker for

degenerating neurons), which maximally appeared between 24 hrs and 1 weeks after SE. While

the Dark Nuclear stain was suggested to detect an early molecular event associated with neuronal

stress (Poirier et al., 2000b), FJ staining was proposed to detect an unknown marker associated

with the later stages of neuronal degeneration (Poirier et al., 2000b; Schmued and Hopkins,

2000).

In the present study, Fluoro-jade B (FJB) was used and it produced results very similar to the

staining pattern produced by its predecessor, FJ (i.e., share the affinity for the same

biomolecule(s), but differ in relative strength of affinity) (Schmued et al., 2000). In pilot studies,

we comparably found that the presence of FJB staining in the hippocampus slowly increased

after the 3 hrs recovery time, and was maximally present by 24 hrs. FJB staining was still

present within the pyramidal cell layer of the dorsal and ventral CA1 subfields at 1 week

following SE, which is consistent with the additional reduction of neuronal densities we

quantified for these regions. With this staining method, however, we found it difficult to

determine whether the presence of FJB was attributed to ongoing degeneration, or debris from

degenerated neurons that had not yet been removed. For instance, although maximal neuronal

110

loss in the CA3 and CA4 subfields were quantified to occur prior to the 24 hrs recovery time,

some FJB staining was still present at 24 hrs, indicating that this staining was from debris of

already degenerated cells. Overall, the spatio-temporal staining pattern of FJB provides

additional evidence that the majority of neuronal damage occurs early and is attributed to SE (see

section 4.4.2).

Because we used an unbiased quantification method (optical dissector method) to assess

neuronal death, and increased the number of early times analyzed, we were able to demonstrate,

in a precise, quantitative manner, that maximum neuronal loss in several brain regions occurred

before 24 hrs of recovery. In the present study, maximal neuronal loss was detected as early as 3

hrs in the hilus, ventral CA3, reticular thalamic nucleus (Rt) and posteromedial cortical

amygdaloid nucleus (PMCo), by 6 hrs in the dorsal CA4 and posterior thalamic nuclei, and by 12

hrs in the dorsal CA3 and posterior piriform cortex (PPC) (see table 4.3). These findings

represent the first detailed, quantitative time-course comparison of SE-induced neuronal death

between brain regions at several times (1, 3, 6, and 12 hrs) preceding 24 hrs recovery.

4.4.2 The relationship between SE, SRSs and delayed neuronal death

Presently, it is difficult to ascertain whether damage occurring after the 24 hr period is the

consequence of SRSs, or the delayed damage occurring from SE as the IPI (Dudek et al., 2002).

For this reason, the effect of SRSs on neurodegeneration remains unclear. While some studies

report no correlation of neuronal death and the frequency of SRSs (Pitkänen et al., 2002; Gorter

et al., 2004), others have demonstrated progressive neuronal loss in chronically epileptic rats

(Roch et al., 2002). Although we did not determine the latency to onset of SRSs in rats

following SE, our findings did demonstrate that by 6 weeks of recovery, all rats were epileptic.

Glien et al., (2001) reported that following 60 minutes of SE induced by the RLDP protocol, the

average latency to the first SRS was 40 days (range 31 – 44 days). It is therefore reasonable to

assume that any neuronal death prior to 40 days or so was likely the consequence of SE, and that

any neuronal loss after this period might result from SE, SRSs or both. The only regions to

exhibit additional loss of neurons after 2 weeks in the present study were the thalamic

somatosensory nuclei (VPM, VPL), although we do not know if this death occurred prior to or

after the initiation of SRSs. We further showed that FJB-stained neurons are present at 24 hrs

111

following SE, but not at the 3 months recovery time in epileptic rats. Overall, our results

indicate that the vast majority of neuronal death is the result of SE and that little, if any

additional death, occurs subsequent to the start of SRSs.

In support of the suggestion that SE primarily contributes to neuronal death, Liu et al., (1994)

used the high-dose pilocarpine procedure and showed a significant decrease in neuronal density

and total neuron number at 3 weeks recovery in the dorsal CA1 and CA3. The authors reasoned

that since SRSs appeared approximately 2 to 2 ½ weeks after SE in these animals, but did not

contribute to any significant additional neuronal loss 6 to 12 weeks later, that neuronal death

primarily resulted from the acute pilocarpine-induced seizures (Liu et al., 1994). At this time,

we are unable to determine whether the delayed neuronal loss between 14 days and 3 months in

the thalamic somatosensory nuclei (VPM, VPL) was caused by delayed damage from SE, or

from damage caused by the development of SRSs. Although Peredery et al., (2000) similarly

showed that the thalamus was the only structure to exhibit some additional loss after 20 days of

recovery, further studies are necessary to determine the cause (e.g., SE and/or SRSs) of this

damage.

4.4.3 Differences in the severity and spatial pattern of neuronal death following SE

In the present study, differences in the severity of SE-induced neuronal death between regions

were detected. For instance, while the dorsal and ventral CA1 were the most severely damaged

regions within the hippocampus, with neuronal loss exceeding 70% of shams, no neuronal death

was detected in the CA2 or dentate gyrus. In the thalamus, the LDDM, LDVL, Po, and VPL

showed severe neuronal death exceeding 75% of shams, while less severe neuronal loss of 60%

and 40% was detected in the VPM and Rt, respectively. Similarly, in the amygdala, severe

neuronal death exceeding 75% of shams was detected in LaDL, LaVM, BLP and BMP, while

only moderate neuronal loss of 28% was detected in the PMCo. Although the specific

mechanisms responsible for these differences remain unclear, several observations regarding the

spatial pattern in severity of SE-induced neuronal death have been made. Studies using 14

C-2-

deoxyglucose functional mapping have shown that brain regions exhibiting the highest metabolic

rates during SE also exhibit the most severe neuronal damage (Ingvar, 1986; Ingvar et al., 1987;

Handforth and Ackermann, 1992; Handforth and Ackermann, 1995; Fernandes et al., 1999;

112

Bouilleret et al., 2000). Similar results were obtained in studies examining Fos expression, a

marker for cellular hyperactivation (Motte et al., 1998; Fernandes et al., 1999). The regional

distribution of glutamatergic receptors (Olney et al., 1986; Fernandes et al., 1999) and regions

exhibiting Ca2+

accumulation (Friedman et al., 2008) were other factors shown to coincide with

the spatial pattern of SE-induced brain damage. These results indicate that the most heavily

damaged brain regions are those that are most strongly activated during SE, and support the

suggestion that SE-induced neuronal death is initiated by excitotoxicity (Fernandes et al., 1999).

Excitotoxicity occurs when glutamate receptors are excessively stimulated, allowing high levels

of Ca2+

to enter the cell and activate cell-death signaling mechanisms (Fujikawa, 2005). Regions

resistant to cell death are proposed to be rich in Ca2+

-binding proteins, such as calbindin, and are

therefore able to buffer the seizure-induced excessive calcium load (Chard et al., 1993). This

idea is based on the findings that seizure-induced damage in the hippocampus displays a

topographical profile that closely resembles the pattern of distribution of Ca2+

-binding proteins in

the hippocampal formation (Sloviter, 1989; Sloviter et al., 1991). In the hippocampus, pyramidal

cells in the CA1 and CA3, which contain no or only small traces of calbindin, are most heavily

damaged while the dentate granular cells and pyramidal cells in the CA2, which are rich in

calbindin, remain resistant to seizure-induced damage (Sloviter, 1989; Sloviter et al., 1991).

This supports the role of Ca2+

in seizure-mediated neuronal death, and is consistent with our own

findings and results from other studies (Turski et al., 1983a; Clifford et al., 1987; Liu et al.,

1994) that showed no overt damage in the dentate gyrus and CA2.

4.4.4 The type of cell death produced by SE

The present data showed that regions within the hippocampus, thalamus, amygdala and piriform

cortex have different temporal patterns of neuronal death following SE. Although our results do

not allow the specific mechanisms responsible for neuronal death to be identified, other studies

indicate that variation in the temporal profile of neuronal loss may reflect varying combinations

of apoptotic and necrotic processes. Several studies demonstrated that dying neurons present by

24 hrs after SE predominately exhibited a necrotic morphology (Fujikawa, 2005; Fujikawa et al.,

1999; Fujikawa et al., 2000b; Kotariya et al., 2010). On the other hand, delayed neuronal death

(3 –to 7 days) exhibited apoptotic features (Narkilahti et al., 2003a; Weise et al., 2005). Several

regions in the present study exhibited early and delayed stages of neuronal death, and this may

reflect different forms of cell death in the same neuronal population. For instance, kainic acid or

113

pilocarpine-induced SE in adult rats have been shown to result in two types of pyramidal cell

death: early necrosis (≤ 1 day) (Araújo et al., 2008; Wang et al., 2008) and delayed cell death

with apoptotic features (≥ 3 days) (Narkilahti et al., 2003a; Weise et al., 2005; Wang et al.,

2008). This finding is consistent with the early and delayed times of pyramidal cell death we

observed within the dorsal and ventral CA1, and may apply to the other neuronal populations in

regions (LaDL, LaVM, BLP and BMP areas of the amygdala, VPM and VPL areas of the

thalamus) exhibiting similar patterns of neuronal death. Further studies are required to determine

if different cell death mechanisms between regions and within the same neuronal populations

underlie different patterns of neuronal death, and if so, what proportion of degenerating neurons

at a specific time point are necrotic versus apoptotic.

4.4.5 Conclusion

The present study demonstrates that the RLDP procedure for the induction of SE results in

widespread neuronal death, and that different regions, even within the same structure, may

exhibit differences in both the temporal profiles and the severity of neuronal loss following SE.

Neuronal loss was detected as early as 1 hr after SE in some brain regions, with significant brain

damage in all regions (except CA2 and dentate gyrus) present by 24 hrs. Although neuronal

death occurred between 1 and 14 days in regions within the hippocampus and amygdala, and

extended beyond 14 days in the thalamic somatosensory nuclei, it only contributed minimally to

the total damage in these brain structures caused by SE. Furthermore, with the possible

exception of the thalamic somatosensory nuclei, the development of SRSs did not appear to

contribute significantly to the neurodegeneration observed in epileptic rats.

114

Figure 4.14: The number of damaged brain regions as a function of increasing recovery

time after 60-min of SE. Grey bars represent the number of brain regions with initial neuronal

loss significantly different from corresponding shams. Black bars represent the number of brain

regions exhibiting maximal neuronal loss. With the exception of CA2, all brain regions

sustained early damage within 24 hours after SE. There is a delay, however, between initial

neuronal loss and maximal neurodegeneration for most brain regions. Brain regions exhibiting

initial neuronal damage and maximal neuronal damage at specific recovery times are listed in

tables 4.2 and 4.3.

115

116

Table 4.2 Temporal progression of brain regions exhibiting initial neuronal loss significantly different from

corresponding shams.

Brain structure 1 hr

Recovery

3 hr

recovery

6 hrs

Recovery

12 hrs

recovery

24 hrs

recovery

Hippocampus Dorsal CA1 Dorsal CA1

Ventral CA3

Hilus

Dorsal CA1

Ventral CA3

Hilus

Dorsal CA4

Dorsal CA1

Ventral CA3

Hilus

Dorsal CA4

Dorsal CA3

Dorsal CA1

Ventral CA3

Hilus

Dorsal CA4

Dorsal CA3

Ventral CA1

Thalamus LDDM LDDM

Rt

LDVL

Po

LDDM

Rt

LDVL

Po

VPM

VPL

LDDM

Rt

LDVL

Po

VPM

VPL

LDDM

Rt

LDVL

Po

VPM

VPL

Amygdala and piriform

cortex

LaDL LaDL

PMCo

BMP

LaDL

PMCo

BMP

LaVM

PPC

LaDL

PMCo

BMP

LaVM

PPC

BLP

LaDL

PMCo

BMP

LaVM

PPC

BLP

All of the following brain regions showed statistically significant neuronal loss compared to shams. Hippocampus:

Dorsal (dCA1) and ventral (vCAL) CA1, CA3, CA4, and hilus. Thalamus: laterodorsal thalamic nucleus,

dorsomedial part (LDDM), laterodorsal thalamic nucleus, ventrolateral part (LDVL), posterior thalamic nuclei (Po),

ventral posteromedial thalamic nucleus (VPM), ventral posterolateral thalamic nucleus (VPL), reticular thalamus

nucleus (Rt). Amygdala: lateral amygdaloid nucleus, dorsolateral part (LaDL), lateral amygdaloid nucleus,

ventrolateral part (LaVM), basolateral amygdaloid nucleus, posterior part (BLP), basomedial amygdaloid nucleus,

posterior part (BMP), posteromedial cortical amygdaloid nucleus (PMCo). Posterior piriform cortex (PPC).

117

Table 4.3 Temporal progression of brain regions exhibiting maximal neuronal death following SE

Brain

structure

3 hr

recovery

6 hrs

recovery

12 hrs

recovery

24 hrs

recovery

3 days

recovery

7 days

recovery

14 days

recovery

3

months

recovery

Hippocampus Hilus

Ventral

CA3

Hilus

Ventral

CA3

CA4

Hilus

Ventral

CA3

CA4

Dorsal

CA3

Hilus

Ventral

CA3

CA4

Dorsal

CA3

Hilus

Ventral

CA3

CA4

Dorsal

CA3

Dorsal

CA1

Hilus

Ventral

CA3

CA4

Dorsal

CA3

Dorsal

CA1

Ventral

CA1

Hilus

Ventral

CA3

CA4

Dorsal

CA3

Dorsal

CA1

Ventral

CA1

Hilus

Ventral

CA3

CA4

Dorsal

CA3

Dorsal

CA1

Ventral

CA1

Thalamus Rt Rt

Po

Rt

Po

Rt

Po

LDDM

LDVL

Rt

Po

LDDM

LDVL

Rt

Po

LDDM

LDVL

Rt

Po

LDDM

LDVL

Rt

Po

LDDM

LDVL

VPM

VPL

Amygdala and

piriform

cortex

PMCo PMCo PMCo

PPC

PMCo

PPC

PMCo

PPC

PMCo

PPC

BLP

BMP

PMCo

PPC

BLP

BMP

LaDL

LaVM

PMCo

PPC

BLP

BMP

LaDL

LaVM

All of the following brain regions showed statistically significant (and maximal) neuronal loss compared to shams.

Hippocampus: Dorsal (dCA1) and ventral (vCAL) CA1, CA2, CA3, CA4, and hilus. Thalamus: laterodorsal

thalamic nucleus, dorsomedial part (LDDM), laterodorsal thalamic nucleus, ventrolateral part (LDVL), posterior

thalamic nuclei (Po), ventral posteromedial thalamic nucleus (VPM), ventral posterolateral thalamic nucleus (VPL),

reticular thalamus nucleus (Rt). Amygdala: lateral amygdaloid nucleus, dorsolateral part (LaDL), lateral

amygdaloid nucleus, ventrolateral part (LaVM), basolateral amygdaloid nucleus, posterior part (BLP), basomedial

amygdaloid nucleus, posterior part (BMP), posteromedial cortical amygdaloid nucleus (PMCo). Posterior piriform

cortex (PPC).

118

Chapter 5

Neuroprotection following status epilepticus by targeting protein interactions with PSD-95

5.1 Introduction

Status epilepticus (SE), defined as 30 min or greater of continuous seizure activity, is a

neurological emergency resulting in high mortality and morbidity (DeLorenzo et al., 1996;

Fountain, 2000). Sustained activation of N-methyl-D-aspartate receptors (NMDARs), which

mediate excitatory neurotransmission in the central nervous system, is necessary for the

induction of SE (Rice et al., 1998; Deshpande et al., 2008). Overactivation of NMDARs

mediates excitotoxicity, causing widespread neurodegeneration in humans (Fujikawa et al.,

2000a) and in animal models of epilepsy, including kindling (Cavazos and Sutula, 1990), and the

administration of chemoconvulsants such as pilocarpine ( Turski et al., 1983b; Nevander et al.,

1985; Cavalheiro et al., 1987) and kainic acid (Ben-Ari, 1985; Fujikawa et al., 2000b).

Consistent with a critical role for NMDARs in SE-induced neuropathology, the systemic

administration of NMDAR antagonists provides substantial neuroprotection in rodent models of

epilepsy, even when given after the onset of SE (Fariello et al., 1989; Clifford et al., 1990;

Fujikawa et al., 1994; Fujikawa, 1995). The clinical efficacy of NMDAR antagonists, however,

is limited because of their associated side effects, including the induction of psychosis in

humans ( Krystal et al., 1994; Lahti et al., 1995; Malhotra et al., 1996; Rowland et al., 2005) and

neurotoxicity in rats (Olney et al., 1989; Olney et al., 1991; Fix et al., 1993). A proposed

alternate approach to preventing NMDAR-mediated neurotoxicity is to prevent excitotoxic

signaling from the NMDAR, such as the production of nitric oxide, by blocking interaction of

the receptor with downstream signaling molecules (Aarts et al., 2002). PSD-95 is a critical

scaffolding protein that links the NMDAR to signaling enzymes within the postsynaptic density,

and suppression of the expression of PSD-95 has selectively attenuated excitotoxicity triggered

via NMDARs (Sattler et al., 1999). Tat-NR2B9c is a synthetic peptide consisting of the C-

terminal 9 amino acids of the NR2B subunit of NMDARs fused to the membrane transduction

domain of the HIV-1-Tat protein, that was designed to disrupt neurotoxic signaling from the

NMDAR by interfering with protein interactions involving the PDZ1 and PDZ2 domains of

PSD-95 (Aarts et al., 2002; Cui et al., 2007). Tat-NR2B9c was reported to provide significant

119

neuroprotection and to preserve cognitive function following transient stroke in rats (Aarts et al.,

2002; Sun et al., 2008).

Because activation of NMDA receptors is critically involved in the neuropathological outcomes

of SE (Rice et al., 1998; Deshpande et al., 2008), we investigated the ability of Tat-NR2B9c to

provide neuroprotection following SE induced by lithium/pilocarpine. The results demonstrate

that Tat-NR2B9c, when administered 3 hrs following the termination of SE, significantly reduces

neuronal cell death in the hippocampus of adult rats.

5.2 Methods


All procedures were approved by the University of Toronto Animal Care Committee and were in

accordance with the guidelines established by the Canadian Council on Animal Care. Male

Wistar rats (Charles River Laboratories, Sherbrooke, Quebec, Canada) weighing between 300

and 350 g were individually housed with free access to food and water for at least 7 days in 12 h

light/dark cycles before experimental use.

For the induction of SE, rats were pretreated with lithium chloride (3mEq/kg, i.p.) 24 hours

before the injection of pilocarpine, and received methylatropine nitrate (10 mg/kg, i.p.) 30 min

prior to pilocarpine. Pilocarpine (10 mg/kg, i.p.) was administered every 30 min as described by

Glien et al., (2001) until the rat experienced a generalized, class 4/5 seizure, since rats generally


4/5 seizure received additional pilocarpine injections at 30-min intervals up to a maximum of 6

injections. Animals that did not develop SE after the sixth injection of pilocarpine were not used

in this study. Duration of seizure activity was controlled by the administration of diazepam (4

mg/kg, i.p.) 1, 3 and 5 hours following the onset of SE. Sham animals underwent identical

procedures but received saline in place of pilocarpine, and did not exhibit any seizures.

Pilocarpine, lithium chloride and methylatropine nitrate were purchased from Sigma (St Louis,

Missouri, USA) and dissolved in 0.9% saline prior to administration. Diazepam was purchased

from CDMV (Saint-Hyacinthe, Quebec, Canada) and used as the commercial solution (5mg/ml).

The behavioural progression of pilocarpine-induced seizures was assessed using a modified

Racine scale (Racine, 1972; Cammisuli et al., 1997), as follows: class 1, facial clonus; class 2,

120

head nodding; class 3, forelimb clonus; class 4, forelimb clonus and rearing; class 5, forelimb

clonus, rearing and one fall (loss of postural control); class 6, forelimb clonus, rearing and

multiple falls; and class 7, running and jumping. Animals were continuously monitored

following the first injection of pilocarpine. The average maximum seizure activity was

determined by averaging the highest class of seizure that occurred in each 5 minute interval

following the initiation of SE.

Following the termination of SE, animals were kept in a quiet room for 3 days. Immediately

following the first injection of diazepam, animals received 5 ml 0.9% saline (3ml i.p. and 2ml

s.c.), and this was repeated in the morning and evening of the following day (5ml i.p.) as

described by Glien et al., (2001). Starting on the second day after SE, animals were tube-fed

softened rat chow mixed with applesauce for 3 days on average. Softened rat chow was also

provided in dishes until the rats commenced to eat hard pellets.

5.2.2 Administration of peptides

Tat-NR2B9c, a synthetic peptide consisting of the C-terminal 9 amino acids of the NR2B subunit

of the NMDA receptor (Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val) fused to the cell membrane

protein transduction domain of the HIV-1-Tat protein (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-

Arg-Arg) disrupts protein interactions involving the PDZ1 and PDZ2 domains of PSD-95 (Cui

et al., 2007; Kornau et al., 1995) and was a generous gift of noNO Inc. (Toronto, ON, Canada).

Control peptide (Tat-NR2BAA) consisted of Tat-NR2B9c in which the COOH-terminal serine

and valine of the NMDAR peptide were changed to alanine residues (Lys-Leu-Ser-Ser-Ile-Glu-

Ala-Asp-Ala). The altered PDZ-binding motif is not expected to bind PDZ domains (Kornau et

al., 1995; Aarts et al., 2002; Cui et al., 2007) and was devoid of neuroprotective properties in a

model of ischemia (Aarts et al., 2002). Peptides were dissolved in saline at a concentration of

3mM and stored at -80o

C until use.

For the administration of peptides, animals underwent femoral vein cannulation and were

allowed at least 4 days recovery prior to SE induction. Briefly, animals were anaesthetized with

a continuous supply of 3 - 5% isoflurane (CDMV, Saint-Hyacinthe, Quebec, Canada) and

oxygen. A 2 cm ventral skin incision was made along the crease formed by the abdomen and

right thigh. The right femoral vein was cannulated with PE10 tubing prefilled with saline and

secured in place with suture. A 0.5 cm dorsal, midline skin incision was made between the

121

scapulae. The PE10 tubing was fed underneath the connective tissue to the dorsal incision using

a sterile stainless metal tube, immobilized, sutured with a surgical button, and occluded with a

pin. Rats received temgesic (0.1 mg/kg, i.p.; CDMV, Saint-Hyacinthe, Quebec, Canada) for

postoperative pain relief. Peptides were administered (3 or 9 nMols/gm) via the cannula over 4

to 5 minutes, either 10 minutes following the onset of SE, or 3 hrs following the termination of

SE. Saline control animals were treated identically except that they received saline (0.3 ml) in

place of peptide. In all cases the experimenter was blinded to the nature of the injection.

5.2.3 NeuN Immunohistochemistry

Two weeks following the termination of SE, animals were anaesthetized with a mixture of

xyaline (26 mg/kg i.p., Rompun, CDMV, Saint-Hyacinthe, Quebec, Canada) and ketamine (174

mg/kg i.p., Ketalar, CDMV, Saint-Hyacinthe, Quebec, Canada), and perfused transcardially

with 90 ml of 0.1 M phosphate-buffered saline, pH 7.4 (PBS) followed by 400-500 ml of 4%

(w/v) paraformaldehyde in PBS. Brains were removed and left overnight at 4˚C in the

paraformaldehyde solution. The following day, brains were soaked for cryoprotection in 30%

(w/v) sucrose in PBS until they sank at room temperature. Next, brains were frozen in 2-

methylbutane at -35˚C and stored at -80˚C. Forty µm coronal sections were prepared with a

freezing microtome. Sections were placed in 24-well culture plates containing antifreeze

solution (0.05 M sodium phosphate buffer, pH 7.4, 30% (w/v) ethylene glycol, 15% (w/v)

glucose), and stored at -20˚C.



edition),





brain, replacing the structures CeC and CeL detected in the initial section. Section were rinsed in

PBS (3 x 5 min washes), reacted overnight at 4˚C with NeuN antibody (1:1000; Chemicon,

Billerica, MA, USA) in 0.2% (v/v) goat serum, 0.3% (v/v) Triton X-100 in PBS followed by 2

hours at room temperature with Cy3 conjugated secondary antibody (1:200; Chemicon, Billerica,

MA, USA) in 0.2% (v/v) goat serum, 0.3% (v/v) Triton X-100 in PBS.

122

Neurons were imaged using a Zeiss LSM510 Laser Scanning Confocal microscope equipped

with a 40x/1.3 oil-immersion objective lens, and neuronal density determined using the unbiased

optical dissector technique as described by West and Gundersen (1990). Briefly, three counting

frames (120 X 60 µM) were positioned in the CA1, CA3 and posterior piriform cortex (PPC),

two counting frames were positioned in the CA4 region, and 1 counting frame was placed in

CA2 of the left brain hemisphere as shown in Figures 5.1 A and C. Right brain structures were


NeuN-positive cells that contained a relatively large (>8µm) soma (Shi et al., 2004).






density occur whether such borders were used or not (Harding et al., 1994; Azcoitia et al., 2005;

Gardella et al., 2003). Unless otherwise specified, cell densities for individual animals represent



5.2.4 Statistical Analysis

Statistical analysis was performed using Statistica 6.0 software. Significant differences were

determined using one-way analysis of variance (ANOVA). The Newman-Keuls post-hoc test

was used to determine difference between treatment groups. Chi-square analysis was performed

to analyze mortality rates. Significance was set at a p-value of 0.05 or less.

5.3 Results


SE was induced by the repeated administration of low doses of pilocarpine (10 mg/kg) to

animals that had been pre-treated with lithium chloride and methyl atropine nitrate as described

by Glien et al., (2001). Of 63 animals that received pilocarpine, 16 (25%) did not develop SE

after 6 injections of pilocarpine, and were not used for the present study. The other animals

developed SE after one (n=2), two (n=23), three (n=16) or four (n=6) pilocarpine injections, with

123

SE developing within 22 – 120 minutes (mean 68 +/- 22 min) following the initial pilocarpine

injection. The overall fatality rate for animals that entered SE was 30% and did not differ among

experimental groups (Table 5.1).

All animals which entered SE displayed stage 4/5 convulsive seizures within the first 10 minutes.

During the 60 minutes of SE, animals experienced seizures varying between 2 and 4/5 on the

Racine scale (1972) and did not regain consciousness. There was no significant difference

between groups with respect to the average maximum seizure activity recorded during SE, which

approximated to 3.0 for all groups (Table 5.1). All groups experienced similar weight gain

during the recovery period (Table 5.1) so that by 14 days there was no difference in mean body

weight between groups (Table 5.1)

124

Table 5.1: Comparison of the effect of treatment on mortality, seizure severity and weight gain

following SE.

Treatment Total Survived1 Died

1 SE

Severity2

Initial

Weight3

(gm)

Final

Weight3

(gm)

SE + Saline

(administered

following SE)

11 8 3

(27.3%)

3.06 ± 0.12 402 ± 15 468 ± 18

(16.6%)

SE + Tat-NR2B9c

(3 nmol/gm,

administered

following SE)

14 10 4

(28.6%)

2.98 ± 0.10 395 ± 12 431 ± 22

(9.2%)

SE + Tat-NR2B-AA

(3 nmol/gm,

administered

following SE)

11 8 3

(27.3%)

2.90 ± 0.12 405 ± 15 462 ± 12

(14.2%)

SE + Tat-NR2B9c

(3 nmol/gm,

administered during

SE)

7 4 3

(42.9%)

3.13 ± 0.14 393 ± 9 441 ± 15

(12.3%)

SE + Tat-NR2B9c

(9 nmol/gm,

administered during

SE)

4 3 1

(25.0%)

3.09 ± 0.18 387 ± 10 430 ± 27

(11.2%)

Sham (no SE) 11 11 0 No SE 379 ± 7 433 ± 11

(14.5%)

1. The number of animals that survived or died within 14 days following SE. Numbers in ( )

represent the % of animals that died. 2. Average maximum seizure activity for all animals

determined as in Methods (mean ± SD). 3. Initial and final group weights evaluated

immediately before, and at 2-weeks following, SE induction (mean ± SD). Values in () indicate

% weight gain. No significant differences in mortality rates (chi-square analysis), seizure

severity (one-way ANOVA) or % weight gain (one-way ANOVA) were detected between

treatment groups.

125

5.3.2 SE induced by repeated low doses of pilocarpine results in neurodegeneration in the hippocampus and piriform cortex

The hippocampus and piriform cortex play critical roles in the development and maintenance of

limbic seizures, and are the most susceptible brain regions to SE-induced damage (Turski et al.,

1983b; Druga et al., 2003; Andre et al., 2007; Chen et al., 2007;). We therefore focused our

analyses on these regions. Preliminary experiments demonstrated that neurodegeneration in both

the dorsal hippocampus and the piriform cortex occurred rapidly within the first few days

following the termination of SE, and was complete within 2 weeks (see chapter 4). The effect of

SE on neuronal cell death was therefore assessed 14 days following the termination of seizures.

Because the effect of SE induced by the repeated administration of low doses of pilocarpine on

neurodegeneration has not previously been described, we initially determined the effect of SE on

neuronal cell density in animals that experienced SE but that did not receive any additional

treatment. Preliminary comparisons of pyramidal cell counts in each of the 3 coronal brain

sections showed that the extent of SE-induced cell death did not vary between sections. For

example, the average cell densities in the CA1 region before and after SE were 110,796 ± 23,841

and 33,654 ± 24,097, 106,271 ±24,551 and 33,234 ± 24,207, 104,692 ± 18,128 and 35,879 ±

21,056 for the 3 sections from the most proximal to the most distal from Bregma. Similar

results were obtained for the other hippocampal subfields. Cell densities were therefore

determined by averaging the densities for all counting frames in all 3 sections for a particular

region. The results presented in Figure 5.1 show that SE resulted in a marked loss of pyramidal

neurons in the CA1, CA3, and CA4 regions of the dorsal hippocampus, and in layer ІІ of the

posterior piriform cortex (PPC). Neuronal densities (cells/mm3 ± SD) prior to and following SE

were 104,993 ± 18,982 and 18,982 ± 22,093 (70% cell loss) in the CA1 region, 64,705 ± 3,575

and 37,616 ± 12,412 (34% cell loss) in the CA3 region, 64,704 ± 9,697 and 31,084 ± 20,344

(58% cell loss) in the CA4 region, and 48,417 ± 12,123 and 5,851 ± 5,103 (88% cell loss) in the

PPC. SE did not result in a significant change in cell density in the CA2 region (81,018 ± 10,195

cells/mm3

in naive animals, 68,297 ± 12,678 cells/mm3

in SE rats), and for this reason the effect

of Tat-NR2B9c on the CA2 region was not assessed. Overall, pyramidal cell loss with the

current procedure for SE induction was similar to neuronal loss reported in previous studies

using other variations of the pilocarpine model (Turski et al., 1983a; Clifford et al., 1987;

Covolan and Mello, 2000; Fujikawa, 1996).

126

We next compared the effect of SE on cell density in individual counting frames to determine if

SE-induced pyramidal cell loss was uniform within discrete structures. The data in Table 5.2

show that the extent of cell loss in individual counting frames was similar regardless of position

within the specific hippocampal subfield or in the PPC, indicating that the effect of SE on

neurodegeneration was independent of cell location within the region. As also illustrated in

Table 5.2, the boundary between hippocampal subfields with respect to neurodegeneration was

quite sharp. For example, in counting frame 3c, on the CA3 side of the border between CA3

and CA4, 29.9 ± 22.4% of the pyramidal cells were lost following SE, whereas in counting

frame 4a, on the CA4 side of the border, the decrease was 64.3 ± 36.4% (p<0.05).

Table 5.2: Comparison of SE-induced pyramidal cell loss in individual counting frames

Brain Region Counting frame a Counting frame b Counting frame c

CA1 71.9 ± 19.9 76.9 ± 17.9 71.6 ± 12.2

CA3 37.4 ± 25.7 30.2 ± 23.3 29.9 ± 22.4

CA4 64.3 ± 36.4 54.2 ± 25.6 ------

PPC 85.3 ± 16.4 89.6 ± 16.7 91.2 ± 20.2

Individual counting frames were positioned in hippocampal subfields and the PPC as depicted in

Figure 5.1 A and C. Numbers represent % decrease of pyramidal cells ± SD (n=5). Pyramidal

cell loss within individual regions was similar regardless of the placement of the counting frame

(one-way ANOVA).

127

Figure 5.1 Neurodegeneration depicted in NeuN-stained coronal sections of the rat dorsal

hippocampus and posterior piriform cortex (PPC) 14 days following status epilepticus (SE).

A, B: Representative images of the left dorsal hippocampus from a sham rat (A) or from a rat 14

days after SE (B). Open arrowheads define the borders of CA1, whereas the dotted line

indicates the border between CA3 and CA4. Pyr depicts the pyramidal cell layer. C, D:

Representative images of the PPC from a sham (C) or from a rat 14 days after SE (D). Boxes

represent placement of counting frames in the CA1 (1a-c), CA2 (2a), CA3 (3a-c) and CA4 (4a,b)

regions of the hippocampus, and in the PPC (a-c). Scale bar = 500 µm. Low-powered images

captured at 25X magnification. E: Cell densities in sham (grey bars, n=5) and SE (black bars,

n=5) animals at 2 weeks recovery (mean density ± SD). Significant pyramidal cell loss occurred

in the CA1, CA3, CA4 and PPC, but not in the CA2. * depicts significant difference from naive

animals (p<0.05).

128

E

* *

*

*

129

5.3.3 Tat-NR2B9c reduces SE-induced neurodegeneration in the hippocampus

We next determined the effect of Tat-NR2B9c on SE-induced neuronal loss. For these

experiments, animals received Tat-NR2B9c, its inactive homologue Tat-NR2B9AA, or saline 3

hrs following the termination of SE, and cell densities were determined 2 weeks later. Results for

individual animals are plotted in Figure 5.2. SE-induced cell loss in animals that received saline

was similar to that reported above (compare Fig 5.2 and Fig 5.1). The administration of Tat-

NR2B9c resulted in a statistically significant overall reduction in pyramidal cell loss in

hippocampal subfields CA1 and CA4 relative to saline treated animals, although there was

considerable animal to animal variation in the efficacy of the peptide. In CA1, the decrease in

pyramidal cell density in animals that received Tat-NR2B9c was 38 ± 35% (mean± SD) as

compared to a decrease of 66 ± 18% in the saline treated animals (p<0.05). Similarly in CA4,

administration of Tat-NR2B9c reduced SE-induced cell loss from 60 ± 28% in saline controls,

to 26 ± 39% in animals that received the peptide (p<0.05). In neither case did Tat-NR2B9AA,

the inactive homologue of Tat-NR2B9c, exhibit any neuroprotective effect, consistent with the

action of the active peptide being mediated via its interaction with PDZ domains. In contrast to

the results for CA1 and CA4, Tat -NR2B9c failed to reduce overall cell loss in either CA3 or the

PPC when compared to saline treated animals (Fig 2).

130

FIGURE 5.2 Tat-NR2B9c reduces pyramidal cell loss in the dorsal hippocampus when

administered 3 hours after SE. Animal received saline, Tat-NR2B9c or Tat-NR2B9AA 3 hr

following the termination of SE, and were sacrificed 14 days later. Coronal sections were

stained with NeuN and cell densities determined as in Methods. A: Representative NeuN

stained images of the pyramidal cell layer of indicated regions of the hippocampus and of layer II

of the posterior piriform cortex (PPC). The scale bar = 20 µm. High-powered images captured

at 400X magnification. B. Administration of Tat-NR2B9c reduced SE-induced cell loss in the

CA1 and CA4, but not in the CA3 or PPC. Cell densities for individual animals were plotted as

open circles. The dash represents the mean cell density for the group. Mean cell densities for

each brain region were as follows. CA1: shams (107253 ± 4339 cells/mm3, mean ± S.E.M.,

n=11), Saline (36699 ± 7002 cells/mm3, n=8), Tat-NR2B9c (66049 ± 11993 cells/mm

3, n=10),

Tat-NR2B9AA (30767 ± 7606 cells/mm3, n=8). CA3: shams (62812 ± 1354 cells/mm

3), Saline

(44618 ± 5702 cells/mm3), Tat-NR2B9c (52916 ± 4124 cells/mm

3), Tat-NR2B9AA (41354 ±

6401 cells/mm3). CA4: shams (67847 ± 2923 cells/mm

3), Saline (27391 ± 7657 cells/mm

3),

Tat-NR2B9c (50173.61 ± 8238 cells/mm3), Tat-NR2B9AA (32118 ± 7012 cells/mm

3). PPC:

shams (49961 ± 3033 cells/mm3), Saline (8611 ± 3991 cells/mm

3), Tat-NR2B9c (9104 ± 3691

cells/mm3), Tat-NR2B9AA (12935 ± 3437 cells/mm

3). * denotes a significant difference from

sham group (p<0.05), # denotes a significant difference from saline and Tat-NR2B9AA groups

(p<0.05), ^ indicates a significant difference from saline group (p<0.05), and approaching

significance with Tat-NR2B9AA group (p=0.08).

131

132

5.3.4 Preferential neuroprotection of Tat-NR2B9c is found within specific regions of the CA1 and CA3

The above results demonstrate that the administration of Tat-NR2B9c 3 hrs following SE

resulted in reduced neurodegeneration in the CA1 and CA4 hippocampal subfields, but not in

CA3 or the PPC. These findings were obtained by averaging the cell densities of all counting

frames placed in the different brain regions. To determine if the neuroprotective effects of Tat-

NR2B9c were uniform within individual regions, we compared the effect of Tat-NR2B9c on

neuronal cell densities in individual counting frames. Consistent with the results presented in

Table 5.2, the extent of SE-induced neurodegeneration was similar in each of the counting

frames placed within CA1, CA3, and CA4 and in the PPC in animals that received either saline

or the inactive peptide, Tat-NR2B9AA (Figure 5.3, grey and oblique stripped bars). However, in

contrast to the uniform effect of SE on cell death, comparison of cell densities within individual

counting frames identified differences in the neuroprotective actions of Tat-NR2B9c within CA1

and CA3. The neuroprotective effect of Tat-NR2B9c varied in the medial to lateral direction

across CA1, increasing with proximity to the subicular border (Figure 5.3A, black bars). Thus,

in the presence of the peptide, 81 ± 13% (86,226 ± 14,027 cells/mm3 in animals that received

Tat-NR2B9c vs 106,691 ± 5051 in control animals, p<0.05) of pyramidal neurons remained in

counting frame 1a, adjacent to the subicular border, following SE, whereas only 44 ± 9%

(47,685 ± 9,707 cells/mm3 in animals that received Tat-NR2B9c vs 109,638 ± 4542 in control

animals) remained in counting frame 1c, adjacent to CA2 (Figure 5.3A, black bars). Similarly,

although Tat-NR2B9c had no overall protective effect in CA3 (Figure 5.2), comparison of

individual counting frames showed that whereas there was no protective effect in the areas

represented by counting frames 3a and 3b, there was nearly complete protection in the area

represented by counting frame 3c, the area proximal to the transitional border with CA4 (Figure

5.3B, black bars) Differential neuroprotective effects of Tat-NR2B9c were not observed in

either CA4 or PPC.

133

FIGURE 5.3 Tat-NR2B9c exhibits differential neuroprotection within different regions of

the CA1 and CA3 subfields of the hippocampus. Neuronal densities in individual counting

frames in hippocampal subfields were compared between treatment groups. Results are

expressed as mean ± SEM. Open bars: shams (n = 11); grey bars: saline (n=8); black bars: Tat-

NR2B9c (n=10); oblique stripped bars: Tat-NR2B9AA (n=8). * denotes significant difference

from sham group, # denotes significant difference from saline and Tat-NR2B9AA groups, ^

indicates a significant difference from saline group (p<0.05), and approaching significance

relative to Tat-NR2B9AA group (p=0.067). ** indicates significant difference between counting

frames a and c.

134

0

40

80

120

cells

/mm

3x

10

-3

0

20

40

60

80

cells

/mm

3x

10

-3

1a 1b 1c 3a 3b 3c

A. CA1

0

20

40

60

cells

/mm

3x

10

-3

0

25

50

75

100

cells

/mm

3x

10

-3

a b c4a 4b

B. CA3

C. CA4 D. PPC

* *

*

* *

*^#

* ***

*

**

**

* ** * *

** *

***#

* *

*

* *

*

**

***

#

135

5.3.5 Tat-NR2B9c did not provide neuroprotection in CA1 when administered during SE

The above findings demonstrate that administration of Tat-NR2B9c 3 hrs following the

termination of SE results in significant protection of pyramidal neurons in the hippocampus. We

also determined if Tat-NR2B9c would provide protection if it was administered during SE. For

these experiments, animals received Tat-NR2B9c after 10 minutes of continuous seizure activity,

and SE was allowed to continue for an additional 50 minutes. Administration of the peptide at

this time had no affect on seizure severity or survival rates (Table 5.1). The results presented in

Figure 5.4 show that under these conditions Tat-NR2B9c offered no neuroprotection in CA1.

Increasing the dosage of peptide from3 nMols/g to 9 Mols/g did not increase the neuroprotective

properties of the peptide under these conditions.

136

FIGURE 5.4 Tat-NR2B9c is not neuroprotective when administered 10 minutes following the onset

of SE. SE was induced as in Methods section 5.2.1 and animals received saline or Tat-NR2B9c (3 or 9

nmol/gm) following 10 minutes of continuous behavioural seizures. Seizures were allowed to continue

and SE was terminated after 60 minutes and animals sacrificed 14 days later. A: Representative images

of NeuN stained pyramidal cells in the CA1 region of the dorsal hippocampus. The scale bar = 20 µm.

High-powered images captured at 400X magnification. B: NeuN stained neurons in the CA1 were

quantified as in Methods section 5.2.3 and cell densities plotted. Cell densities for individual animals

were plotted as open circles for sham (n=10) and saline (n=8) groups. Solid shapes denote cell densities

for individual animals that received Tat-NR2B9c at doses of 3 nMols/gm (circles, n=4), or 9 nMols/gm

(triangles, n=3). The dash represents the mean cell density for the group. Mean cell densities were:

shams (107253 ± 4339 cells/mm3, mean ± S.E.M.), Saline (36996 ± 7002 cells/mm

3), Tat-NR2B9c at

3nMol/gm (26427 ± 5847 cells/mm3), Tat-NR2B9c at 9nMol/gm (28340 ± 10290 cells/mm

3). * denotes

significant difference when compared to sham group (p<0.05).

137

138

5.4 Discussion

In the current study we determined the ability of Tat-NR2B9c, a synthetic peptide designed to

selectively disrupt the NMDAR signaling complex (Aarts et al., 2002), to reduce SE-induced

neuropathology. Previously, it was reported that Tat-NR2B9c markedly reduced brain damage

following an ischemic challenge (Aarts et al., 2002; Sun et al., 2008), while maintaining function

of the NMDAR ion channel (Sattler et al., 1999; Aarts et al., 2002). Cui et al (Cui et al., 2007),

analyzed the interaction of Tat-NR2B9c with all known potential human binding partners and

found that it was highly specific for the PDZ2 domain of PSD-95, suggesting that its

neuroprotective action is due to the disruption of protein interactions involving PSD-95.

Although NMDAR antagonists are highly neuroprotective in rodent models of epilepsy, even

when administered after the onset of SE (Fariello et al., 1989; Clifford et al., 1990; Fujikawa et

al., 1994; Fujikawa, 1995), clinical trials with these compounds have failed because of poor

tolerance and efficacy (Dyker et al., 1999; Davis et al., 2000; Albers et al., 2001; Ikonomidou

and Turski, 2002; Muir, 2006). Tat-NR2B9c offers an alternative approach to disrupting

NMDAR-mediated pro-death signaling, while sparing signaling pathways linked to survival and

plasticity (Aarts et al., 2002; Arundine and Tymianski, 2003; Aarts and Tymianski, 2004).

5.4.1 Tat-NR2B9c provided significant neuroprotection in the hippocampus

The primary finding of the present study is that Tat-NR2B9c significantly attenuated

hippocampal damage induced by an episode of SE. Importantly, substitution of the C-terminal

ser and val of Tat-NR2B9c by alanine residues to prevent interaction with the PDZ domains of

PSD-95 (Kornau et al., 1995; Aarts et al., 2002; Cui et al., 2007), completely eliminated the

neuroprotective effect of the peptide, supporting the hypothesis that neuroprotection is due to

disruption of protein interactions involving the PDZ domains of PSD-95. We initially reported

that Tat-NR2B9c preferentially reduced the co-immunoprecipitation of NR2B and PSD-95 from

rat brain extracts (Aarts et al., 2002). More detailed binding analysis subsequently demonstrated

that Tat-NR2B9c has high affinity for the PDZ2 domain of PSD-95, and inhibits the binding of

NR2 subunits, as well as nNOS, to this domain with IC50 values in the low uM range (Cui et al.,

2007). Neuronal nitric oxide synthase (nNOS) is a component of the NMDAR complex and is

activated by the influx of Ca2+

ions via the NMDAR ion channel (Forder and Tymianski, 2009).

NO production is enhanced following SE (Gupta and Dettbarn, 2003), and inhibition of nNOS is

139

neuroprotective (Montecot et al., 1998; Murashima et al., 2000) and antiepileptogenic

(Rajasekaran et al., 2003; Sardo and Ferraro, 2007). Taken together, these findings support a

model in which the neuroprotective actions of Tat-NR2B9c result from disruption of the

NMDAR signaling complex, and in particular dissociation of nNOS from PSD-95, by

competing for PDZ domain binding sites on PSD-95, and are consistent with known

mechanisms of SE-induced cell death (Arundine and Tymianski, 2003; McNamara et al., 2006).

5.4.2 Regional specificity of neuroprotection by Tat-NR2B9c within CA1 and CA3

An unexpected observation was the regional specificity of neuroprotection by Tat-NR2B9c

within CA1 and CA3, the greatest protection of pyramidal neurons within these regions

occurring proximal to the CA1/subicular border and CA3/CA4 border, respectively. The

molecular basis for the differential neuroprotective effects of Tat-NR2B9c remains unclear but

the finding suggests the presence of previously unidentified differences in the properties of these

neurons. The severity of SE-induced neurodegeneration was similar across all counting frames

within CA1 or CA3, indicating that the differential effects of Tat-NR2B9c cannot be explained

by variable sensitivities to the initial insult. The projection patterns of pyramidal cells change

gradually as one moves across CA3 from the border with CA2 to that with CA4 (Lorente De Nó,

1934; Li et al., 1994) and it is tempting to speculate that the increase in neuroprotection from

CA3a (essentially no protection) to CA3c (nearly complete protection), may be related to the

varying pattern of connectivity of CA3 pyramidal neurons. Alternatively, subunit composition is

an important determinant of NMDAR function (Nakanishi et al., 1994; Cull-Candy et al., 2001)

and may affect association of the receptor with members of the PSD-95 family of membrane

associated guanylate kinases (Cousins et al., 2008; Cousins et al., 2009) as well as with

downstream signaling molecules, such as nNOS (Lynch and Guttmann, 2002; Al-Hallaq et al.,

2007). The results therefore suggest the occurrence of differences between protected and non-

protected neurons in subunit composition of the NMDAR and/or in protein interactions involving

PSD-95 within the NMDAR signaling complex. Although differences in the subunit

composition of NMDARs between hippocampal subfields and between dorsal and ventral CA1

have been reported (Coultrap et al., 2005; Pandis et al., 2006; Papatheodoropoulos, 2007), we are

not aware of any studies examining either NMDAR subunit composition or the NMDAR

140

signaling complex within different regions of the same hippocampal subfield, and this remains

an important subject for future investigation.

5.4.3 Neuroprotective effect of tat-NR2B9c is dependent on time of administration

Although the administration of Tat-NR2B9c significantly reduced neuronal loss when the

peptide was given after the termination of SE, it was ineffective if given during SE. Previous

studies have shown that forty to sixty minutes of continuous seizure activity is required before

neuron loss occurs (Nevander et al., 1985; Fujikawa, 1996), suggesting that molecular

mechanisms that occur in the earlier stages of SE are distinct from the pro-death signaling that

occurs following prolonged SE. In general accord with this suggestion, SE results in numerous

changes in the composition and properties of the postsynaptic density, including the recruitment

and activation of several signaling molecules (Niimura et al., 2005; Wyneken et al., 2001),

enhanced phosphorylation of the NMDA receptor and other proteins (Niimura et al., 2005; Huo

et al., 2006), and changes in protein interactions or molecular organization (Moussa et al., 2001).

These seizure-induced changes to the composition and structure of the postsynaptic signaling

apparatus may alter the way in which Tat-NR2B9c interacts with its binding partners and render

the neuron more susceptible to the neuroprotective actions of the peptide. Alternatively,

although peptides fused to the Tat protein transduction domain readily penetrate the blood brain

barrier (Schwarze et al., 1999; van Vliet et al., 2007; Fabene et al., 2008) and enter neurons

(Aarts et al., 2002), enhanced efficacy of Tat-NR2B9c at later times may also result from

increased accessibility to neurons as a result of seizure-related damage to the blood brain barrier

(Fabene et al., 2008). In contrast to the present findings, NMDA receptor antagonists were

reported to provide protection to several brain regions, including the hippocampus and piriform

cortex, when administered only 15 min into SE induced with pilocarpine (Fujikawa, 2004), or

when administered 90 min into SE induced by kainic acid (Ebert et al., 2002; Brandt et al.,

2003b). The difference between these results and those described here with Tat-NR2B9c

presumably relate to the different mechanisms of action of the drugs, the antagonists preventing

entry of Ca2+

ions via the NMDA receptor ion channel and Tat-NR2B9c affecting signalling

downstream of the receptor.

141

5.4.4 Conclusion

In summary, the present study demonstrates that the use of Tat-NR2B9c for the targeted

disruption of the NMDAR signaling complex represents a viable approach for limiting the

neuropathological consequences of SE. Because preservation of neurons within the

hippocampus reduces the behavioural consequences of SE (Brandt et al., 2006), we subsequently

investigated whether Tat-NR2B9c also offered long-term protection against SE-induced

neurological deficits (see chapters 6 and 7).

142

Chapter 6

Long-lasting behavioural and anxiolytic changes in rats following status epilepticus

6.1 Introduction

Depression and anxiety disorders, followed by psychoses, are the most common interictal

behavioural disturbances observed in patients with epilepsy (Boro and Haut, 2003; Devinsky,

2004a; Gaitatzis et al., 2004; Garcia-Morales et al., 2008). Moreover, several studies have found

that these comorbid psychiatric disorders are an independent risk factor for a poor quality of life

in epileptic patients (Cramer, 2002; Gilliam, 2002; Cramer et al., 2003; Boylan et al., 2004;

Johnson et al., 2004). The prevalence rates of psychiatric comorbidities are significantly higher

in temporal lobe epilepsy (TLE) than in other types of epilepsies (Perini et al., 1996; Matsuura et

al., 2003; Garcia-Morales et al., 2008), with up to 50% of patients with TLE experiencing

profound interictal disturbances in emotional behaviour (D. Blumer, G. Montouris and B.

Hermann, 1995). The control of seizure activity with pharmacologic treatment does not always

alleviate these disorders (Engel et al., 1991) and the causal relationship between TLE and

psychiatric disorders is poorly understood (Devinsky, 2003; Swinkels et al., 2005).

The lithium/pilocarpine model of SE in rats recapitulates most clinical and neuropathological

features of human TLE (Curia et al., 2008). In adult rats, the injection of lithium and pilocarpine

leads to status epilepticus (SE) and subsequent development of spontaneous recurrent seizures

(SRSs) within 2 to 6 weeks. Recent literature suggests that the ‗seizure-free‘ period following

SE, also referred to as epileptogenesis, is when neuropathological changes occur to develop an

epileptic brain (McNamara et al., 2006; Curia et al., 2008; Pitkänen and Lukasiuk, 2009).

During epileptogenesis, neuronal loss, gliosis, and synaptic reorganization participate in the

constitution of a hyperexcitable circuit that underlies the occurrence of SRSs (Fujikawa, 2005;

Acharya et al., 2008). The affective symptoms associated with epilepsy are believed to be

secondary to seizures, and are also derived from these neuropathological changes (Sayin et al.,

2004). Previous studies have shown that SE in rats causes extensive neuronal loss in the

hippocampus, cortex, amygdala and thalamus (Honchar et al., 1983; Turski et al., 1983b;

Covolan and Mello, 2006). These structures are known to be functionally important in anxiety

and exploration (Davis, 1992; Degroot and Treit, 2002; Moreira et al., 2007; Carvalho et al.,

143

2008), and damage to them following SE is likely to produce behavioural impairment. This idea

is supported by findings that neuroprotection within these regions attenuates SE-induce

anxiolytic changes in rats when tested in the elevated plus-maze and open field (dos Santos et al.,

2005; Brandt et al., 2006).

While administration of epileptogenic agents is known to produce long-term behavioural

disruption (Rice et al., 1998; Narkilahti et al., 2003b; dos Santos et al., 2005; Detour et al., 2005;

Szyndler et al., 2005; Brandt et al., 2006; de Oliveira et al., 2008; Cardoso et al., 2009), the

relationship between behavioural disruption and the development of status epilepticus remains

unclear. In chapter 4, we demonstrated that the majority of SE-induced neuronal death occurs

within 3 days after the termination of SE. Because neurodegeneration is associated with

cognitive and behavioural morbidity (Milgram et al., 1988; Mikati et al., 2001; Wu et al., 2001;

dos Santos et al., 2005; Brandt et al., 2006), our first objective was to examine the time course of

behavioural changes following SE. We assessed short-term behavioural disturbances at 1 and 2

weeks following SE, and long-term behavioural disturbances at 12 weeks to determine if these

changes were persistent or were affected by the development of SRSs.

Rats treated with the repeated low-dose pilocarpine (RLDP) protocol exhibited less severe status

and a lower mortality rate compared to rats treated with the LDP protocol (see Results sections

3.3.2 and 3.3.3). Although neuropathology in rats treated with the two seizure-inducing

protocols was similar (see Results section 3.3.5), it is possible that differences in other factors

not assessed (i.e., changes in synaptic plasticity, neurogenesis, synaptic reorganization) may be

present and as a result, cause differences in behavioural responses in rats treated with the two

procedures. Therefore, our second objective was to compare the subsequent behavioural effects

of the two seizure-inducing protocols. Because we demonstrated protection within the CA1,

CA3 and CA4 subfields of the dorsal hippocampus following administration of tat-NR2B9c (see

Results 5.3.3), our third objective was to determine the effect of tat-NR2B9c on behaviour. Our

behavioural assessment protocol consisted of two common tests for anxiety-like behaviour, the

open-field (Prut and Belzung, 2003) and the elevated plus maze (File, 1993), and four

hyperexcitability tests (Rice et al., 1998).

144

6.2 Methods

6.2.1 Animals



Animal Care. Male Wistar rats (Charles River Laboratories, Sherbrooke, Quebec, Canada)

weighing between 300 and 350 g were individually housed with free access to food and water for

at least 7 days in 12 h light/dark cycles before experimental use.

6.2.2 Induction of status epilepticus and administration of peptides

Two separate studies were performed. In the first, the effects of the low-dose pilocarpine (LDP)

and the repeated low-dose pilocarpine (RLDP) seizure-inducing protocols on behaviour were

compared. SE was induced in adult Wistar rats (320 – 350 g) using either the LDP or RLDP

methods exactly as described in Methods section 3.2.2. SE was terminated by the administration

of diazepam (4 mg/kg, i.p.) at 1, 3 and 5 hours following the onset of SE. Ten animals

developed SE following the LDP procedure and were used for the subsequent behavioural

studies (Table 6.1).

For the RLDP method, rats were pretreated with lithium chloride (3mEq/kg, i.p.) approximately

24 hours before and methylatropine nitrate (10 mg/kg, i.p) 30 min before the initial injection of

pilocarpine. Pilocarpine (10 mg/kg, i.p.) was administered every 30 min as described by Glien et

al., (2001) until the rat experienced a generalized, class 4/5 seizure, since rats generally


4/5 seizure, received additional pilocarpine injections at 30-min intervals up to a maximum of 6

injections. Diazepam (4 mg/kg, i.p.) was administered at 1, 3 and 5 hours following onset of SE

to terminate seizure activity. Twenty-seven animals developed SE using the RLDP procedure

(Table 6.1)

The second study assessed the effect of the peptide tat-NR2B9c on behavioural outcomes in rats

following SE. SE was induced with the RLDP procedure and peptides, tat-NR2B9c and tat-

NR2BAA, were administered as described in Methods section 5.2.2. Two control groups were

used: (1) Sham rats (n=19) received a mock femoral vein cannulation and were treated

identically to SE animals except that they received saline in place of pilocarpine; they did not

145

develop SE. In a pilot study, sham rats were found to behave identically in the behavioural tests

(open field, elevated-plus maze, and hyperexcitability tests) to naïve rats not subjected to the

mock surgery, indicating that femoral vein cannulation had no effect on the behavioural

outcome. (2) ‗Pilo no-SE‘ rats (n=22) were treated with the RLDP protocol but did not develop

SE. Figure 6.1 summarizes the SE-inducing protocols and experimental animal groups used in

the behavioural studies (chapters 6 and 7).

Animals were continuously monitored following the first injection of pilocarpine, and seizure

activity was recorded as described in Methods section 3.2.3. All rats received post-seizure care

as described in Methods section 3.2.4.

146

Figure 6.1 Schematic of SE-induction protocols and treatments in SE and non-SE groups

(see Methods section 6.2.2).

147

148

6.2.3 Behavioural tests

6.2.3.1 Pre-observational period to behavioural testing

All animals were observed for 1 hr prior to testing. If the rat was seizure-free within the

observation period, the animal was tested as described below. If a rat exhibited recurrent

seizures within the observation period, than the test was postponed until the following day. Any

trial in which a rat exhibited SRSs was excluded from the final analyses of the data. The open

field test and hyperexcitability tests were performed 1, 2 and 12 weeks following SE. The

elevated plus maze was performed 12 weeks after SE. The elevated plus maze test was

performed on separate days from the open field and hyperexcitability tests.

In the behavioural studies, all SRSs observed during handling, during the testing or observation

periods, or by direct observation of the rats in their home cages were recorded. A rat was

considered epileptic after exhibiting one or more SRSs.

6.2.3.2 Open-field test

The open field test measures anxiety-like behaviour and locomotor activity (Prut and Belzung,

2003). Rats were placed in the center of a square open field (diameter 100 cm) enclosed with

walls 36 cm high, and created from clear Plexiglas. The apparatus was divided into 16 equally

spaced squares, with the 4 centre squares defined as the centre arena. The open field was placed

inside a light- and sound-attenuated room. Before each trial, the field was cleaned with 70%

alcohol solution. Behaviour was recorded with a video camera for 10 min, and later analyzed

independently by 2 experimenters blinded to animal treatment. Experimenters were not present

in the room during the test. For each rat, the total number of squares crossed (with four paws) in

the peripheral and center regions of the open field, the number of entries (with four paws) into

the centre arena, and the frequency and duration of rearing activity was assessed.

6.2.3.3 Elevated plus maze test

The elevated-plus maze is used to assess anxiety in rodents (Hogg, 1996). The procedure

consists of allowing rats to freely explore two elevated open and two elevated closed arms. In

such a situation, rats spontaneously prefer the darker closed arms and avoid the anxiety-

provoking open arms. The apparatus consisted of painted black wood in the form of two open

149

arms (50 X 10 cm), and two enclosed arms (50 X 10 X 40 cm), annexed to a central platform (10

X 10 cm) to form a plus sign. Grip was facilitated by lining the enclosed and open arms with

black textured rubber. The apparatus was elevated 50 cm above the floor level. The test was

conducted inside a light- and sound-attenuated room, without the presence of an experimenter.

Before each trial, the maze was cleaned thoroughly with 70% alcohol. At the beginning of the

trial, rats were placed on the central platform always facing the same closed arm. Behaviour was

recorded with a video camera for 5 min, and later analyzed independently by 2 experimenters.

For each rat, the time spent and number of entries (with four paws) in different sections of the

maze (open and closed arms), frequency of risk assessments (with two paws in the open arm,

head extended over the ledge), and rearing frequency was assessed. Occasionally, SE groups fell

from the ledge of the outer arm to the floor of the elevated plus maze. In these cases, the SE

groups were retested the following day; if the animal fell during the second trial, the data was

eliminated from final analyses.

6.2.3.4 Hyperexcitability tests

Rice et al., (1998) described four behavioural tests that potentially discriminate hyperexcitability

differences between SE groups and non-SE rats. These tests were taken from the functional

observational battery described by Moser et al., (1988). The four tests are described below:

1. Approach-response test: A pen held vertically is moved slowly toward the face of the

animal. Responses were scored as 1, no reaction; 2, the rat sniffs at the object, 3, the rat

moves away from the object; 4, the rat freezes; 5, the rat jumps away from the object; and

6, the rat jumps at or attacks the object.

2. Touch-response test: The animal is gently prodded in the rump with the blunt end of a

pen. Responses were scored as 1, no reaction; 2, the rat turns toward the object; 3, the rat

moves away from the object; 4, the rat freezes; 5, the rat jerks around toward the touch; 6,

the rat turns away from the touch; and 7, the rat jumps with or without vocalizations.

3. Finger-snap test: A finger snap several inches above the head of the animal is performed.

Responses were scored as 1, no reaction; 2, the rat jumps slightly or flicks the ear (normal

reaction); and 3, the rat jumps dramatically.

4. Pick-up test: The animal is picked up by grasping around the body. Responses were

scored as 1, very easy; 2, easy with vocalizations; 3, some difficulty, the rat rears and

150

faces the hand; 4, the rat freezes (with or without vocalizations); 5, difficult, the rat

avoids the hand by moving away; and 6, very difficult, the rat behaves defensively, and

may attack the hand.

A minimum of three observers independently recorded the behaviour for each rat, and the means

of their scores were calculated for each animal in each test. The tests were accomplished in the

home cage, and were conducted at least 30 min apart.


Analysis of variance (ANOVA) and chi-square analysis were performed using Statistica 9.0

software. Non-parametric testing was performed using GraphPad Prism 5 software.

Significance was set at a p-value of 0.05 or less.

151

6.3 Results

6.3.1 SE induction

SE induction rates and mortality rates between groups were assessed using chi-square analysis.

The number of pilocarpine injections administered in the RLDP protocol and weight gain

between groups was assessed by one-way ANOVA. As demonstrated in Table 6.1, there was no

significant difference between groups with respect to seizure susceptibility, as assessed by the

number of repeated low-dose pilocarpine injections (10 mg/kg) administered in the RLDP

protocol, and by the percentage of rats entering SE. By 3 months after SE, however, rats treated

with the LDP protocol exhibited a higher mortality rate compared to rats treated with the RLDP

protocol. All experimental groups experienced similar weight gain at 3 months following SE.

By 3 months, all rats that developed SE were observed to exhibit at least 1 recurrent seizure

(stage 4/5), and therefore were considered to be epileptic.

Table 6.1: Comparison of experimental groups in seizure susceptibility and mortality at 3 months

following SE Treatment Total #

of rats

per

group

# of

pilocarpine

injections for

RLDP

protocol1

Latency to SE

onset for LDP

protocol2

(min)

SE

induction3

Survived4 Died

4 Weight

5

(g)

Saline RLDP

(Saline SE)

54 3.8 ± 1.3 – 36 (67%) 27 9 (25%)* 550 ± 43

Tat-NR2B9c

SE

33 3.6 ± 1.0 – 23 (70%) 17 6 (26%)** 548 ± 29

Tat-

NR2BAA SE

45 3.5 ± 1.3 – 30 (66%) 23 7 (23%)* 545 ± 41

Wistar LDP 25 – 48 ± 16 17 (68%) 10 7 (41%) 552 ± 60

Sham 19 – – – – – 556 ± 49

Pilo non-SE 22 – – – – – 561 ± 46

1. Number of low dose pilocarpine injections (10 mg/kg) administered in the RLDP protocol (average ± SD).

2. Latency to SE onset in the LDP protocol (average ± SD). 3. Number of animals that developed SE for 60 min.

Numbers in () represent % of animals that entered SE. 4. Number of animals that surivived or died within 3 months

following SE. Numbers in () represent the % of animals that died. * Lower mortality rate detected in rats treated

with the RLDP protocol compared with the LDP protocol p<0.005, **p<0.10. 5. Weight in grams (average ± SD) at

3.5 months after SE. Data assessed using one-way ANOVA or chi-square analysis.

152

6.3.2 Open field test

The open field data was first assessed by repeated measures ANOVA followed by the Newman-

Keuls post-hoc test to determine group differences. No significant effect in locomotor activity as

assessed by the total number of squares crossed between SE and non-SE groups, week at which

the test was conducted, and no significant interaction between test week and group was detected.

These results indicate no presence of motor impairment between groups or between testing

sessions. However, there were group differences in rearing. SE groups exhibited a lower

frequency and a lower duration of rearing activity when compared to non-SE groups within the

first week after SE, and this difference in behaviour remained unchanged in the second and

twelfth week (Figure 6.2, A,B). No difference was detected between SE groups and non-SE

groups in the number of entries into the centre, or the number of centre squares crossed (Figure

6.2, C,D). Instead, all groups showed substantially higher locomotor activity in the peripheral

region of the open field (Figure 6.2, E). Rats treated with either the LDP or the RLDP seizure-

inducing protocol exhibited similar performance in the open field test (Figure 6.3).

153

Figure 6.2: The effect of SE on behaviour in the open field. SE was induced with either the

RLDP (n=27) or the LDP (n=10) procedure and terminated after 60 min as in Methods section

6.2.2. Non-SE control groups included ‗pilo no-SE‘ rats (n=22) treated with pilocarpine and did

not develop SE, and ‗sham‘ rats (n=19) treated with saline instead of pilocarpine. Behaviour in

the open field assessed 1 week (black bars), 2 weeks (grey bars) and 12 weeks (oblique-striped

bars) after SE (see Methods section 6.2.3.2). (A) Illustrates the frequency of rearing during the

10-min trial. (B) Illustrates the total duration of rearing activity (sec). (C) Illustrates the

frequency of entries (with four paws) into the centre square. (D) Illustrates number of centre

squares crossed (with four paws). (E) Illustrates number of peripheral squares crossed (with four

paws). (F) Illustrates total number of squares crossed. * Significant difference from non-SE

control groups across all three times assessed (p<0.05). Bars represent group average ± SEM.

Data analyzed by one-way ANOVA followed by Newman-Keuls post-hoc test.

154

155

6.3.3 Hyperexcitability tests

Because behaviour in the hyperexcitability tests was assessed using a 3- to 6- grade ordinal scale,

data from each week assessed was first analyzed using the non-parametric Kruskall-Wallis

ANOVA by ranks, followed by the Dunn‘s test to determine individual group differences.

Among the four hyperexcitability tests described by Rice et al., (1998), SE groups and non-SE

groups exhibited similar responses on the approach-response test and the finger-snap test (Figure

6.3, A, C). By contrast, a clear difference was observed in the touch-response test in that the SE

groups were significantly more touch-sensitive than the non-SE groups (Figure 6.3, B). In

addition, the SE groups reacted more aggressively than the non-SE groups in the pick-up test

(Figure 6.3, D). The difference in responses between SE and non-SE groups was detected within

the first week after SE, and persisted in the second and twelfth week. The nonparametric

Friedman‘s test was used to measure the performance stability for each group all along the 3

testing sessions. This analysis showed that for each group, animal responses in the first week did

not change in the second and twelfth week in any of the four hyperexcitability tests. The

behaviour in the hyperexcitability tests of the RLDP SE group was indistinguishable from the

LDP SE group.

156

Figure 6.3 The effect of SE on behaviour in the four hyperexcitability tests. SE was induced

with either the RLDP (n=27) or the LDP (n=10) procedure and terminated after 60 min as in

Methods section 6.2.2. Non-SE control groups included ‗pilo no-SE‘ rats (n=22) treated with

pilocarpine and did not develop SE, and ‗sham‘ rats (n=19) treated with saline instead of

pilocarpine. Behaviour in the hyperexcitability tests assessed 1 week (black bars), 2 weeks (grey

bars) and 12 weeks (oblique-striped bars) after SE. See Methods section 6.2.3.4 for behavioural

responses assessed in the following tests: (A) approach-response test; (B) touch-response test;

(C) finger-snap test; (D) pick-up test. * Significant difference from non-SE control groups

across all three times assessed (p<0.05). Bars represent average ± SEM. Data analyzed by

Kruskal-Wallis (ANOVA for non-parametric data) test followed by Dunn‘s test for individual

differences.

157

158

6.3.4 Elevated-plus maze:

Behaviour in the elevated-plus maze was determined 12 weeks following SE and data was first

analyzed using one-way ANOVA. The Newman-Keuls post-hoc test detected differences

between SE groups and non-SE groups. Similar to the findings in the open field test, SE groups

showed attenuated rearing activity when compared to non-SE groups in the elevated plus maze

(Figure 6.4, A). The frequency of risk assessments was also decreased in the SE groups,

indicating decreased exploratory behaviour (Figure 6.4, B). Non-SE groups spent most of their

time within the protected closed arms of the elevated-plus maze (Figure 6.4, C-F). However,

unlike controls, the SE groups spent a greater amount of time in the open unprotected arms, and

had fewer entries into the closed arms of the maze (Figure 6.4, C-F), indicating decreased

anxiety-like behaviour. Rats treated with either the LDP or the RLDP protocol had similar

behavioural responses in the elevated-plus maze.

Some SE groups failed to remain on the elevated-plus maze, and fell off the ledge of the open

arm in two separate 5-min trials. The results from these incomplete trials were eliminated from

the final analyses. Chi-square analysis revealed no statistically significant difference between

SE groups in terms of the number of rats excluded: 3 from the ‗LDP SE‘ experimental group, 6

from the ‗RLDP SE‘ (or ‗saline SE‘) group, 2 from the ‗tat-NR2B9c SE‘ group, and 3 from the

‗tat-NR2BAA SE‘ group. All rats in the non-SE groups remained on the elevated-plus maze

during the duration of testing.

159

Figure 6.4 The effect of SE on behaviour in the elevated-plus maze. SE was induced with

either the RLDP (grey bar, n=21) or the LDP (checker-patterned bar, n=7) procedure and

terminated after 60 min as in Methods section 6.2.2. Non-SE control groups included ‗pilo no-

SE‘ rats (white bar, n=22) treated with pilocarpine and did not develop SE, and ‗sham‘ rats

(black bar, n=19) treated with saline instead of pilocarpine. Behaviour in the elevated-plus maze

assessed 12 weeks after SE (see Methods section 6.2.3.3). (A) illustrates the frequency of

rearing during the 5-min trial. (B) illustrates the frequency of lookouts (with two paws in the

open arm, head extended over the ledge). (C) illustrates the frequency of entries (with four paws)

into the open arm. (D) illustrates the frequency of entries (with four paws) into the closed arm.

(E) illustrates time spent in the open arm (sec). (F) illustrates time spent in the closed arm (sec).

* Significant difference from non-SE control groups (p<0.05). ** Significant difference from

sham group. Bars represent average ± SEM. Data assessed by one-way ANOVA followed by

Newman-Keuls multiple comparison post-hoc test.

160

161

6.3.5 The effect of Tat-NR2B9c on behaviour following SE

In chapter 5, we demonstrated that tat-NR2B9c is neuroprotective within the CA1, CA3 and CA4

pyramidal cell layers of the dorsal hippocampus. Because the dorsal hippocampus is involved in

behaviours such as anxiety and exploration (Davis, 1992; Moreira et al., 2007; Carvalho et al.,

2008; Degroot and Treit, 2002), we determined whether the neuroprotection provided by tat-

NR2B9c would mitigate SE-induced behavioural morbidity. The results showed that

administration of tat-NR2B9c did not alter the behavioural outcomes in the open field test (figure

6.5), the hyperexcitability tests (figure 6.6) or the elevated-plus maze test (figure 6.7). Data for

each test was analyzed as described above.

162

Figure 6.5 Tat-NR2B9c had no effect on behaviour in the open field. In drug-treated

animals, tat-NR2B9C (n=16), tat-NR2BAA (n=19) or saline (n=27) was administered 3 hr

following the termination of SE induced by the RLDP procedure (see Methods section 6.2.2).

Shams (n=19) received saline instead of pilocarpine. Behaviour in the open field assessed 1

week (black bars), 2 weeks (grey bars) and 12 weeks (oblique-striped bars) after SE (see section

6.2.3.2). (A) illustrates the frequency of rearing during the 10-min trial. (B) illustrates the total

duration of rearing activity (sec). (C) illustrates the frequency of entries (with four paws) into the

centre square. (D) illustrates number of centre squares crossed (with four paws). (E) illustrates

number of peripheral squares crossed (with four paws). (F) illustrates total number of squares

crossed. * Significant difference from shams across all three times assessed (p<0.05). Bars

represent average ± SEM. Data analyzed by one-way ANOVA followed by Newman-Keuls

post-hoc test.

163

164

Figure 6.6 Tat-NR2B9c had no effect on behaviour in the four hyperexcitability tests. In

drug-treated animals, tat-NR2B9C (n=16), tat-NR2BAA (n=19) or saline (n=27) was

administered 3 hr following the termination of SE induced by the RLDP procedure (see Methods

section 6.2.2). Shams (n=19) received saline instead of pilocarpine. Behaviour in the

hyperexcitability tests assessed 1 week (black bars), 2 weeks (grey bars) and 12 weeks (oblique-

striped bars) after SE. See section 6.2.3.4 for behavioural responses assessed in the following

tests: (A) approach-response test; (B) touch-response test; (C) finger-snap test; (D) pick-up test.

* Significant difference from shams across all three times assessed (p<0.05). Bars represent

average ± SEM. Data analyzed by Kruskal-Wallis (ANOVA for non-parametric data) test

followed by Dunn‘s test for individual differences.

165

166

Figure 6.7 Tat-NR2B9c had no effect on behaviour in the elevated-plus maze. In drug-

treated animals, tat-NR2B9C (black bars, n=14), tat-NR2BAA (oblique-striped bars, n=16) or

saline (grey bars, n=21) was administered 3 hr following the termination of SE induced by the

RLDP procedure (see Methods section 6.2.2). Shams (white bar, n=19) received saline instead

of pilocarpine. Behaviour in the elevated-plus maze assessed 12 weeks after SE (see section

6.2.3.3). (A) illustrates the frequency of rearing during the 5-min trial. (B) illustrates the

frequency of lookouts (with two paws in the open arm, head extended over the ledge). (C)

illustrates the frequency of entries (with four paws) into the open arm. (D) illustrates the

frequency of entries (with four paws) into the closed arm. (E) illustrates time spent in the open

arm (sec). (F) illustrates time spent in the closed arm (sec). * Significant difference from shams

(p<0.05). Bars represent average ± SEM. Data assessed by one-way ANOVA followed by

Newman-Keuls multiple comparison post-hoc test.

167

168

6.4 Discussion

In the present study, the behavioural responses of rats were assessed at 1 week, 2 weeks and 12

weeks following SE in the open field test and in four hyperexcitability tests, and at 12 weeks in

the elevated plus maze test (described in Methods section 6.2.3). The study showed (1) that SE

in rats causes anxiolytic changes in behaviour when assessed in the open field and elevated-plus

maze, and increased hyperexcitability in the touch-response test and in the pick-up test; (2) these

behavioural changes appear within the first week after SE and persist with the occurrence of

SRSs and; (3) treatment with tat-NR2B9c did not modify the post-SE behavioural response.

These findings are discussed below.

6.4.1 SE causes anxiolytic changes in behaviour and increased hyperexcitability

In the present study, we demonstrated that rats following SE showed anxiolytic changes in

behavior and increased hyperexcitability. Although previous findings have reported behavioural

changes in rats following SE (Detour et al., 2005; dos Santos et al., 2005; Szyndler et al., 2005;

Brandt et al., 2006; Cardoso et al., 2009; Groticke et al., 2008), there are some differences

between our observations and previously published results. In the open field and elevated-plus

maze, we observed decreased rearing activity following SE. This observation is similar to the

findings reported in Dos Santos et al., (2006) with Wistar rats using high-dose pilocarpine (320

mg/kg), and in Groticke et al., (2008) with mice using intrahipppocampal injection of kainate,

and has been interpreted as indicating that rats following SE display hyporeactivity to a stressful

environment (dos Santos et al., 2005; Groticke et al., 2008). On the contrary, elevated rearing

activity in Sprague Dawley rats was reported after SE induced by the low-dose pilocarpine

procedure (Detour et al., 2005), and after self-sustaining status epilepticus (SSSE) induced by

electrical stimulation of the basolateral amygdaloid nucleus (Brandt et al., 2006); in these

studies, increased rearing activity was suggested to reflect disinhibited hyperactive behaviour

(dos Santos et al., 2005). Although our results and previous findings demonstrated anxiolytic

behavioural changes in the elevated-plus maze, with SE groups spending a greater amount of

time in the unprotected open arm (dos Santos et al., 2005; Detour et al., 2005; Brandt et al.,

2006; de Oliveira et al., 2008; Cardoso et al., 2009), Groticke et al., (2008) failed to find

behavioural changes with this task in SE mice. Additional behavioural responses not observed in

the current study but previously reported include increased locomotor activity (Milgram et al.,

169

1988; Santos et al., 2000; Kubova et al., 2004; Sayin et al., 2004; Detour et al., 2005; dos Santos

et al., 2005; Szyndler et al., 2005; Brandt et al., 2006; Müller et al., 2009; Cardoso et al., 2009;

Sun et al., 2009), and elevated activity in the central square of the open field (Detour et al., 2005;

Szyndler et al., 2005; Brandt et al., 2006).

For the hyperexcitability tests, our results are similar to Rice et al., (1998) with Sprague-Dawley

rats using high-dose pilocarpine (320 mg/kg), demonstrating that SE groups scored significantly

higher on the touch-response and pick-up tests. In contrast, Brandt et al.,(2006) only detected a

significant difference in the touch-response test in Sprague-Dawley rats after SSSE induced by

electrical stimulation of the basolateral amygdaloid nucleus. Because rat strain or use of

different seizure protocols have been shown to result in differences of behavioural outcomes

(Cardoso et al., 2009; Hort et al., 2000), these factors may account for the differences between

our findings and previously published results. An alternative possibility is that our results differ

from others because the rats were retested multiple times in the open field and in the

hyperexcitability tests. Previous studies have shown that repeated testing in the open field can

result in decreased open field activity directed to searching the environment, and is considered an

adaptive response (Stafstrom et al., 1993; Erdogan et al., 2005). However, we detected no

behavioural differences between test times in SE and non-SE animal groups, and consider that

the interval between each test session was long enough not to cause habituation in the open field

or in the hyperexcitability tests.

In chapter 3, we demonstrated that rats treated with the RLDP protocol exhibited a lower

mortality rate and less severe seizure activity compared to rats treated with the LDP protocol.

Despite these differences, we found that the RLDP SE groups and the LDP SE groups exhibited

similar behavioural responses in the elevated-plus maze, open field and hyperexcitability tests.

The significance of these findings is discussed in greater detail in section 7.4.1, but in general

they support the conclusion that the RLDP protocol is a better method for generating seizures.

Although the open field and elevated-plus maze both measure behavioural responses to anxiety

and stress in rodents, we found that the elevated-plus maze was the more sensitive test in

distinguishing SE groups from controls. As discussed above, we found SE groups to be different

from non-SE groups in several behavioural measures assessed with the elevated-plus maze.

Briefly, these differences consisted of SE groups exhibiting a decrease in rearing activity, a

lower frequency of outlooks, less entries and time spent in the closed arm, and increased entries

170

and time spent in the open arm. Although rearing activity in SE groups was attenuated in the

open field, other parameters assessed (i.e., entries and activity in the center of the maze) were

found to be similar to non-SE groups. In general, we found that with the lithium/pilocarpine

model, the elevated-plus maze was more sensitive in detecting behavioural alterations in rats

caused by SE than the open field.

6.4.2 Behavioural changes in rats following SE are long-lasting

SE-induced behavioural alterations in the hyperexcitability tests and in the open field test

appeared within the first week after SE and were still apparent 12 weeks later, indicating that

these changes were unaltered by development of SRSs. Our results are in agreement with Rice

et al., (1998) who detected behavioural changes in the touch-response and pick-up tests as early

as 2 days after SE. Severe and persistent behavioural changes are also reported in other seizure

models. For instance, kainic acid-treated rats showed increased hyperreactivity in response to

handling and increased activity in the open field 24-hours after SE, with behavioural changes

present 2 months later (Milgram et al., 1988). Many authors have suggested that early

behavioural changes are caused by neuronal injury resulting from continuous seizure activity

persisting longer than 30 minutes (Milgram et al., 1988; dos Santos et al., 2005; Detour et al.,

2005; Szyndler et al., 2005; Brandt et al., 2006; Groticke et al., 2008). The hippocampus and

amygdala strongly mediate the unconditioned fear response resulting from exposure to a

threatening situation (Pitkanen et al., 2000; Kjelstrup et al., 2002). The ventral hippocampus

projects to the prefrontal cortex and is closely connected to the bed nucleus of the stria terminalis

and the amygdala, as well as other subcortical structures which are associated with the

hypothalamic-pituitary-adrenal axis (Pitkanen et al., 2000; Bannerman et al., 2004). Most of the

amygdaloid nuclei have some reciprocal projections with the hippocampal formation, although

this is most pronounced for the basal and lateral nuclei (Pitkanen et al., 2000; Bannerman et al.,

2004). Detour et al., (2005) suggested that disruption of these networks which are involved in

fear expression causes misevaluation of threatening situations, which could in turn reduce

anxiety (as we observed in the open field and elevated-plus maze), and enhance impulsive

maladapted behaviour (as we observed in the hyperexcitability tests).

171

Because we conducted an intensive time-course evaluation of SE-induced neurodegeneration

(refer to chapter 4), we were able to compare the progression of neuron loss with the occurrence

of behavioural changes in rats following SE. We demonstrated that extensive neuronal loss

occurs within the hippocampus, amygdala, thalamus and piriform cortex as early as 3 hrs after

SE, with the majority of damage present by 3 days (see chapter 4, section 4.3.2). The acute loss

of neurons we observed in rats after SE is consistent with the early development of behavioural

deficits described by Rice et al., (1998), by Milgram et al., (1988), and with our results in the

present study (see above). Although we are unable to link neuropathology with behavioural

disruption, our data suggest that the occurrence of neuronal loss caused by SE is accompanied by

early behavioural disturbances, and that these behavioural changes are not altered by the

occurrence of SRSs.

6.4.3 Treatment with tat-NR2B9c did not have neuroprotective effects as assessed behaviourally.

In Chapter 5, we reported that administration of tat-NR2B9c resulted in decreased loss of

pyramidal cells in the CA1, CA3, and CA4 subfields of the dorsal hippocampus. In the present

study, we investigated whether this protection was associated with reversal of SE-induced

behavioural changes. Although traditionally associated with learning and memory, recent

studies show that the dorsal hippocampus also exhibits a modulatory role in anxiety-related

behaviours. For instance, pharmacological stimulation of the GABAergic, serotonergic or

cholinergic receptor systems in the dorsal hippocampus produces robust anxiolytic effects in

several animal models of anxiety (Engin and Treit, 2007). Similarly, lesion studies by

Bannerman et al., (1999) in either dorsal or ventral hippocampus produces anxiolytic effects in

the elevated-plus maze (Bannerman et al., 1999). More specifically, Menard et al., (2001)

demonstrated that the pyramidal cells within the dorsal hippocampus sends glutamatergic

efferents to the septal nucleus, and that these regions act in concert to regulate some (open arm

avoidance in elevated-plus maze test) but not all (e.g. shock-probe burying test) anxiety-related

behaviours (Menard and Treit, 2001). Despite the evidence that the dorsal hippocampus is

involved in anxiety-related behaviours, we found that the degree of neuroprotection provided by

tat-NR2B9c within this region had no effect on any of the behavioural tests analyzed.

While some studies support a role for neuroprotection in the improvement of behavioural

functioning, other studies have failed to establish this connection. Congruent with our findings,

172

Narkilahti et al., (2003b) demonstrated that although inhibition of caspase-3 after SE provided

partial protection to CA1 and CA3c, it did not modify behavioural deficits as assessed in the

open field and elevated-plus maze. In contrast, Brandt et al., (2006) demonstrated that nearly

complete neuroprotection within the hippocampus in valporate-treated rats reduced anxiolytic

effects in the open field, in the elevated-plus maze and in the touch-response test. Dos Santos et

al., (2005) also demonstrated that treatment with cycloheximide four hours after SE prevented

tissue shrinkage of the dorsal hippocampus, and spared behavioural functioning in the open field

and in the elevated-plus maze. A possible explanation for the difference in findings between

these studies may be related to variations in the amount of protection under the different

conditions. While Brandt et al., (2006) and dos Santos et al., (2005) observed nearly complete

preservation of the dorsal hippocampus, only partial protection was described by Narkilahti et

al., (2003b) and by us (see chapter 5). It is also possible that the various treatments resulted in

differential protection of other brain regions relevant to the behaviours analyzed, and/or affected

other neurpathological factors that may contribute to altered behaviour (i.e., neurogenesis and

synaptic plasticity).

The administration of Tat-NR2B9c did not prevent the development of SRSs, in spite of its

neuroprotective effect. All rats that developed SE were determined to be epileptic within 3

months regardless of whether or not they received the peptide. This result is in line with several

other studies in which neuroprotection did not prevent the development of epilepsy (Andre et al.,

2001; Brandt et al., 2003b; Brandt et al., 2006), and indicates that other factors such as axonal

sprouting, neurogenesis and/or synaptic reorganization may play a more important role in the

development of SRSs (Curia et al., 2008). The effect of tat-NR2B9c on these processes has yet

to be assessed.

6.4.4 Conclusion

In the present study, we found that treatment of rats with lithium and pilocarpine produced long-

lasting behavioural modifications that appeared within the first week after SE, and remain

unchanged by development of SRSs at 3 months. These modifications consisted of decreased

exploratory behaviour as assessed in the open field test, and increased hyperreactivity as assessed

in the pick-up test and in the touch-response test. SE also produced anxiolytic effects as

measured in rats by the elevated-plus maze at 3 months following treatment. The behavioural

173

effects were accompanied by histopathological changes in the brain structures (i.e.,

hippocampus, thalamus, amygdala, and piriform cortex) involved in the control of exploration

and anxiety. However, further research is necessary to establish the link between early SE-

induced histopathological changes and behavioural alterations.

In sum, the obtained results indicate that lithium/pilocarpine-induced SRSs are also accompanied

by profound behavioural disturbances, and thus could model anxiety-related disorders observed

in humans with TLE.

174

Chapter 7

The effect of SE on performance in the Morris water maze and use of exploratory strategies

7.1 Introduction

Impaired learning and memory is common in patients with epilepsy (Motamedi and Meador,

2003; Vingerhoets, 2006). The type of neuropathology associated with epilepsy may affect the

type of cognitive dysfunction. For instance, temporal lobe epilepsy (TLE) with hippocampal

sclerosis is often associated with memory impairment (Motamedi and Meador, 2003). The most

reliable observations in TLE patients are deficits in declarative memory (ability to acquire facts

and events related to one‘s personal past, which is often compared with visual-spatial learning in

rats; (Guerreiro et al., 2001) and in the performance of visuospatial tasks (Hermann et al., 1997;

Gleissner et al., 1998; Abrahams et al., 1999)). When administered systemically pilocarpine

results in a condition similar to human TLE (Honchar et al., 1983; Turski et al., 1989; Persinger

et al., 1993; Curia et al., 2008). Rats receiving pilocarpine undergo convulsive status epilepticus

(SE), which is followed by development of spontaneous recurrent seizures (SRSs), as well as

memory and learning impairment (Persinger et al., 1993; Rice et al., 1998; Curia et al., 2008).

The Morris water maze (MWM) has frequently been used to investigate spatial learning and

memory (Morris, 1984; Vorhees and Williams, 2006). During testing, a rat is placed into a pool

of water and swims to find a hidden (submerged) platform. It is now recognized that acquisition

of the water maze task has two main components: (1) procedural–strategy learning and (2)

spatial learning (Morris and Frey, 1997; Inglish and Morris, 2004). The first component

involves learning behavioural strategies that allow the rat to move around in its spatial

environment and to learn the most effective strategies for finding and reaching its target. To

accomplish this, the rat must learn to search away from the pool wall (where the escape platform

is never placed), realize that the escape platform offers the only refuge in the pool, and suppress

attempts to climb out of the pool by clawing at the wall (Whishaw, 1989). During spatial

learning the rat builds a spatial cognitive map correlating context information (extramaze cues)

with platform location (Morris, 1984; DiMattia and Kesner, 1988; Fenton and Bures, 1993).

Once this map is created, the rat is able to swim directly to the escape platform from any point of

the circumference of the tank. Success in the spatial learning component requires prior learning

175

of the procedural-strategy component (Morris, 1989;; Whishaw, 1989; Saucier and Cain, 1995;

Bannerman et al., 1995; Baldi et al., 2003).

Although previous studies have showed impaired spatial learning during MWM testing in rats

following SE (Persinger et al., 1993; Rice et al., 1998; Hort et al., 1999; Hort et al., 2000; Wu et

al., 2001; McKay and Persinger, 2004; dos Santos et al., 2005; Frisch et al., 2007; Zhou et al.,

2007; Cunha et al., 2009;), little attention has been given to the effect of SE on search strategy

use. In the present study, we conducted a more refined analysis that included assessment of the

acquisition and application of search strategies in SE rats for several reasons. First, there appear

to be separate neural systems required for spatial learning and procedural-strategy learning

(McDonald and White, 1994; McDonald and White, 1993; Miranda et al., 2006), and both

systems are involved in acquisition of the MWM task. Evidence strongly supports the role of the

hippocampus and posterior parietal cortex in spatial mapping and memory (Cain et al., 2006).

Conversely, the prefrontal cortex, caudate nucleus, cerebellum and medial thalamus appear to be

involved in acquisition of procedural-search strategies (Packard and McGaugh, 1996; Leggio et

al., 1999; Cain et al., 2006). Because all of these brain regions are damaged in rats following SE

(Turski et al., 1983b; Clifford et al., 1987; Fujikawa, 1996;), the impaired spatial learning of

these animals is most likely attributed not only to poor spatial memory, but also to impaired use

of search strategies. Second, Brody et al., (2006) demonstrated that poor search strategy use

largely contributed to impaired MWM performance in transgenic mice following traumatic brain

injury (TBI). Since TBI results in diffuse brain damage comparable to the pattern of injury we

reported in rats following SE (see chapter 4), use of less efficient search strategies may similarly

underlie impaired MWM performance in SE animals. Finally, the clinical relevance of this

analysis is supported by impaired cognitive strategy use detected in humans following traumatic

brain injury (Kennedy et al., 2003; Burke et al., 2004; Salmond et al., 2005) and in patients with

TLE (Kennedy et al., 2003; Burke et al., 2004; Salmond et al., 2005; Butman et al., 2007;

Labudda et al., 2009). Our first objective was therefore to investigate the effect of SE on

performance in the MWM, with particular emphasis on search strategy use. We analyzed the

predominate search strategy used in each trial during hidden platform testing according to the

schema proposed by Graziano et al., (2003).

In chapter 3, we demonstrated that rats treated with the RLDP protocol exhibited less severe

seizures and had a lower mortality rate compared to rats treated with the LDP protocol. To

176

determine whether the advantage in using the RLDP protocol to reduce mortality results in

similar cognitive and behavioural changes as compared to the LDP protocol, our second

objective was to compare the MWM performance of rats following SE induced by the two

procedures. Finally, studies have reported that neuroprotection within the hippocampus

following SE improves performance in visual-spatial learning (Rice et al., 1998; Yang et al.,

2007; Cunha et al., 2009; Jun et al., 2009). Because we demonstrated neuroprotection within the

CA1, CA3 and CA4 subfields of the dorsal hippocampus following administration of tat-

NR2B9c (see chapter 5), brain regions implicated in spatial learning (Broadbent et al., 2004;

Gaskin et al., 2009), our third objective was to determine whether protection within these regions

would improve MWM performance in SE rats.

7.2 Methods

7.2.1 Animals



Animal Care. Male Wistar rats (Charles River Laboratories, Sherbrooke, Quebec, Canada)

weighing between 300 and 350 g were individually housed with free access to food and water for

at least 7 days in 12 h light/dark cycles before experimental use. All experiments were

conducted during daylight hours between 7 a.m. and 7 p.m.

7.2.2 Induction of status epilepticus and administration of peptides

SE was induced using both the low-dose pilocarpine (LDP) and the repeated low-dose

pilocarpine (RLDP) procedures, and peptides (tat-NR2B9c or tat-NR2BAA) were administered

as described in chapter 6 (Methods section 6.2.2). Saline control rats were treated identically

except that they received saline (0.3 ml) following SE in place of the peptide. The non-SE

control groups consisted of the following: ‗Pilo no-SE‘ rats (n=22) were treated with the RLDP

protocol and did not develop SE. Shams (n=19) received saline instead of pilocarpine. SE-

inducing protocols and experimental animal groups are shown in Figure 6.1. SE and non-SE

groups used in the present study are the same animals used in chapter 6, and are illustrated in

Figure 6.1. Animals were tested in behavioural tasks (i.e., elevated plus maze, open field,

hyperexcitability tests) described in chapter 6 on separate days and prior to commencement of

MWM testing.

177

7.2.3 Morris water maze testing

7.2.3.1 Pre-observational period to behavioural testing

MWM testing began 3 months after SE induction. All rats were observed for recurrent seizures

at least 1 hr prior to testing. If the rat was seizure-free within the observation period, the animal

was tested as described below. If a rat exhibited recurrent seizures within the observation period,

than the test was postponed for at least 3 hours, at which time the rat was again observed for

recurrent seizures. On the rare occasion that a rat exhibited a seizure during testing, the trials for

that day were removed from the final analysis. No difference in the number of trials removed

from each experimental group was detected: in RLDP (or saline-treated) SE rats, a total of 24

trials (or 1% of trials) were removed; in LDP SE rats, 18 trials (or 2.1% of trials); in tat-

NR2B9c-treated SE rats, 24 trials (or 1.8% of trials); in tat-NR2BAA-treated SE rats, 18 trials

(or 1.2% of trials).

7.2.3.2 Apparatus

The water maze was a blue circular pool (2 m diameter) located in the centre of a room, with

distal visual cues (geometric shapes, posters) on the walls and features (doors, cabinets, shelving,

etc.) that served as visual cues. The pool was filled with water (19 - 22°C) and made opaque by

adding non-toxic white paint. Four regions surrounding the pool were designated as north (N),

east (E), south (S) and west (W), dividing the pool into four quadrants: NE, SE, NW and EW

(not true compass headings). A clear Plexiglas escape platform (16.5 cm height, 5.5 cm

diameter) placed in the centre of a quadrant was used to allow the rat to escape from the water.

7.2.3.3 Visible platform (cued) testing

This portion of the test controls for differences between groups in motivation to escape from the

water, swimming ability, and visual acuity (Vorhees and Williams, 2006). On the first day of

testing, a 60-s free swim with no platform was performed to familiarize the animals to the pool.

During visible platform learning, the escape platform was elevated 2.5 cm above the water and

cued with a multi-coloured ‗flag‘ that was extended by 12 cm above the water. The platform

was rotated between the NE, NW and SE quadrants of the pool during subsequent trials in a

pseudorandom order, as determined by the Research Randomizer Form V4.0 computer program.

Each rat was tested with 6 trials per day, for up to 3 consecutive days. At the beginning of each

trial, the rat was gently grasped around the midsection and lowered into the pool facing the SW

178

wall. The experimenter subsequently left the room and was not present during testing. During

the 60-s inter-trial intervals and following testing, rats were towel dried and placed next to a

space heater. Each trial lasted a maximum of 90 s, and at the end of each trial, the rat was guided

to the platform and/or allowed to stay on the platform for 15 s. The criteria to pass visual acuity

consisted of the following: 1) the rat located the platform three consecutive times in less than 30

seconds, or 2) the rat located the platform in less than 30 seconds in 4 out of the 6 trials, and

located the platform in the remaining 2 trials. Once the rat passed visual acuity, it was not

required to repeat the task on the following day. Animals that failed to pass this test were

removed from the study. Both the latency to platform (i.e. the amount of time necessary for the

rat to reach the platform) and swimpaths were recorded as described in Methods 7.2.3.7.

7.2.3.4 Hidden platform testing

During spatial acquisition, the rats must learn to use distal cues to navigate a direct path to the

hidden platform (Vorhees and Williams, 2006). For these tests, the platform was submerged 2.5

cm beneath the surface of water, and remained in a constant position. The platform was placed

in the SW quadrant of the pool for 56% of the rats, and in the NE quadrant for the remaining

animals. A total of 6 trials per day for each animal were conducted over 14 consecutive days.

Rats were placed twice into each of the 3 quadrants that did not contain the platform in a

pseudorandom order. Both the path taken and latency to platform were recorded (see Methods

section 7.2.3.7). Each trial lasted a maximum of 60 s, and at the end of each trial, the rat was

guided to the platform and/or allowed to stay on the platform for 15 s. The score for each animal

was calculated as the average time of the 6 trials.

Spatial reversal evaluates the ability of the rat to extinguish its initial learning of the platform

path (spatial acquisition), and acquire a direct path to the new goal position (Vorhees and

Williams, 2006). The protocol was the same as described above for spatial acquisition, except

that the platform was relocated to the opposite quadrant, and testing was conducted over 5, rather

than 14, consecutive days. Spatial reversal was conducted the day following the probe test for

spatial acquisition (see Methods 7.2.3.5)

179

7.2.3.5 Probe trials

Probe trials assess reference memory and are determined by preference for the platform area

when the platform is absent (Vorhees and Williams, 2006). The probe trials were administered

24 hr following the last day of the spatial acquisition test and the spatial reversal test. Rats were

tested for only a single 60 s trial, and were lowered into the opposite quadrant from where the

platform was located during spatial learning. The number of crossings in the area where the

platform was previously located in each trial was recorded.

7.2.3.6 Search strategy analysis

Explorative strategies were classified by two independent investigators, blinded to treatment of

the animals, into seven prototypical behavioural categories as defined by Graziano et al., (2003)

(Figure 7.1). The data analyzed in the present study were based on the swim paths categorized

by the principle investigator, and had a 96% inter-observer agreement on strategy classification.

In reanalysis of a subset of trials by the principle investigator 1 month later, the intra-observer

agreement on strategy classification was 98%. Consequently, we were confident that the swim

paths could be reliably sorted into one of the seven search strategies (Graziano et al., 2003). As

illustrated in Figure 7.1, the seven behavioural categories were divided into 3 broader categories

that included spatial strategies, systematic non-spatial strategies and repetitive looping-based

strategies (Brody and Holtzman, 2006).

Spatial strategies include the following categories: 1) spatial direct, which consists of swimming

directly to the platform, 2) approaching, characterized by adjusting trajectory while approaching

the platform, and 3) self-orienting, characterized by approaching and missing the platform by

several degrees, followed by returning to the start point and making another attempt to reach the

platform, or briefly re-orienting elsewhere in the pool and then reaching the platform. Spatial

strategies rely on distal cues to efficiently reach the platform.

Non-spatial, systematic strategies do not use distal cues to locate the platform, but instead

involve procedural strategies (i.e. swimming away from the pool wall) that increase the

likelihood of locating the platform. The following categories are defined as non-spatial,

systematic strategies: 1) scanning, whereby the searching pattern is focused primarily on the

center of the pool, and the subject swims back to the centre of the pool immediately if it touches

180

the edge and 2) random searching, in which the rat moves in jagged paths through the entire

pool, with sudden changes in direction.

Strategies involving repetitive looping do not involve the use of distal cues and are the least

efficient in locating the platform, They consisted of the following two categories: 1) circling, in

which the rat moves away from the wall forming circular trajectories, but often remains too close

to the periphery and often fails to locate the platform, and 2) thigmotaxis, characterized by

swimming almost exclusively at the periphery and repeatedly searching for contact with the tank

wall. Swim paths in which the rat formed tight circles with some net directional movement were

also categorized as thigmotaxis.

181

Figure 7.1 Behavioural categories. Each row illustrates a few prototypical tracks for each

search strategy.

182

183

7.2.3.7 Data Acquisition

A digital video camera mounted to the ceiling directly above the center of the pool was relayed

to a tracking system (SMART, San Diego Instruments) in a separate room. During Morris water

maze testing, escape latency (s) and swim speed (cm/s) were recorded. Swim paths were traced

with a mouse on the monitor during testing in a separate room from where the water maze tank

was located, and later classified as described in section 7.2.3.6.


Statistical analysis was performed using Statistica 6.0 software. For Morris water maze

performance data, repeated measures ANOVAs were used. For probe trial data, assessment was

performed by one-way ANOVAs. The Newman-keuls post-hoc test was used to determine

difference between treatment groups. For comparison of strategy use between groups, chi-square

analysis was performed. Significance was set at a p-value of 0.05 or less.

7.3 Results

7.3.1 SE induction

See Results section 6.3.1 and Table 6.1.

7.3.2 Visible platform testing

As illustrated in Figure 7.2, SE rats required a greater number of trials to pass the visible

platform test when compared to rats in both the ‗pilo no-SE‘ and ‗sham‘ control groups. Eight

SE animals failed to locate the platform by the end of 3 days and were eliminated from the study.

There were no differences between SE groups in terms of the number of rats excluded: 0 from

the ‗LDP SE‘ experimental group, 4 from the ‗RLDP SE‘ (or ‗saline SE‘), 1 from the ‗tat-

NR2B9c SE‘, and 3 from the ‗tat-NR2BAA SE‘ (p=0.47). Of the disqualified SE rats, some

consistently clung to the wall of the pool (classified as thigmotaxis), which may suggest sensory

or motor impairment in these animals, and/or exhibited frequent recurrent seizures during testing.

In contrast, thigmotaxis and circling in SE rats during hidden platform testing did not appear to

reflect a visual or motor deficit or a lack of motivation, because they performed successfully

during visible platform testing, during which they displayed incentive to escape from the pool,

and exhibited similar swim speed to corresponding non-seizure controls (Figure 7.4 C).

184

Figure 7.2 Number of trials performed to reach criterion during visible platform testing.

Criterion to pass visible acuity was assessed as described in Methods section 7.2.3.3. SE was

induced with either the RLDP (n=23) or the LDP (n=10) procedure and terminated after 60 min.

In drug-treated animals, tat-NR2B9C (n=16) or tat-NR2BAA (n=19) was administered 3 hr

following the termination of SE induced by the RLDP procedure (see Methods section 6.2.2).

‗Pilo no-SE‘ rats (n=22) were treated with the pilocarpine and did not develop SE. Shams

(n=19) received saline instead of pilocarpine. * different from shams and ‗pilo no-SE‘ control

groups (p<0.05). Data expressed as average ± SEM and analyzed by one-way ANOVA followed

by Newman-Keuls post-hoc.

185

186

7.3.3 The effect of SE on spatial acquisition

The position of the platform (i.e., SW or NE quadrant) during testing had no impact on the final

performance within any of the experimental groups. As illustrated in Figure 7.3, both SE groups

showed significantly greater latencies than the non-SE control groups over the 14-day testing

period. The sham and ‗pilo no-SE‘ control groups performed equally, with average escape

latencies of 21.0±2.2 s and 20.2±2.0 s in the initial 4 days of testing, reduced to 9.2±1.0 s and

9.9±1.2 s in the final 4 days, respectively. No significant difference in performance was detected

between the LDP and the RLDP SE rats (p=0.17), with the average escape latencies of 42.7± 2.8

s and 41.1± 3.6 s in the initial 4 days of testing, improving to 32.9± 4.0 s and 34.3± 4.7 s in the

final 4 days, respectively (Figure 7.3).

7.3.4 The effect of SE on spatial reversal

During spatial reversal, the hidden platform was placed in the opposite quadrant from the

quadrant in which it was located during spatial acquisition (Figure 7.3). In SE and non–SE

groups, an increase in latencies was observed on the first day of spatial reversal when compared

to the last day of spatial acquisition (Figure 7.3, compare day A14 with day R1); however,

reversal latencies remained lower than those for day 1 of spatial acquisition (Figure 7.3, compare

day R1 with day A1). Whereas both non-SE control groups exhibited reduced latencies by the

second day of reversal training, no improvement in performance was detected in either of the SE

experimental groups.

187

Figure 7.3 The effect of SE on hidden platform testing in the Morris water maze. SE was

induced with either the RLDP (red circles, n=23) or the LDP (blue circles, n=10) procedure and

terminated after 60 min as in Methods. ‗Pilo no-SE‘ rats (black diamonds, n=22) were treated

with the pilocarpine and did not develop SE. Shams (green triangles, n=19) received saline

instead of pilocarpine. Each data point represents the average of 6 trials per day (± SEM). Each

group was tested for 14 days of spatial acquisition (A1 – A14) and for 5 days of spatial reversal

(R1 – R5). # Performance in SE groups different from non-SE control groups (p<0.05).

* During acquisition training, latencies in final 4 days (A11-A14) were reduced compared to

initial 4 days (A1-A4) in SE groups and in non-SE control groups (p<0.05). ** Latencies

reduced in day R1 compared to day A1 in the RLDP SE group and in both non-SE control

groups (p<0.05). *** Latencies increased in day R1 compared to day A14 in SE and non-SE

control groups (p<0.05). ^ During reversal training, latencies reduced in day R5 compared to

day R1 in non-SE control groups (p<0.05). Data was analyzed by repeated-measure ANOVA.

Dashed lines represent the predicted performance of corresponding groups based solely on

search strategy use (see appendix III, section 3.1).

188

189

7.3.5 Acquisition and reversal probe tests

SE rats exhibited impaired spatial memory in the acquisition and reversal probe tests. As

illustrated in Figure 7.4 (A, B), rats following SE exhibited a lower number of platform crossings

compared to both non-SE control groups.

190

Figure 7.4 Number of platform crossings in spatial acquisition (A) and spatial reversal (B)

probe trials and swim speed (C). SE was induced with either the RLDP (n=23) or the LDP

(n=10) procedure and terminated after 60 min as in Methods section 7.2.2. In drug-treated

animals, tat-NR2B9C (n=16) or tat-NR2BAA (n=19) was administered 3 hr following the

termination of SE induced by the RLDP procedure (see Methods section 7.2.2). ‗Pilo no-SE‘

rats (n=22) were treated with the pilocarpine and did not develop SE. Shams (n=19) received

saline instead of pilocarpine. A, B: SE groups exhibit poor retention in location of the hidden

platform: * different from shams and ‗pilo no-SE‘ control groups (p<0.05). C: Swim speed was

similar across all animal groups during the probe tests. Black bars depict swim speed during the

spatial acquisition probe trial. Grey bars depict swim speed during the spatial reversal probe

trial. Data expressed as average ± SEM. Data analyzed by one-way ANOVA followed by

Newman-Keuls post-hoc.

191

192

7.3.6 Effect of SE on search strategy use during spatial acquisition

Because differences in escape latencies may reflect the type of search strategy used (Brody and

Holtzman, 2006), we next analysed the use of different search strategies by the various groups.

The distribution of each search strategy used in SE and non-SE groups is illustrated in Figure

7.5, and a comparison in search strategy use between groups is demonstrated in Figure 7.6. In

control groups, both the sham and the ‗pilo no-SE‘ rats employed similar strategies at all times

assessed: on day 1 of spatial acquisition, these animals used a high proportion of spatial

strategies (sham: 37%, pilo non-SE: 34%) and non-spatial systematic strategies (sham: 52%,

non-SE: 59%, Figure 7.6A, B), and a low fraction of repetitive looping-based strategies (Sham:

11%, Non-SE: 8%, Figure 7.6C). As their performance improved, control rats used spatial

strategies nearly exclusively (Figure 7.6, A). In contrast to non-SE rats, rats following SE

employed a lower proportion of spatial strategies and a greater proportion of non-spatial,

systematic and/or repetitive looping-based strategies on all days tested (p<0.05, Figure 7.6).

Over the 14-days of testing, RLDP SE and LDP SE rats increased their use of spatial strategies

and decreased their use of non-spatial, systematic strategies and repetitive-looping based

strategies (Figure 7.6).

7.3.7 Effect of SE on search strategy use during spatial reversal

The above results show that SE rats exhibit impaired use of search strategies when compared to

non-SE groups, but still increased the use of more efficient strategies over 14-days of spatial

acquisition. We next assessed the effect of the spatial reversal task on search strategy use by the

various groups. Following relocation of the submerged platform to the opposite quadrant, both

SE and non-SE groups exhibited a decrease in the use of spatial strategies, and an increase in the

use of non-spatial, systematic strategies (Figure 7.6, compare days A14 and R1).

During spatial reversal, non-SE rats used more efficient search strategies (i.e., a greater

proportion of spatial strategies and a lower proportion of non-spatial, systematic strategies and

repetitive looping-based strategies) compared to SE rats across all days assessed (Figure 7.6,

p<0.05). Both the sham and the ‗pilo no-SE‘ rats showed similar improvement in strategy use

over 5-days of testing, with spatial strategies being used nearly exclusively by day 5 (Figure

7.6A). Although RLDP SE rats exhibited an increase use of spatial strategies over this period

(Figure 7.6A), more than half of the strategies selected still consisted of non-spatial, systematic

193

and repetitive-based looping strategies on day 5 (Figure 7.6B, C). In contrast to RLDP SE rats,

LDP SE rats showed no change in search strategy use (Figure 7.6A).

194

Figure 7.5 The effect of SE on the distribution of search strategies used during spatial

acquisition and spatial reversal testing. SE was induced with either the RLDP (n=23) or the

LDP (n=10) procedure and terminated after 60 min as in Methods section 7.2.2. ‗Pilo no-SE‘ rats

(n=22) were treated with the pilocarpine and did not develop SE. Shams (n=19) received saline

instead of pilocarpine. Animals tested in the MWM and search strategies assessed as in Methods

section 7.2.3on day 1 (A1), day 7 (A7) and day 14 (A14) of spatial acquisition, and on day 1

(R1) and day 5 (R5) of spatial reversal. Results are presented as the percentage of trials and as

the total number of trials that all animals in each group used a given strategy.

195

196

Figure 7.6 Summary of search strategy use between groups during Morris water maze

testing. SE was induced with either the RLDP (n=23) or the LDP (n=10) procedure and

terminated after 60 min as in Methods section 7.2.2. ‗Pilo no-SE‘ rats (n=22) were treated with

the pilocarpine and did not develop SE. Shams (n=19) received saline instead of pilocarpine.

Animals tested in the MWM and search strategies assessed. Results presented as the percentage

of trials that all animals in each group used a given strategy: (A) spatial strategies, (B) non-

spatial, systematic strategies, and (C) repetitive looping-based strategies. * Search strategy use

different from day A1 (p<0.05). ** Search strategy use on day R5 different from day R1

(p<0.05). ^ Search strategy use on day R1 different from day A14 (p<0.05). # Search strategy

use in SE rats different from non-SE control groups on corresponding day (p<0.05). Chi-square

analysis was used for each comparison

197

198

7.3.8 Quantitative assessment of the contribution of search strategy to overall performance

The above results demonstrating relatively poor performance of the SE rats in the probe test (a

measure of spatial memory) suggested that the reduction in escape latencies during acquisition

testing in the MWM might primarily reflect improved use of more efficient search strategies by

these animals. We therefore determined the extent to which the shift in search strategies

contributed to improved performance. An alternative possibility was that the rats became better

at using one or more of the individual strategies over the 14-days of testing and that this

contributed to improved performance. We first calculated the average escape latency for each

individual strategy on the first and last day of acquisition learning. As shown in Table 7.1, both

of the non-SE control groups exhibited improved performance in the use of both spatial

strategies and non-spatial, systematic strategies. In contrast, improved performance in the RLDP

SE rats occurred only with the scanning strategy, and no significant improvement in any of the

strategies was detected in the LDP SE rats.

199

Table 7.1: Performance as a function of strategy use in hidden platform Morris water maze testing

Strategy RLDP SE

day 1

RLDP SE

day 7

RLDP SE

day 14

LDP SE

day 1

LDP SE

day 7

LDP SE

day 14

Repetitive Looping 58.5 ± 2.5

n = 36

53.3 ± 2.3

n = 32

53.9 ± 4.1

n = 14

55.3 ±

1.9

n = 29

56.2 ± 3.4

n = 15

56.4 ± 6.0

n = 10

Thigmotaxis 59.2 ± 0.3 n

= 26

54.3 ± 3.4

n = 18

51.9 ± 5.4

n = 10

56.2 ±

1.7

n = 28

56.3

±18.7

n = 13

54.7 ± 5.3

n = 6

Circling 56.7 ± 0.7

n = 10

52.8 ± 2.9

n = 14

49.9 ± 5.9

n = 4

29.6

n = 1

53.2

±16.8

n = 2

32.2

±20.3

n = 2

Non-spatial,

systematic

51.2 ± 3.9

n = 73

43.3 ± 2.6

n = 47*

41.6 ± 2.5

n = 53*

43.4 ±

3.9

n = 25

42.4 ± 2.8

n = 30

40.2 ± 3.4

n = 23

Random Searching 57.4 ± 1.7

n = 42

56.9 ± 2.8

n = 20

51.2 ± 4.1

n = 22

60.0

n = 10

55.4 ± 2.4

n = 11

57.8 ± 2.0

n = 8

Scanning 42.9 ± 2.7

n= 31

33.2 ± 2.8

n = 27*

34.9 ± 2.7

n = 31*

32.2 ±

4.7

n = 15

34.8 ± 3.0

n = 19

32.3 ± 3.4

n = 15

Spatial 13.2 ± 1.5

n = 23

8.3 ± 0.8

n = 52

9.2 ± 0.9

n = 63

5.9 ± 1.2

n = 6

14.1 ±

3.6

n = 15

14.7 ± 3.1

n = 24

Orienting 18.4 ± 1.0

n = 13

12.6 ± 1.5

n = 17

15.9 ± 1.6

n = 20

13 .0

n = 1

11.0 ± 1.9

n = 7

17.8 ± 3.8

n = 15

Approaching 7.3 ± 3.2

n = 8

6.3 ± 0.6

n = 34

6.8 ± 0.8

n = 37

5.4 ± 1.2

n = 4

16.8 ± 6.5

n = 8

10.0 ± 5.3

n = 9

Direct Find 2.7 ± 0.7

n= 2

3.6

n=1

3.1 ± 0.2

n = 2

1

n = 1

N/A N/A

Latency in seconds (mean ± standard error). n: number of individual trials during hidden platform testing where a

given strategy was used. N/A: not applicable since no example of this search strategy was found. Shaded rows:

classes of strategies, values reflect the pooled results from the individual strategies listed below each shaded row.

* Performance significantly different from day 1 (p<0.05). Data assessed using one-way ANOVA.

200

Table 7.1: continued

Strategy Sham

day 1

Sham

day 7

Sham

day 14

Pilo no-

SE

day 1

Pilo no-

SE

day 7

Pilo no-

SE

day 14

Repetitive Looping 42.6 ± 5.2

n = 12

N/A N/A 54.5 ±

3.5

n = 10

N/A N/A

Thigmotaxis 42 ± 5.2

n = 12

N/A N/A 54.5 ±

3.5

n = 10

N/A N/A

Circling N/A N/A N/A N/A N/A N/A

Non-spatial,

systematic

42.5 ± 2.3

n = 59

18.6 ± 4.0

N = 9 *

24.9 ± 3.6

n=16 *

47.6 ±

1.9

n = 72

24.4 ±

3.3

n = 27 *

19.5 ± 3.8

n = 12 *

Random Searching 58.3 ± 1.7

n = 20

N/A N/A 59.9 ±

0.1

n = 31

60

n = 2

N/A

Scanning 34.4 ± 2.4

n = 39

18.6 ± 4.0

n = 9 *

24.9 ± 3.6

n = 16 *

37.8 ±

2.5

n = 40

21.6 ±

2.7

n = 25 *

19.5 ± 3.8

n = 12 *

Spatial 11.4 ± 0.1

n = 42

5.8 ± 0.6

n = 104*

7.0 ± 0.6

n = 97*

11.8 ±

1.0

n = 44

6.3 ± 0.5

n = 98*

5.3 ± 0.3

n = 114*

Orienting 15.9 ± 1.7

n = 13

11.5 ± 2.2

n = 19

13.8 ± 1.5

n = 25

17.0 ±

1.4

n = 19

11.3 ±

1.2

n = 20*

8.4 ± 0.5

n = 21*

Approaching 9.6 ± 1.0

n = 28

4.9 ± 0.6

n = 69*

5.0 ± 0.5

n = 62*

8.0 ± 0.8

n = 24

5.4 ± 0.6

n = 67*

5.0 ± 3.4

n = 74*

Direct Find 3.4

n = 1

2.8 ± 0.02

n = 16

2.3 ± 0.2

n = 10

1.0

n = 1

6.3 ± 0.5

n = 98

2.7 ± 0.2

n= 19

Latency in seconds (mean ± standard error). n: number of individual trials during hidden platform testing where a

given strategy was used. N/A: not applicable since no example of this search strategy was found. Shaded rows:

classes of strategies, values reflect the pooled results from the individual strategies listed below each shaded row.

* Performance significantly different from day 1 (p<0.05). Data assessed using one-way ANOVA.

201

We next compared the importance of the shift in strategies relative to the improved efficiency

when using each strategy. Quantitative analyses were based on calculations described by Brody

et al., (2006). As demonstrated in section 3.1 of Appendix III, convolution analysis was used to

determine the predicted performance of the RLDP SE rats based on shifts in strategy use for all

days of spatial acquisition, while removing the changes in the performance of each strategy from

the assessment. The predicted performance based on this analysis closely matched the actual

data collected in the RLDP SE and the LDP SE groups (Figure 7.2, dashed lines). In RLDP SE

rats, the convolution analysis predicted that performance would be improved by 33% from day 1

to day 14, a value that is close to the 40% improvement observed. A similar analysis conducted

in LDP SE rats predicted a 34% improvement in performance from day 1 to day 14, and is close

to the 32% improvement observed.

The converse analysis was also performed. To obtain the predicted performance, the change in

performance of each strategy was used in the assessment, while the effect of shift in strategy use

was removed. Calculations are illustrated in section 3.2 of Appendix III. This analysis predicted

an improved performance of only 11% from day 1 to day 14 in the RLDP SE rats, and an

improved performance of only 0.1% in the LDP SE rats. These values markedly underestimated

the actual improvement in performance of 40% and 32% in the RLDP SE and the LDP SE rats,

respectively. Based on this analysis, we concluded that the improved performance s between

days 1 and 14 was mainly accounted for by a shift in strategy use.

A similar analysis was conducted for the control groups. For shams, the convolution analysis

based on strategy shift predicted a 68% improvement over the 14 days of testing, similar to the

observed improvement of 74%. When the analysis was based on improved performance within

each strategy, the predicated improvement was 48%. Predicted values were similar in pilo no-

SE rats, with a 73% improvement attributable to a shift in strategy use, and a 53% improvement

based on increased efficacy in performance within each strategy. The predicted value based on

shift in strategy use was closest to the observed improvement of 72% in pilo no-SE rats.

Accordingly, in non-SE rats, shifts in strategy use predominately contributed to improved

performance during spatial acquisition. However, improvements in the use of each individual

strategy also played an important role in improved performance.

202

7.3.9 SE results in differential impairment in Morris water maze performance and search strategy use

A closer examination in the performance of individual RLDP SE rats during spatial acquisition

showed that SE differentially affects MWM performance, despite each rat receiving similar

treatment for SE induction. In this analysis, individual rats were partitioned into groups based on

escape latencies in the initial 4 days compared to the final 4 days of spatial acquisition testing.

Here, performances of individual rats clearly fell into three discrete groups. As illustrated in

Figure 7.7, 8 of the RLDP SE rats (group 1) showed no improvement in performance over 14-

days of testing, and were significantly different from 10 of the RLDP SE rats (group 2) that

showed significant reduction in latencies within this period. These 2 groups also exhibited

differences in search strategy use (Figure 7.8). The 8 RLDP SE rats that showed no

improvement in MWM performance also had no shift in strategy use during testing. In contrast,

the 10 RLDP SE rats that exhibited improved performance also showed an increase in use of

spatial strategies (Figure 7.8A), and a decrease in use of non-spatial, systematic strategies

(Figure 7.8B). All of these animals showed impaired performance and use of less efficient

strategies when compared to shams. Five of the RLDP SE rats (group 3) exhibited no

impairment during MWM testing, with performance (Figure 7.7) and selection of search

strategies (Figure 7.8) similar to shams.

As shown in Table 7.2, there was no significant difference between groups with respect to

seizure susceptibility and seizure severity. All groups exhibited similar swim velocities when

compared to shams, indicating no presence of motor impairment. Reference memory, as

assessed by the number of platform crossings in the spatial acquisition and spatial probe tests,

was different between the three RLDP SE groups. Although group 1 had a lower number of

platform crossings when compared to group 2, both groups performed poorly when compared to

shams. On the other hand, the number of platform crossings in group 3 RLDP SE rats was

similar to shams in the two probe tests.

203

Table 7.2: Comparison of RLDP SE rats that exhibit differences in Morris water maze performance

Treatment

/group #

# of

pilocarpine

injections for

RLDP

protocol1

Seizure

Severity2

Swim velocity

during spatial

acquisition

probe test3

# of platforms

crossed during

spatial

acquisition

probe test4

Swim velocity

during spatial

acquisition

probe test3

# of platforms

crossed during

spatial

acquisition

probe test4

1. RLDP

SE (n=8)

3.6 ± 1.1 2.8 ± 0.3 25.6 ± 2.5 0.3 ± 0.5* ^ 27.0 ± 3.8 0.4 ± 0.7* ^

2. RLDP

SE (n=10)

3.9 ± 1.4 2.9 ± 0.3 25.3 ± 2.1 1.2 ± 0.8* 24.6 ± 2.1 1.3 ± 1.3*

3. RLDP

SE (n=5)

3.8 ± 1.5 2.9 ± 0.4 25.4 ± 3.2 3.6 ± 2.0 27.4 ± 2.1 3.4 ± 0.9

Shams

(n=19)

---- ---- 25.0 ± 3.1 3.4 ± 1.3 27.0 ± 3.0 3.1 ± 1.3

1. Number of repeated low-dose pilocarpine injections (10 mg/kg) administered in the RLDP protocol (average ±

SD). 2. Average maximum seizure activity for all animals determined as in Methods (average ± SD). 3. Average

swim velocity (cm/s) (average ± SD). 4. # of platforms crossed in probe trial (average ± SD). * different from

shams (p<0.05). ^ different from group 2 RLDP SE rats (p<0.05). Data analyzed by one-way ANOVA followed

by Newman-Keuls post-hoc.

204

Figure 7.7 SE results in differential impairment in Morris water maze performance. SE

was induced with the RLDP (n=23) procedure and terminated after 60 min as in Methods section

7.2.2. RLDP SE animals were subdivided into 3 groups based on performance (see section

7.3.9). Shams (black circles, n=19) received saline instead of pilocarpine. Each data point

represents the average of 6 trials per day (± SEM). * During acquisition training, latencies in

final 4 days (A11-A14) reduced compared to initial 4 days (A1-A4) in RLDP SE groups 2 and 3

and in shams (p<0.05). # Performance in RLDP SE groups 1 and 2 are different from RLDP SE

group 3 and shams (p<0.05). Data analyzed by repeated-measure ANOVA.

205

206

Figure 7.8 SE results in differential use of search strategies during Morris water maze

testing. SE was induced with the RLDP (n=23) and terminated after 60 min as in Methods

section 7.2.2. SE animals were subdivided into 3 groups based on performance (see section

7.3.9). Shams (n=19) received saline instead of pilocarpine. Animals tested in the MWM and

search strategies assessed as in Methods section 7.2.3. Results presented as the percentage of

trials that all animals in each group used a given strategy: (A) spatial strategies, (B) non-spatial,

systematic strategies, and (C) repetitive looping-based strategies. * Search strategy use different

from day A1 (p<0.05), ** p=0.1. # Search strategy use different from shams on corresponding

day (p<0.05). Chi-square analysis was used for each comparison.

207

208

7.3.10 The effect of Tat-NR2B9c on visual-spatial learning and use of search strategies following SE

In chapter 5, we demonstrated that tat-NR2B9c is neuroprotective within the CA1, CA3 and CA4

pyramidal cell layers of the dorsal hippocampus. Because the dorsal hippocampus is involved in

visual-spatial learning, we determined whether the neuroprotection provided by tat-NR2B9c

would improve spatial learning and search strategy use following SE. Rats treated with tat-

NR2B9c following SE had similar swim velocities when compared to both non-SE control

groups, indicating no presence of motor impairment (Figure 7.4C). Figure 7.9 demonstrates that

administration of tat-NR2B9c did not improve escape latencies during either spatial acquisition

or spatial reversal testing, and did not improve reference memory as assessed in the probe tests

(Figure 7.4A, B). Furthermore, search strategy use as a function of time was not altered by the

drug treatment during spatial acquisition (Figure 7.10), with all SE experimental groups using

less efficient search strategies when compared to shams.

209

Figure 7.9 Tat-NR2B9c did not improve performance in SE rats during hidden platform

learning. Escape latencies (mean ± SEM) during spatial acquisition (A1 – A14) and spatial

reversal testing (R1 – R5) in tat-NR2B9c (n = 16; purple squares), tat-NR2BAA (n=17, orange

triangles) and saline-treated (n = 24; red circles) rats 3 months following 60-minutes SE and in

shams (n=19, black diamonds) were determined as in Methods. Each data point represents the

average of 6 trials per day: # Performance in SE rats different from shams (p<0.05). Data

analyzed by repeated-measure ANOVA followed by Newman-Keuls post-hoc.

210

211

Figure 7.10 Tat-NR2B9c has no effect on the distribution of search strategies used during

spatial acquisition in rats following SE. SE was induced with the RLDP procedure and

terminated after 60 min as in Methods. Tat-NR2B9C (n=16), Tat-NR2BAA (n=19) or saline

(n=23) was administered 3 hr following the termination of SE. Shams (n=19) received saline

instead of pilocarpine. Search strategies were assessed as in Methods. Results are presented as

the percentage of trials for which all rats in a group used a given strategy. Similar shift in search

strategy use was detected in rats treated with saline, tat-NR2BAA and tat-NR2B9c (p=0.51). *

Search strategy use different from day A1. # Search strategy use different from shams. Chi-

square analysis was used for each comparison.

212

213

7.4 Discussion

Although many studies have examined the impact of SE on spatial learning during MWM testing

(Persinger et al., 1993; Rice et al., 1998; Hort et al., 1999; Hort et al., 2000; Wu et al., 2001;

McKay and Persinger, 2004; dos Santos et al., 2005; Frisch et al., 2007; Zhou et al., 2007), little

attention has been given to the effect of SE on the use of search strategies. In the present study,

we analyzed the effect of SE on strategy use during spatial acquisition and spatial reversal

testing. The main findings include: (1) SE groups treated with either the RLDP or LDP seizure-

inducing protocol exhibit impaired performance in the MWM, and show some improvement in

performance over prolonged training. (2) SE groups use less efficient search strategies than non-

SE groups. (3) Increased use of more efficient search strategies, rather than improvement in

performance of individual strategies, most closely predicted the improved performance in both

the SE and non-SE groups. (4) Within the same group of animals with SE, variability in

behaviour of individual rats during MWM testing occurred. While the majority of rats show

impaired performance and use of poor search strategies, but differed in the ability to show

improvement over prolonged training, some rats exhibited no behavioural impairment when

compared to non-SE rats. (5) Finally, neuroprotection within the dorsal hippocampus provided

by tat-NR2B9c did not modify MWM performance in rats following SE. These results are

discussed below.

7.4.1 SE rats exhibit impaired performance in the MWM and improve during prolonged training

In agreement with previous studies, we found that MWM performance in rats following SE was

markedly impaired (Rice et al., 1998; Hort et al., 2000; Wu et al., 2001; McKay and Persinger,

2004; Frisch et al., 2007; Zhou et al., 2007; dos Santos et al., 2005; Cunha et al., 2009).

However, we found that with prolonged training, SE rats were capable of improving their

performance. This finding is generally similar to that of dos Santos et al., (2006) but differs

from that of several other groups that found no improvement in performance (Rice et al., 1998;

Hort et al., 2000; Wu et al., 2001; McKay and Persinger, 2004; Zhou et al., 2007; Cunha et al.,

2009); in these latter studies, MWM testing was conducted for up to a maximum of 5 days.

Since we did not observe a significant change in performance within the first 5 days of testing,

this duration may not have been long enough for improved performance to occur in SE rats

described in the other studies (Rice et al., 1998; Hort et al., 2000; Wu et al., 2001; McKay and

214

Persinger, 2004; Zhou et al., 2007; Cunha et al., 2009). In general, our results suggest that rats

following SE retain the ability to improve performance in MWM testing with extended training.

We previously demonstrated that rats treated with the RLDP protocol exhibited a lower mortality

rate and less severe status compared to rats treated with the LDP protocol (see chapter 3).

Despite these differences, we found that the MWM performance was similar between the RLDP

SE and the LDP SE groups. These results are congruent with our previous findings that the two

seizure-inducing protocols also cause similar behavioural impairments in rats when assessed in

the open field, elevated plus maze, and hyperexcitability tests (see chapter 6). These results are

not unexpected, since we previously found that neuronal loss in the hippocampus and piriform

cortex was similar in RLDP SE and LDP SE rats, brain regions associated with cognitive (see

section 7.1) and behavioural performance (see section 6.4.2). Our general finding is that the

RLDP protocol has the advantage to reduce mortality in Wistar rats following SE, without

affecting SE-induced morbidity and neuropathology, and therefore serves as a more beneficial

method in generating seizures for this rat strain (see section 7.4.6).

7.4.2 SE rats use less efficient strategies in the MWM

One of our main findings was that rats following SE showed impaired search strategy use in the

spatial acquisition and spatial reversal tasks. SE groups displayed a greater proportion of less

efficient search strategies (repetitive-looping based strategies and non-spatial, systematic

strategies) while using fewer spatial strategies compared to non-SE groups. Nonetheless, with

increased training times, epileptic rats learned how to use more efficient strategies and the

increased use of these strategies was able to account for the improved performance. The

observation that SE rats showed poor spatial memory compared to non-SE rats (as assessed by

the probe tests) further supports the idea that the reduced latencies during spatial acquisition

resulted from improvements in the procedural-strategy component of the MWM task as oppose

to the spatial component. The prolonged training necessary for improved performance in SE

groups is also congruent with the fact that procedural learning is a form of habit-like learning

that can take weeks rather than days to acquire, and does not involve cognitive factors (e.g.,

memory and expectancy) that occur during place learning (Kirsch et al., 2004; Bayley et al.,

2005). In the present study, once the more efficient strategies were learned by SE rats, they were

retained, as indicated by the observation that performance and strategy selection was better on

215

initial day of spatial reversal than at the beginning of spatial acquisition. These findings indicate

that in spite of the initial poor performance on the MWM, the rats following SE were able to

form and retain a procedural memory of newly acquired search strategies.

During the spatial reversal task, no improvement in overall performance was observed in rats

treated with either induction protocol. Although there was no change in strategy use by the LDP

SE rats, RLDP SE rats increased their use of more efficient strategies over the 5 days of reversal

training. It is not clear why the change in strategy use in RLDP SE rats did not coincide with

improved performance during reversal training, or why strategy use was different in rats treated

with the LDP protocol compared to the RLDP protocol. Because spatial reversal assesses

different factors (i.e., cognitive flexibility) than those assessed during spatial acquisition

(Vorhees and Williams, 2006), the differences in the procedures may account for the differences

in behavioural responses of SE rats detected during spatial acquisition when compared to spatial

reversal. Another possibility is that testing in the spatial reversal task was only 5-days, and that

this duration was not long enough to detect improvement in performance in SE rats, as was

detected over 14-days of testing in the spatial acquisition task.

7.4.3 Improvement in search strategy selection contributed to improved performance epileptic rats

In the present study, we found that the RLDP SE group and the non-SE groups improved their

performance in using individual search strategies. To further assess the importance of a change

in strategy use relative to improved efficiency in use of individual strategies in contributing to

improved performance, we conducted the convolution analyses as described by Brody et al.,

(2006). The general finding was that changes in strategy use more closely predicted and

therefore contributed mostly to the change in performance observed in the SE groups. A shift in

strategy use was also found to predominately contribute to improved performance in non-SE

groups. Brody et al., (2006) found similar results in transgenic mice following traumatic brain

injury. This emphasizes that the use of less efficient search strategies in rats following SE, and

perhaps in brain trauma more generally (Brody et al., 2006), along with poor spatial memory, are

important factors underlying the impaired performance in these animals.

216

7.4.4 The pathological effects of SE may interfere with the selection of more efficient search strategies

Our results indicate that search strategy use does not operate based on a simple hierarchical

hypothesis as was suggested by Sutherland et al (1982) (Sutherland et al., 1982). In this

hypothesis, Sutherland et al., (1982) proposed that mice capable of using spatial information will

proceed to exclusively use spatial strategies, whereas those incapable of using spatial

information will predominately use nonspatial, systematic strategies. We found, however, that

SE rats showing little evidence of true spatial memory in the probe tests showed improvement in

use of spatial strategies during spatial acquisition. This finding is similar to that previously

reported for transgenic and wild type mice subjected to traumatic brain injury (Brody and

Holtzman, 2006). In addition, when the platform was changed to the opposite quadrant, SE rats

exhibited a decrease in performance that correlated with the use of fewer spatial strategies, and

the use of more non-spatial, systematic strategies (in Figures 7.2 and 7.4, compare day 16 with

day 14). These results suggest that SE rats are capable of acquiring spatial memory, but

nevertheless continue to predominately use less efficient search strategies and to perform poorly

in the spatial probe tests. In this respect, they behave similarly to caudate-putamen lesioned rats,

which although capable of using spatial strategies, exhibit a preference for alterative, possibly

simpler strategies (Whishaw et al., 1987). Wishaw et al., (1987a) hypothesized that damage to

the caudate-putamen, which has previously been reported in rats following SE (Fujikawa, 1996;

Clifford et al., 1987), may disrupt the ability of the animal to select the most appropriate

behavioural strategy.

Although the physiological basis for determining strategy choice during spatial navigation tasks

is not fully understood, studies have indicated that: 1) the cholinergic system is involved

(Sutherland et al., 1982; Whishaw and Petrie, 1988; Whishaw, 1989; Chang and Gold, 2003;

McIntyre et al., 2003), and that 2) different neural systems control whether spatial or non-spatial

strategies are employed (McDonald and White, 1993; McDonald and White, 1994; Miranda et

al., 2006). Muscarinic blockade with atropine does not disrupt performance in a non-spatial

version (visible platform) of the Morris water maze, but impairs strategies in searching for the

platform in a subsequent spatial version (hidden platform) (Whishaw and Petrie, 1988; Whishaw,

1989). In addition, the level of acetylcholine in the striatum and in the hippocampus determines

whether tasks are solved using either response (egocentric) or place (allocentric) mechanisms,

217

respectively (Chang and Gold, 2003; McIntyre et al., 2003). These results suggest that when

spatial information is required for the solution of a task the hippocampus is more active,

whereas when stimulus-response learning is required the striatum is more active (Miranda et al.,

2006). Several authors have suggested that the striatal and hippocampal neural systems interact

through competitive mechanisms, so that in determining which strategy is best suited to solve a

task, the more efficient system is used (McDonald and White, 1993; McDonald and White, 1994;

Epp et al., 2008). Previous studies have demonstrated that widespread neurodegeneration

(Honchar et al., 1983; Turski et al., 1983b; Peredery et al., 2000) and changes in the cholinergic

system (Jope and Gu, 1991; Mingo et al., 1997; Baran et al., 2004; Mendes de Freitas et al.,

2005) occur in rats following SE. Consequently, these and other pleiotropic effects of SE on the

nervous system most likely contribute to the impaired selection of search strategies by SE rats.

7.4.5 Rats following SE exhibited variability in behaviour during MWM testing

One of the main findings was that variability in behaviour during MWM testing occurred within

the same group of rats with SE. Although the majority of rats following SE showed impaired

performances during MWM testing, there were differences in their ability to improve

performances over prolonged training. Within the RLDP SE group, 44% of the rats learned how

to use more efficient strategies, and the increased use of these strategies was able to account for

the improved performance in these animals. On the other hand, 34% of the SE rats exhibited no

improvement in escape latencies or changes in strategy use with extended training. Since these

rats simply swam around the pool and rarely located the platform, this behaviour can account for

why these animals had a worst performance in the probe tests when compared to SE rats that

showed improved performance, and utilized strategies that increased their likelihood of

encountering the platform. Five out of 23 rats exhibited no behavioural impairment, with

performances in the MWM and probe tests indistinguishable from controls.

We previously demonstrated that rats following SE sustain major neuronal loss in brain regions

associated with MWM performance (i.e., hippocampus and thalamus, see chapters 4 and 5).

However, variability in the severity of brain damage between individual SE rats was also

observed. Therefore, it is possible that differences in the pattern of brain damage involved in

learning of the MWM task may contribute to the variability in performance between rats

following SE. In support of this idea, McKay and Persinger (2004) found that ketamine-treated

218

SE rats performed equivalently to nonseized controls in spatial and non-spatial water maze tasks,

whereas acepromazine-treated SE rats performed poorly. Despite cognitive sparing in ketamine-

treated SE rats, extensive brain damage was still present. However, the pattern of brain damage

(i.e., amygdala, hippocampus and thalamus) was different between the two groups, and may have

contributed to the differences in MWM performance between the ketamine-treated SE rats and

the acepromazine-treated SE rats (McKay and Persinger, 2004). Further research is necessary to

determine whether the variability in MWM performance we observed in rats following SE is

correlated with differences in neuropathology.

7.4.6 Neuroprotection of the dorsal hippocampus by tat-NR2B9c did not modify performance in the MWM

Administration of tat-NR2B9c resulted in neuroprotection within the CA1, CA3 and CA4

subfields of the dorsal hippocampus (see Chapter 5). In spite of its protective action, the

administration of Tat-NR2B9c did not result in improved performance in the MWM, or in the

use of more efficient search strategies during MWM testing. In contrast to our results, several

studies have reported a connection between neuroprotection and improved cognitive functioning.

Cunha et al., (2009) found that repeated daily treatments of diazepam, carbamazipine and

phenytoin immediately following SE provided substantial neuroprotection, resulting in less than

30% neuronal loss in the CA1, CA3 and dentate gyrus; this protective effect correlated with

improved spatial learning during MWM testing. Similarly, Yang et al., (2007) found that

treatment of rats with erythropoietin prior to SE induction resulted in neuroprotection of the CA1

and CA3 (although the extent was not specified), as well as improved cognitive functioning and

use of spatial strategies in the MWM (Jun et al., 2009). Rice et al., (1998) also showed that pre-

treatment of rats with MK-801 resulted in almost complete protection of the CA1 and greatly

improved spatial learning. A possible explanation for the difference in findings from these

studies compared to our results may be the amount of protection that was afforded in the

hippocampus following the different treatments. For instance, Broadbent et al., (2004)

demonstrated that spatial learning was impaired by bilateral dorsal hippocampal lesions that

encompassed 30 to 50 percent of the total hippocampal volume (Broadbent et al., 2004).

Consequently, the partial neuroprotection of the hippocampus by tat-NR2B9c may not have been

enough to result in cognitive sparing.

219

An alternative possibility is that other damaged brain regions not affected by tat-NR2B9c may

contribute to cognitive impairment during MWM testing. As previously discussed, McKay and

Persinger (2004) demonstrated that different patterns of brain damage (i.e., amygdala,

hippocampus, thalamus) following SE can differentially affect performance in the MWM (see

section 7.4.5). Although our results suggest that neuroprotection within the dorsal hippocampus

does not modify SE-induced alterations in spatial learning and behavioural strategies, it is also

possible that there was not enough protection within the dorsal hippocampus in the present study

to have a noticeable behavioural effect.

7.4.7 Conclusion

The present study demonstrates that suboptimal search strategy use, along with poor spatial

learning and memory, contributes to the impaired performance of epileptic rats during spatial

learning tasks in the MWM. With extended training, the majority of rats following SE were able

to improve their performance by learning how to use more efficient search strategies during

spatial acquisition. Partial neuroprotection within the dorsal CA1 provided by tat-NR2B9c was

insufficient to improve spatial learning, suggesting either that the amount of neuroprotection in

the hippocampus was insufficient to rescue behaviour or that other damaged brain regions not

affected by Tat NR2B9c may contribute to the decrease in learning ability. In general, our

results indicate that systematic assessment of behavioural strategies and their contribution to

overall performance is an important factor to consider when assessing morbidity in animal

models of epilepsy. Patients with TLE may have analogous poor use of cognitive strategies.

220

Chapter 8

General Discussion

8.1 The lithium/pilocarpine model of mesial temporal lobe epilepsy

The lithium/pilocarpine rodent model recapitulates the main clinical and neuropathological

features of mesial temporal lobe epilepsy with hippocampal slerosis (MTLE-HS), the most

common form of human epilepsy (see section 1.1). Both in the rodent pilocarpine model and in

the human condition the seizure disorder is initiated by an initial precipitating injury (IPI)

followed by a latent period, whereby the subjects appear neurologically normal with subsequent

development of unremitting epileptic seizures (Wieser, 2004; Curia et al., 2008). In both

conditions, widespread neuronal loss is observed in many brain regions including the

hippocampus, neocortex, piriform and entorhinal cortices, septum, amygdaloid and thalamic

structures (Moran et al., 2001; Jutila et al., 2002; Bernasconi et al., 2003; Bonilha et al., 2010a;

Bonilha et al., 2010b). A high prevalence of cognitive impairment and behavioural disturbances

(e.g., aggression, anxiety) is also reported in human MTLE-HS ( Boro and Haut, 2003; Gaitatzis

et al., 2004; Devinsky, 2004a; Swinkels et al., 2005; Cornaggia et al., 2006; Marcangelo and

Ovsiew, 2007; Garcia-Morales et al., 2008) and is observed after SE in rodents (Rice et al.,

1998; Hort et al., 1999; Hort et al., 2000). Nonetheless, the causal relationship between neuronal

death, epileptogenesis, and cognitive and behavioural morbidity following SE remains unclear

(see sections 1.5 and 1.6). Our general hypothesis is that genesis of SRSs, cognitive impairment

and behavioural alterations are caused by SE-induced neuronal death. This thesis focused on

five specific hypotheses that further characterized neuronal death and behavioural/cognitive

morbidity in rats following SE induced by the lithium/pilocarpine model (see chapter 2). The

causal relationship between neuronal death, epileptogenesis and cognitive and behavioural

morbidity was investigated by: (1) comparing the temporal onset between neuronal death and

behavioural alterations following SE (section 8.6), and (2) assessing the effect of partial

neuroprotection within the hippocampus on the incidence of SRSs, behavioural alterations and

cognitive deficits after SE in rats (section 8.7). In the subsequent sections, we summarize our

main findings and discuss the significance of these results to clinical human data.

221

8.2 Comparison of the low-dose (LDP) and repeated low dose lithium/pilocarpine (RLDP) procedures

High mortality in rodents is a major drawback in using the lithium/pilocarpine model (reviewed

in section 1.3.4). Glien et al., (2001) showed that when compared to a single dose of pilocarpine

(30 mg/kg, LDP protocol), repeated administration with reduced doses of pilocarpine (10 mg/kg,

RLDP protocol) at 30-min intervals until SE onset significantly reduced mortality in Wistar rats.

Still, it is difficult to determine a ‗best-of protocol‘ since a comparison of the effect of the LDP

and the RLDP procedures on neurodegeneration, behavioural alterations and cognitive

impairment has not been completed. This type of analysis becomes complex since most studies

have used multiple outbred rat strains such as Sprague-Dawley, Wistar, and Long-Evans hooded

(LEH) in epilepsy research. Interstrain differences have been detected in post-status epilepticus

models, including kainic acid (Sanberg and Fibiger, 1979; Golden et al., 1995; Xu et al., 2004),

pentylenetetrazol (Becker et al., 1997a) or pilocarpine (Xu et al., 2004; Hort et al., 2000).

Nonetheless, the potential interstrain differences in the LDP and RLDP lithium/pilocarpine

procedures have not been determined. In chapter three, we compared the effect of the LDP and

RLDP procedures on the rates of SE induction, intensity of behavioural seizures and mortality

rates in Wistar rats and in LEH rats. Our findings showed that the RLDP protocol reduced

mortality and severity of SE in the Wistar rats when compared to the LDP protocol, but had no

significant effect in LEH rats. Consequently, Wistar rats were used in all other experiments

presented in this thesis to minimize the number of experimental animals used. Next, we showed

that in spite of the differences in mortality rates and in intensity of behavioural seizures between

Wistar rats treated with either the LDP or the RLDP protocols, severity of pyramidal cell death

in the hippocampus and in the piriform cortex was similar at 3 months of recovery (reviewed in

section 3.4). Likewise, we demonstrated animals that developed SE, whether from the LDP or

the RLDP protocol, did not differ in anxiolytic behavioural assessments (reviewed in section

6.4.1) or in Morris water maze (MWM) performance (reviewed in section 7.4.1). Although our

results suggest that the RLDP procedure is the most effective in reducing mortality in Wistar

rats, it is not necessarily the best option to use in other strains as it had no effect in LEH rats. We

were able to validate that the RLDP protocol produced reliable neuronal loss and behavioural

and cognitive deficits in Wistar rats that were similar to the LDP protocol; these results are

discussed in the subsequent sections.

222

8.3 The severity and pattern of neuronal death in the lithium/pilocarpine model

We provided the first analysis of neurodegeneration with the RLDP lithium/pilocarpine protocol

for the induction of SE, and found that the extent of neuronal death in regions of the

hippocampus, amygdala, thalamus and piriform cortex was comparable to that previously

reported in the LDP lithium/pilocarpine protocol, and in the high-dose pilocarpine model

(Honchar et al., 1983; Turski et al., 1983a; Turski et al., 1983b; Clifford et al., 1987; Fujikawa,

1996; Motte et al., 1998; Covolan and Mello, 2000; Peredery et al., 2000; Poirier et al., 2000)

(see section 4.4.1).

8.3.1 Pattern of neuronal death in the hippocampus

The pattern of hippocampal pyramidal cell death we report in rats following SE closely replicates

classical hippocampal sclerosis in humans, with more severe neuronal death occurring in the

CA1 compared to the CA3, CA4 and hilus (Mathern et al., 1996; Wieser, 2004; Löscher and

Brandt, 2010). In addition, we detected no neuronal death in the CA2 or the granule cell layer

of the dentate gyrus. The CA2 and dentate gyrus are often referred to as the ‗resistant‘ sectors

since these areas are also relatively spared in classical human hippocampal sclerosis (Mathern et

al., 1996; Wieser, 2004). Although the lithium/pilocarpine model closely replicates classic

hippocampal sclerosis in humans, this pattern of neuronal loss is not comparable across all post-

status epilepticus models (see section 1.2.2). For instance, when compared to the

lithium/pilocarpine and pilocarpine models, SE elicited by kainic acid results in preferential

pyramidal cell death to the CA3 compared with CA1 (Schwob et al., 1980; Golden et al., 1995;

Tokuhara et al., 2007), and probably reflects the higher distribution of hippocampal kainate

receptors in CA3 (Dudek et al., 2006).

Recent findings have suggested that hippocampi from patients with MTLE can be assigned to

several distinct groups based on the severity and distribution of neuronal loss (Mathern et al.,

2002; de Lanerolle et al., 2003; Blümcke et al., 2007; Mueller et al., 2009; Thom et al., 2010). In

these studies, a classic pattern of hippocampal sclerosis with severe pyramidal cell loss in CA1

and moderate neuronal loss in all other subfields excluding CA2 ranged between 19 to 70

percent of cases evaluated (Mathern et al., 2002; de Lanerolle et al., 2003; Thom et al., 2010;

Blümcke et al., 2007; Mueller et al., 2009) . Other frequently observed sub-categories included

223

severe to varying degrees of pyramidal cell loss in all hippocampal subfields, neuronal loss

restricted to the CA1 area, and neuronal loss restricted to the hilar region (de Lanerolle et al.,

2003; Thom et al., 2005; Blümcke et al., 2007; Mueller et al., 2009; Thom et al., 2010). Up to

19 percent of cases evaluated were reported to have an absence of hippocampal sclerosis (and

therefore, were referred to as paradoxical TLE) (de Lanerolle et al., 2003; Thom et al., 2005;

Blümcke et al., 2007; Mueller et al., 2009; Thom et al., 2010). The different patterns of

hippocampal neuronal loss have been suggested to reflect different pathoetiologies and clinical

subtypes (de Lanerolle et al., 2003; Thom et al., 2005; Blümcke et al., 2007; Mueller et al., 2009;

Thom et al., 2010). For instance, clinicopathological correlations have been demonstrated

between different patterns of HS and the age of an IPI (Van Paesschen et al., 1997; Blümcke et

al., 2007; Thom et al., 2010). Furthermore, different subcategories of HS have been found to be

predictive of postsurgical outcome; patients exhibiting a classic HS, or varying damage within all

hippocampal subfields, had the best outcome of remaining seizure-free up to 2 years following

surgery when compared with patients exhibiting damage restricted to the hilar region, the CA1

region, or with no HS (de Lanerolle et al., 2003; Thom et al., 2005; Blümcke et al., 2007;

Mueller et al., 2009; Thom et al., 2010). Further research is necessary to assess the significance

of different patterns of HS in determining the causes and potential treatment of what may be

different ‗subcategories‘ of MTLE. Although the lithium/pilocarpine model closely replicates

classic HS in humans, it remains unknown how effective this or other post-status epilepticus

models are in representing other potential patterns of HS in human MTLE.

8.3.2 Pattern of neuronal death in extrahippocampal structures

Although the quintessential pathology of human MTLE has been HS, a combination of neuronal

loss, synaptic reorganization and gliosis (see section 1.5.1.1), these pathological alterations have

also been reported in extra-hippocampal structures (Hudson et al., 1993; Du et al., 1993; Wolf et

al., 1997; Juhász et al., 1999; Yilmazer-Hanke et al., 2000; Bernasconi et al., 2003; Natsume et

al., 2003; Bernasconi et al., 2005; Dawodu and Thom, 2005; Thivard et al., 2005). We

comparably showed a broad distribution of neuronal loss within the hippocampus, thalamus,

amygdala and piriform cortex in rats after SE. Previous studies have additionally reported other

pathological changes including synaptic reorganization, gliosis and cell genesis in regions of the

hippocampus, thalamus, amygdala, and piriform and entorhinal cortices (Bertram and Scott,

2000; Roch et al., 2002; Andre et al., 2007; Jung et al., 2009; Ben-Ari and Dudek, 2010). These

224

pathological changes are purported to cause the increased seizure propensity of these regions in

human MTLE ( Babb, 1986; Swanson, 1995; Masukawa et al., 1996; Pitkanen et al., 1998;

Aliashkevich et al., 2003; Bartolomei et al., 2005; Koch et al., 2005; Majores et al., 2007;

Williamson and Patrylo, 2007; Wittner et al., 2009) and in rats after SE (Bertram et al., 1998;

Mangan et al., 2000; Bertram et al., 2001; Aroniadou-Anderjaska et al., 2008). Based on the

broad distribution of the brain pathology, Bertram et al., (2009) suggested that seizure onset is

not just focused in the hippocampus, but involves widespread neuronal circuitry of independent

limbic structures.

Additional data from animal studies further support the concept that seizures arise within a

distributed limbic system. First, electrophysiological recording of multiple limbic sites in

chronically epileptic rats showed that the epileptogenic zone is broad, and indicated that the

substrate for seizure generation is distributed over several brain structures (e.g., hippocampus,

amygdala, and piriform cortex) (Bertram, 1997). Second, kindling studies have demonstrated

that the same type of behavioural seizure may be induced by stimulation of a number of different

limbic sites (Goddard et al., 1969; Bertram, 2007). However, the ease with which the seizure is

induced and the rapidity with which the seizure spread depends on which site is stimulated. For

instance, although kindling of limbic seizures occurred with stimulation of the medial dorsal

thalamic nucleus, the threshold for electric current to induce the seizure was significantly higher

than the threshold in the amygdala and hippocampus (Bertram et al., 2008). Furthermore, the

seizures generalized more rapidly from the medial dorsal thalamic nucleus compared to the

limbic structures (Bertram et al., 2008). Further studies showed that inactivating the medial

dorsal thalamic nucleus pharmacologically or manipulating the excitability of this region had a

profound effect on seizure activity in the hippocampus (Bertram et al., 2001; Bertram et al.,

2008). In general, while the hippocampus, amygdala and entorhinal and piriform cortices are

proposed to act as multiple independent generators of seizures, the thalamus displays

characteristics that suggest this structure may act as a physiological synchronizer (Bertram et al.,

1998; Avoli et al., 2002; Bertram, 2009). Other sites (although unknown) are proposed to act as

neuromodulatory input to the epileptogenic network that affects the foci propensity to seize

(Bertram, 2009).

Similar findings supporting the involvement of distinct epileptogenic structures in human MTLE

have been reported: (1) in pathological studies that showed a broad distribution of

225

neuropathological changes (e.g., neuronal loss, synaptic reorganization, neurogenesis) in

hippocampal and extrahippocampal structures (discussed above), (2) in electrophysiological

studies that have demonstrated multifocal or synchronized regional seizure onset (Spencer and

Spencer, 1994; Blumenfeld et al., 2004; Kobayashi et al., 2009; Bartolomei et al., 2010;

Wendling et al., 2010), or the presence of multiple epileptogenic zones (Maillard et al., 2004;

Bartolomei et al., 2008a; Bartolomei et al., 2008b; Chassoux et al., 2008; Wendling et al., 2010),

(3) in functional imaging studies (PET scans) that have shown broad regional hypometabolism

(indicative of an epileptogenic zone) in the mesial temporal structures (Henry et al., 1990;

Sperling et al., 1990; Ryvlin et al., 1991; Duncan, 1997; Benedek et al., 2004), and (4) in studies

assessing surgical outcomes of seizure control in patients with MTLE-HS. For instance,

although removal of limbic areas of the medial temporal lobe has led to a high rate of seizure

control (varying 50 to 93% between studies), there exist a significant number of patients for

whom seizure control is incomplete, indicating the presence of other seizure focii (Guénot, 2004;

Téllez-Zenteno et al., 2005; Schramm, 2008; Bertram, 2009). This idea is further supported by

studies which demonstrated that the greater extent of resection of mesial temporal structures

correlated with a better prognosis of remaining seizure-free (Nayel et al., 1991; Bonilha et al.,

2004; Vinton et al., 2007); however, others have failed to establish this relationship ( McKhann

et al., 2000; Schramm, 2008). Finally, the presence of extra-temporal epileptogenic zones (e.g.,

thalamus and neocortex) has been found to decrease the effectiveness of surgical intervention in

patients with refractory complex partial seizures (Newberg et al., 2000; Choi et al., 2003; Téllez-

Zenteno et al., 2005; Treiman, 2010). Altogether, these data emphasize the importance of

assessing neuropathological changes not only in the hippocampus, but also in extrahippocampal

structures, with the ultimate goal of improving surgical outcomes and developing other non-

surgical therapies to improve seizure control.

8.3.3 Differences in the pattern of neuronal loss between human MTLE and rats after SE

The symmetry of neuronal death between the cerebral hemispheres is different in human MTLE-

HS compared to rats following SE. In patients with MTLE-HS, atrophy of the hippocampus is

often bilateral but asymmetrical, with the side ipsilateral to the seizure focus exhibiting more

severe sclerosis (Mintzer et al., 2004; Wieser, 2004). The same pattern of neuronal loss also

occurs in extrahippocampal structures, with greater atrophy detected on the side ipsilateral to the

226

seizure focus (Bonilha et al., 2010a; Pail et al., 2010). In contrast, our findings and others noted

bilateral and symmetrical neurodegeneration of hippocampal and extrahippocampal structures in

rats following SE (Ben-Ari et al., 1980; Turski et al., 1983b; Clifford et al., 1987; Covolan and

Mello, 2000). The difference in pattern of neuronal loss may be contributed to species

differences in neural connectivity between the left and right cerebral hemispheres (Engel et al.,

2008). For instance, a major difference exists in the progression of seizure activity in rodents

compared to primates. In the rat, focally evoked seizures appear bilaterally symmetrical from

time of onset, as evident by both behavioural and cortical manifestations (Ben-Ari et al., 1979;

Peterson et al., 1992); seizure activity can spread rapidly mostly via commissural pathways such

as those in the anterior commissure and hippocampus (Engel et al., 2008). In contrast, in

primates, as in humans, behavioural and electrophysiological seizure manifestations remain

lateralized longer, and typically stay asymmetrical even as the seizure progress to engage

brainstem circuitry (Loddenkemper and Kotagal, 2005; Engel et al., 2008; Götz-Trabert et al.,

2008; Jan et al., 2010; Napolitano and Orriols, 2010); this occurs since the path of least

resistance involves seizure activity spreading down the neuraxis rather than across the forebrain

commissures (e.g., corpus collosum, hippocampal commissure, and anterior commissure) (Lieb

et al., 1987; Lieb et al., 1991; Engel et al., 2008).

8.4 The effect of increasing survival time on SE-induced neuronal death

8.4.1 Majority of neuronal death occurs early in rats following SE

Because previous studies have been limited by the semi-quantitative assessment of tissue damage

and by the limited number of recovery times assessed (Fujikawa, 1996; Motte et al., 1998;

Covolan and Mello, 2000; Peredery et al., 2000; Poirier et al., 2000) (see sections 1.5.1.2 and

4.4.1), our knowledge remains restricted on how SE-induced neuronal death in different brain

regions evolves over time. In chapter 4, we extended these findings by providing a detailed,

quantitative time-course comparison of SE-induced neuronal death in 19 brain regions within the

hippocampus, amygdala, thalamus and piriform cortex. Stereological estimates of NeuN positive

neurons were assessed at ten different time intervals after SE (from 1 hour to 3 months). Our

results demonstrated the the majority of neuronal death evolved rapidly after SE in rats, with the

majority of neuronal loss in all brain structures present by 24 hours. We were the first to

demonstrate that maximal neuronal loss occurred as early as 3 hours after SE in the hilus, ventral

227

CA3, reticular thalamic nucleus (Rt) and posteromedial cortical amygdaloid nucleus (PMCo), by

6 hours in the dorsal CA4 and posterior thalamic nuclei, and by 12 hours in the dorsal CA3 and

posterior piriform cortex (PPC) (see Table 4.3). The time course of this degeneration was

different for individual regions of the hippocampus, amygdala, thalamus and piriform cortex.

These findings are extensively discussed in section 4.4.

Clinical studies have provided evidence that the acute neuronal loss we observe in rats following

lithium/pilocarpine-induced SE may closely resemble the evolution of brain damage observed in

humans following an IPI. In studies using magnetic resonance imaging, evidence of acute

neuronal injury was detected in patients when assessed within 1 to 5 days after a prolonged

febriles seizure (Scott et al., 2002; Sokol et al., 2003; Scott et al., 2003). An elevation of serum

neuron-specific enolase, a marker for acute neuronal injury, was also elevated in adult patients

when assessed within 24 hours following SE (DeGiorgio et al., 1996; DeGiorgio et al., 1999).

8.4.2 Majority of neuronal death is the consequence of SE and not of spontaneous recurrent seizures (SRSs)

Because it is difficult to determine whether delayed neuronal loss is caused by SE, the

occurrence of SRSs, or both, the literature remains unclear as to the effect of SRSs on neuronal

loss (Dudek et al., 2002) (see section 1.5.1.2). Although we did not determine the latency to

onset of SRSs in rats after SE, our findings did demonstrate that by 3 months of recovery, all rats

were considered epileptic, defined as having exhibited at least ≥ 1 SRSs. Glien et al., (2001)

demonstrated that following 60 minutes of SE induced by the RLDP protocol in Wistar rats, the

average latency to the first SRS was 40 days (range 31 - 44 days). Thus, it is reasonable to

assume that any neuronal death preceding 40 days after SE is the consequence of SE and not of

SRSs. This suggestion is supported by our findings that: (1) with the exception of the thalamic

somatosensory nuclei (VPM and VPL), all other brain regions assessed showed no further

progression of neuronal loss 2 weeks after SE, a period in which SRSs were observed to occur in

rats analyzed at 3 months of recovery, and (2) Fluoro-Jade B staining, a marker for degenerating

neurons, was not detected in epileptic rats analyzed at 3 months after SE. In support of the

suggestion that SE primarily contributes to neurodegeneration, Liu et al., (1994) used the high-

dose pilocarpine procedure and showed neuronal death at 3 weeks recovery in the dorsal CA1

and CA3. The authors reasoned that since SRSs appeared approximately 2 to 2 ½ weeks after

SE in these animals, but did not contribute to any significant additional neuronal loss 6 to 12

228

weeks later, that neuronal death primarily resulted from the pilocarpine-induced SE (Liu et al.,

1994). In our studies, the thalamic somatosensory nuclei were the only regions to show

additional neuronal death between 2 weeks and 3 months after SE, and therefore, may have

resulted from SE, SRSs, or both. Overall, our results indicate that the vast majority of neuronal

death is the result of SE and that little, if any additional death, occurs subsequent to the start of

SRSs. These results are further discussed in section 4.4.3.

Our findings suggest that SE as the IPI, and not the subsequent development of SRSs, primarily

contributes to neuronal death in rats. This appears to parallel findings in human data that support

the existence of brain damage prior to development of human MTLE-HS (Mathern et al., 1996;

Wieser et al., 2004), and the lack of evidence of substantial neuropathology evolving during the

chronic stage of SRSs (Cendes et al., 1993; Bower et al., 2000; Moran et al., 2001). Regarding

the causes of brain damage, previous studies have suggested a high incidence of an IPI, which

includes febrile convulsions, SE, encephalitis, stroke or traumatic brain injury, as a predisposing

factor in human MTLE-HS (Mathern et al., 1996; Mathern et al., 2002). The most common type

of IPI recognized is prolonged febrile seizures reported in 30 to 80% of MTLE-HS patients

(French et al., 1993; Lewis, 1999; Cendes and Andermann, 2002; Mathern et al., 2002). Clinical

data determining whether MTLE-HS is a progressive disease remains ambiguous. There is

evidence from some (Briellmann et al., 2001; Briellmann et al., 2002; Fuerst et al., 2003), but not

all (Liu et al., 2002; Holtkamp et al., 2004), longitudinal MRI studies that chronic epileptic

seizures result in progressive hippocampal changes suggesting cumulative damage.

Furthermore, while some studies found increased hippocampal neuronal loss in patients with a

longer duration of temporal lobe epilepsy (Mathern et al., 1996; Mathern et al., 2002; Fuerst et

al., 2003), or those with a greater total seizure number (Kalvianinen and Salmenpera, 2002),

others have failed to establish these relationships (Cendes et al., 1993; Moran et al., 2001; Thom

et al., 2005). Still, there appears to be a general consensus that in patients with MTLE-HS, the

majority of neuronal loss occurs prior to the onset of habitual seizures, and that less substantial

neuronal loss may occur with an increased duration of the epileptic syndrome (Mathern et al.,

1996; Mathern et al., 2002; Wieser, 2004).

229

8.5 Cognitive and behavioural alterations following lithium/pilocarpine-induced SE

MTLE-HS in humans is often associated with co-morbid interictal disorders, including anxiety,

depression and psychosis (Boro and Haut, 2003; Gaitatzis et al., 2004; Devinsky, 2004a, Garcia-

Morales et al., 2008), as well as learning and memory impairments (Motamedi and Meador,

2003; Vingerhoets, 2006) (see section 1.6). Studies have shown that co-morbid psychiatric

disorders are an independent risk factor for a poor quality of life in human epilepsies, and can be

equal or more detrimental to the individual‘s overall function than the seizures themselves

(Cramer, 2002; Gilliam, 2002; Cramer et al., 2003; Boylan et al., 2004; Johnson et al., 2004).

Consequently, it is important that animal models not only recapitulate the neuropathology and

genesis of SRSs as observed in human MTLE-HS, but also allow researchers to characterize and

to identify treatments that can potentially mitigate disease-related cognitive and behavioural

morbidity (Stafstrom et al., 2006). In chapter 6, we demonstrated that SE in rats reduced

exploratory behaviour as assessed in the open field (defined in section 1.6.6.2), and increased

hyperreactivity (or aggression) as assessed in the pick-up test and in the touch-response test

(defined in section 1.6.6.3). These behavioural alterations appeared within the first week after

SE and remained unchanged by development of SRSs at 3 months (see section 8.6). SE also

produced anxiolytic effects as measured in rats by the elevated-plus maze (defined in section

1.6.6.1) at 3 months of recovery. In chapter 7, we demonstrated that rats 3 months after SE

exhibited impaired performance in the MWM (i.e., poor spatial learning and use of behavioural-

search strategies) (defined in section 1.6.7.1). These results are extensively discussed in sections

6.4 and 7.4, respectively. While we are the first to report cognitive changes (e.g., spatial learning

and memory) in rats treated by the RLDP procedure, Detour et al., (2005) is the only other group

to have characterized behavioural changes with this protocol (discussed in section 6.4.1). In

general, our data show that lithium/pilocarpine-induced SE and/or SRSs are accompanied by

profound behavioural and cognitive alterations, and thus could model affective and cognitive

disorders observed in human MTLE-HS. Specific aspects of these alterations are discussed in

the sections that follow.

8.5.1 The effect of SE on spatial learning and memory

The lithium/pilocarpine model allows us to not only investigate how seizures alter behaviour, but

also which aspect(s) of behaviour are most at risk for impairment (Stafstrom et al., 2006).

230

Acquisition of the hidden platform version of the Morris water maze (MWM) task has two main

components, behavioural strategies learning and spatial learning (see section 1.6.7.1). Although

previous studies have reported impaired spatial learning and memory in rats after SE, little

attention has been given to determine the effect of SE on behavioural strategies (see section

1.6.7.2). In chapter 7, we extended previous findings by completing a systematic assessment of

behavioural search strategies, and quantifying their contribution to overall cognitive performance

in rats after SE. We first showed that SE in rats exhibited impaired spatial learning and memory.

This finding is supported by the observations that rats after SE (1) still reached criterion in the

visual acuity test and exhibited swim speeds comparable to controls, indicating an absence in

gross motor or visual impairment, (2) performed poorly on the spatial probe tests, and (3)

predominately used repetitive-looping based search strategies and non-spatial, systematic search

strategies; these strategies do not rely on use of spatial information. Our results are consistent

with previous studies that have demonstrated poor spatial learning and memory in rats after SE

(see section 1.6.7.2).

Comparable deficits in visual-spatial learning and memory are reported in human MTLE-HS

(Hermann et al., 1997; Gleissner et al., 1998; Abrahams et al., 1999; Glikmann-Johnston et al.,

2008). For example, spatial and non-spatial, working and reference memory in MTLE-HS

patients were evaluated using a Nine-Box Maze (Abrahams et al., 1997; Abrahams et al., 1999),

a task analogous to the radial-arm maze used in rodents (reviewed in: Lanke et al., 1993;

Hodges, 1996). Results showed that MTLE-HS patients exhibited specific deficits in the

working and reference spatial memory components of the task (Abrahams et al., 1997; Abrahams

et al., 1999). Furthermore, spatial memory errors significantly correlated with volumetric

measures (e.g., degree of atrophy) of mesial temporal lobe structures, indicating a role of the

hippocampal region in spatial memory (Abrahams et al., 1999).

In another study, Glikmann-Johnston et al., (2008) assessed three measures of spatial learning in

patients with MTLE-HS: navigation, object location and plan drawing. The authors

demonstrated that MTLE-HS patients exhibited compromised behavior in all 3 measures. These

results indicate that the poor spatial learning and memory we observed in rats following SE are

comparable to cognitive deficits reported in human MTLE-HS.

231

8.5.2 The effect of SE on use of behavioural search strategies

8.5.2.1 Impaired selection of behavioural search strategies

We next showed that SE in rats dramatically impaired search strategy use in the spatial

acquisition and spatial reversal MWM tasks. Rats following SE displayed a greater proportion

of less efficient search strategies (repetitive-looping based strategies and non-spatial, systematic

strategies) while using fewer spatial strategies compared to controls (see section 7.4.2). Several

specific findings indicate that the application of poor search strategies in the SE group occurred

as a result of behavioural inflexibility (e.g., the inability to select appropriate behavioural

strategies):

(1) Although the SE group showed impaired MWM performance over the entire 14 days of

acquisition training compared to controls, the majority of these animals were able to

reduce escape latencies to finding the platform within this period (discussed in section

7.4.1). By conducting the convolution analyses (described in section 7.3.8), we showed

that changes in search strategy use primarily contributed to improved MWM performance

in these animals. This finding is further discussed in section 7.4.3, and suggests that the

higher escape latencies in rats after SE may not be solely attributed to an inability to

acquire behavioural strategies. Although rats after SE required more trials to pass the

visible platform test, indicating impairment in behavioural strategies, these animals were

still capable of acquiring the necessary skills of swimming away from the wall of the

pool and climbing onto the escape platform.

(2) We noted that although the SE group was capable of acquiring spatial memory, these

animals continued to predominately select less efficient search strategies and to perform

poorly in the spatial probe tests. This observation was supported by the fact that the SE

group showed improvement in use of spatial strategies during spatial acquisition.

Moreover, when the platform was changed to the opposite quadrant, these animals

exhibited an increase in escape latencies that correlated with the use of fewer spatial

strategies, and the use of more non-spatial, systematic strategies. These findings are

further discussed in section 7.4.4.

Altogether, our results suggests that rats after SE are capable of acquiring behavioural strategies,

and are even capable of using spatial search strategies. However, SE elicited in rats may cause

deficits in the ability to inhibit non-efficient escape strategies (i.e., behavioural inflexibility).

232

These results are comparable to previously reported observations in rats treated with

anticholinergic drugs (Whishaw, 1989; Whishaw and Tomie, 1987; Day and Schallert, 1996), in

rats and mice following traumatic brain injury (Brody and Holtzman, 2006; Thompson et al.,

2006), and in rats with lesions to specific brain structures (i.e., hippocampus, caudate-putamen)

(Whishaw and Petrie, 1988; Day et al., 1999). These studies used different testing paradigms in

the MWM task (see chapter 9) to dissociate spatial learning, behavioural-strategy learning and

the ability to switch between behavioural strategies. Although the experimental rats in these

studies were capable of behavioural strategy learning and spatial learning, in adaptive testing

paradigms (i.e., where animals were required to switch use of behavioural strategies), they

perseverated in using the least efficient strategy (see section 9.2).

8.5.2.2 Impaired acquisition of behavioural search strategies

One of our main findings was that although the majority of rats with SE exhibited impaired

MWM performance there were between animal differences in the ability to improve performance

over prolonged training, and we were able to subdivide rats into three categories based on

performance during spatial acquisition. These results are discussed in section 7.4.5. Of the

animals that showed impaired MWM performance compared to controls, 44% learned how to

use more efficient search strategies, and the increased use of these strategies accounted for their

improved performance, suggesting that they were capable of acquiring new behavioural

strategies. In contrast, 34% of the rats showed no improvement in escape latencies or changes in

search strategy use with extended training.

As noted above, acquisition of the MWM has two main components: behavioural strategy

learning and spatial learning. The animal must first acquire behavioural strategies before success

in spatial learning can be achieved. Acquisition of behavioural strategies involves the rat

learning how to navigate in its spatial environment and using the most effective strategies for

searching and reaching its target (see section 1.6.7.1). Failure to swim away from the wall to

search the inner region of the pool can inflate escape latencies, and reduce the usefulness of

escape latencies as a measure of spatial learning (Schenk and Morris, 1985; Whishaw and

Tomie, 1987; Cain, 1997). Although elevated escape latencies are most often interpreted as a

spatial learning impairment, others have reported dissociations between this measure and more

specific and accurate measures of spatial learning (Schenk and Morris, 1985; Cain, 1997).

233

Consequently, escape latencies should be interpreted cautiously, preferably in conjunction with

behavioural strategy analyses, and other more specific and accurate measures of spatial memory

(e.g., probe test analyses). Several authors have suggested that under conditions of marked

thigmotaxic swimming, elevated escape latencies are best interpreted as impairment in

behavioural strategy learning (Schenk and Morris, 1985; Morris, 1989; Whishaw, 1989; Cain,

1997). The subset of rats in our study that exhibited no improvement during spatial acquisition

had parallel impairments in both escape latencies and thigmotaxic swimming, indicating that

they were also impaired in the acquisition of behavioural strategies.

In sum, we showed that both impairment in search strategy use, and poor spatial learning and

memory, contribute to the impaired performance of epileptic rats during spatial learning tasks in

the MWM. The ability to both learn behavioural strategies and to select the most appropriate

behavioural strategy appears to operate on overlapping and separable neural systems. For

instance, the ability to select between allocentric (e.g., based on distal cues) and egocentric (e.g.,

based on placement in the environment) search strategies relies on a competitive interaction

between the hippocampal and the striatal neural systems, respectively (see section 7.4.4). The

amygdala has also been shown to influence search strategy selection (Packard, 2009; Hawley et

al., 2011). In contrast, behavioural strategy learning has been shown to require multiple neural

systems, including the prefrontal cortex, striatum, cerebellum, medial thalamus, and

hippocampus (see section 1.6.7.1). Although in the present study it appears that SE in rats

impaired the ability to select the most appropriate search strategy, a subset of these rats may

instead exhibit deficits in behavioural strategy learning. Future studies using different testing

paradigms are necessary to more directly delineate the effect of SE on spatial learning,

behavioural strategy learning, and behavioural flexibility (see chapter 9).

8.5.3 Impaired use of behavioural strategies in human MTLE-HS

Patients with MTLE-HS may have analogous poor use of behavioural strategies. Recent studies

indicate that MTLE may affect decision-making under ambiguity (Labudda et al., 2009; Delazer

et al., 2010; Delazer et al., 2011; Yamano et al., 2011;), which uses implicit information and

requires processing of feedback and reward-based, adaptive learning (Brand et al., 2006). In

these studies, decision-making under ambiguity was assessed by performance in the Iowa

Gambling Task (IGT). The IGT is proposed to simulate decision-making in real-life situations in

234

which premises, outcomes, rewards or punishments are uncertain (Brand et al., 2006). In the

IGT, MTLE-HS patients were reported as having shifted more often between advantageous and

disadvantageous selections than controls, and had difficulties in establishing a consistent

response pattern (Labudda et al., 2009; Delazer et al., 2010; Yamano et al., 2011; Delazer et al.,

2011). The authors suggested that MTLE-HS patients selected decks randomly and were not

capable of adapting their strategies from feedback.

It has been suggested that decision-making abilities under ambiguity, as measured by the IGT,

are separable from cognitive abilities, including executive functions and intelligence (Brand et

al., 2006; Delazer et al., 2010; Toplak et al., 2010). For instance, recent studies compared

decision-making under ambiguity and decision-making under risk in MTLE-HS patients. Risk

refers to the form of uncertainty where probabilities are known, as opposed to ambiguity where

outcome probabilities are incompletely known (Brand et al., 2006). While MTLE-HS patients

showed severe deficits in decision-making under ambiguity in the IGT, they perform normally in

decision-making under risk as assessed by the game of dice task (GDT) (Labudda et al., 2009;

Delazer et al., 2010), or by the probability-associated gambling task (PAG) (Delazer et al.,

2010), both of which offers explicit probabilities for gains and losses. Brand et al., (2006)

suggested that decision-making under ambiguity and decision-making under risk both require a

feedback route, in which experience in terms of gains and losses is processed. However,

decision-making under risk also uses a cognitive route, in which executive functions use explicit

information to plan and modify decision-making behaviour. This is supported by the fact that

GDT performance has been shown to correlate with executive functions (Brand et al., 2006), but

not with decision-making under ambiguity as assessed by the IGT (Brand et al., 2007; Brand et

al., 2008). Likewise, Toplak et al., (2010) reviewed 43 studies and showed that the majority of

these found no positive correlation with IGT performance and other cognitive abilities, including

measures of inhibition, working memory, and set-shifting as indices of executive functions, as

well as measures of verbal, nonverbal and full-scale IQ as indices of intelligence. Based on this

literature, Labudda et al., (2009) and Delazer et al., (2010) suggested that brain regions

underlying executive functions are preserved, and that this enabled MTLE-HS patients to make

decisions based on explicit, but not implicit, information.

Several lines of evidence suggest that impaired decision-making abilities in MTLE-HS patients

are caused by lesions specific to the temporo-limbic system. First, a recent study showed that

235

IGT performance in patients with MTLE-HS was worse than patients with neocortical TLE

(characterized by lesions in the lateral, basal or polar parts of the temporal lobe) (Delazer et al.,

2011). Although patients with neocortical TLE and controls showed a significant learning effect

over the blocks of the IGT, performance of patients with MTLE-HS showed no improvement

from the first to the last block of trials. These results suggest that TLE patients with lesions

associated with the mesial temporal lobe have greater deficits in decision-making compared to

TLE patients with lesions associated with the neocrotex. (Delazer et al., 2011). Previous studies

have similarly found impaired IGT performance in patients with MTLE who have lesions in the

amygdalo-hippocampal complexes (Bonatti et al., 2009; Labudda et al., 2009; Delazer et al.,

2010). Second, the IGT detected deficits with decision-making abilities in individuals with

lesions in the amygdala and/or hippocampus (Bechara et al., 1999; Gutbrod et al., 2006; Gupta et

al., 2010). Finally, a brain imaging study using functional connectivity analyses provided

evidence that the amygdala and hippocampus are involved in reward-based learning, and the

strength of the connection between these two structures predicts flexibility in adapting decisions

(Cohen et al., 2008).

In sum, we showed that rats after SE used suboptimal search strategies during spatial learning

tasks in the MWM. This finding may be analogous to specific deficits in decision-making under

ambiguity observed in human MTLE-HS. In both conditions, acquisition of behavioural (or

decision-making) strategies relies on use of implicit information, and requires acquisition and

adaption of behavioural strategies based on experience. Furthermore, the neural systems

involved in acquisition and selection of behavioural strategies appear to rely on neural networks

dissociable from cognitive abilities (e.g., spatial learning in rats; executive functions and

intelligence in humans). Finally, human and rodent studies suggest that lesions within limbic

structures may contribute to impaired use of behavioural strategies.

8.6 The temporal relationship between neuronal death, behavioural alterations and cognitive impairment following status epilepticus

Because we conducted a detailed time-course evaluation of SE-induced neuronal death (see

section 8.4), we were able to compare the progression of neuronal death with the occurrence of

behavioural alterations in rats following SE. Our findings showed that neuronal loss occurs

236

within the hippocampus, amygdala, thalamus and piriform cortex as early as 3 to 12 hours

following SE, with the majority of neuronal death present by 24 hours (see section 8.4).

Although we demonstrated SE-induced behavioural changes in the hyperexcitability tests and in

the open field within the first week after SE that persisted for at least 12 weeks (see chapter 6),

previous studies have demonstrated the onset of behavioural alterations and cognitive

impairment as early as 24 to 48 hours (see section 6.4.2). For example, pilocarpine-treated rats

exhibited increased aggression to handling and increased hyperexcitability as early as 2 days

after SE (Rice et al., 1998), and kainic acid-treated rats showed increased hyperreactivity in

response to handling and increased activity in the open field 24-hours after SE (Milgram et al.,

1988). In both studies, behavioural alterations persisted 6 to 8 weeks later (Milgram et al., 1988;

Rice et al., 1998). Similar results were obtained in cognitive tasks (see Table A1-2). Chauvière

et al., (2009) recently demonstrated impaired performance in the displaced object-recognition

task at 4 days following pilocarpine-induced SE, the earliest time assessed, with deficits in

spatial memory and object recognition still present at 40 days. Others reported impaired visual-

spatial learning in the MWM when testing was conducted 5 to 7 days following SE induced by

either kainate or pilocarpine (Milgram et al., 1988; Rice and DeLorenzo, 1999; Cunha et al.,

2009; Sun et al., 2009). Even though Hort et al., (2000) pre-trained rats for 8 days prior to SE

onset, animals still exhibited impaired spatial memory 3 and 6 days following pilocarpine-

induced SE (Hort et al., 1999). Because SE animals are in very poor physical condition

immediately following SE, it is difficult to assess the behavioural and cognitive ability of rats

less than 1 or 2 days following SE so that the time of the first appearance of impaired behavior

remains unknown. In these studies, cognitive impairments persisted for up to 2 months,

indicating no recovery of behavioural function (Milgram et al., 1988; Rice and DeLorenzo, 1999;

Cunha et al., 2009; Sun et al., 2009). These findings, together with our data, indicate that

neurodegeneration, behavioural alterations and cognitive impairment develop within the same

timeframe and are consistent with a functional relationship between these processes.

237

8.7 The effect of neuroprotection on epileptogenesis, behavioural alterations, and cognitive impairment

8.7.1 Neuroprotection within the hippocampus

8.7.1.1 Effect of neuroprotection on epileptogenesis

Human and animal studies suggest hippocampal sclerosis is involved in the development of TLE

and in the genesis of behavioural and cognitive deficits (Engel et al., 1991; Engel, 1996; Mathern

et al., 1996; Sloviter, 1999; Acharya et al., 2008). In chapter 5, we showed that tat-NR2B9c

provided partial neuroprotection of hippocampal pyramidal cells (CA1, CA3 and CA4) when

administered 3 hours following SE. This result allowed us to determine whether partial

neuroprotection within the hippocampus reduced the incidence of SRSs, or modified behavioural

and cognitive alterations in rats following lithium/pilocarpine-induced SE. In spite of its

neuroprotective effect, tat-NR2B9c had no effect on the number of rats developing SRSs

(discussed in section 6.4.3), on behaviour as assessed by the open field, elevated plus maze or

hyperexictability tests (discussed in 6.4.3), or on MWM performance (see section 7.4.6). Our

data show that partial neuroprotection of CA1, CA3 and CA4 pyramidal cells within the dorsal

hippocampus was insufficient to prevent development of SRSs, or to modify behavioural and

cognitive alterations in rats following SE.

Table 8.1 provides a comparison of literature that investigated the effect of various

neuroprotective drugs on the development and occurrence of SRSs, behavioural alterations, and

learning and memory impairments in post-status epilepticus models. Numerous other studies are

in agreement with our findings that partial neuroprotection of the hippocampus does not prevent

the development of epilepsy (Rice et al., 1998; Halonen et al., 2001; Capella and Lemos, 2002;

Ebert et al., 2002; Brandt et al., 2003b; Rigoulot et al., 2003; Rigoulot et al., 2004; dos Santos et

al., 2005; Francois et al., 2006; Frisch et al., 2007; Chu et al., 2008). Even with nearly complete

neuroprotection of the hippocampus, SRSs in rats following SE still occurred (Rice et al., 1998;

Andre et al., 2007; Ebert et al., 2002; Brandt et al., 2006; Nehlig, 2007). In contrast to our

findings and these studies, Bolanos et al., (1998) reported partial neuroprotection of the

hippocampus and a decrease in the incidence of SRSs in rats treated with valproate (AED) for

forty days following kainate-induced SE; however, because the occurrence of SRSs was

monitored during the administration of valproate, it is difficult to determine whether the reduced

238

incidence of SRSs in these animals resulted from the partial neuroprotection of the hippocampus,

or from the anticonvulsant effect of the drug. Jung et al., (2006) also reported that treatment of

rats following lithium/pilocarpine-induced SE with celecoxib (a COX-2 inhibitor) offered partial

neuroprotection of the hippocampus and reduced the incidence of SRSs; however, celecoxib also

reduced the generation of ectopic granule cells in the hilus and reduced gliosis in the CA1,

making it difficult to determine which if any of these drug effects were responsible for reducing

the incidence of SRSs. The development of SRSs in the absence of overt brain damage in other

seizure models further suggests that neuronal loss is not a prerequisite for epileptogenesis (see

section 1.5.1.6), and indicates that other factors such as synaptic reorganization (see section

1.5.2), reactive gliosis (see section 1.5.3) and/or neurogenesis (see section 1.5.4) may play a

more important role in epileptogenesis.

Although we did not assess the effect of tat-NR2B9c on neuronal loss within the dentate hilus,

others have demonstrated that most neuroprotective agents either have no effect or result in only

partial neuroprotection of this region (Halonen et al., 2001; Rigoulot et al., 2003; Chu et al.,

2008; Pitkänen et al., 2004; Rigoulot et al., 2004; Pitkänen et al., 2005; Francois et al., 2006;

Jung et al., 2006; Zhou et al., 2007), even if other hippocampal subfields (e.g., CA1, CA3 and

CA4) are completely protected (Rice et al., 1998; Ebert et al., 2002; Brandt et al., 2003b; Andre

et al., 2007) (see Table 8.1). These results indicate that neurodegeneration in the dentate hilus is

more resistant compared with other hippocampal regions to neuroprotective agents (Löscher and

Brandt, 2010). Loss of dentate hilus cells is a characteristic finding in most post-status

epilepticus rodent models (Loscher, 2002; Martin and Pozo, 2006; Sharma et al., 2007; Löscher

and Brandt, 2010), and is the most consistent cell loss reported in human MTLE-HS (Sloviter,

1994; Lowenstein, 2001; Nadler, 2003; Blümcke et al., 2007; Thom et al., 2009). Hilar cell loss

involves both excitatory mossy cells and inhibitory peptide-containing interneurons (Sloviter,

1987). There are presently two controversial explanations for how hilar cell loss may result in

hyperexictability of the dentate granule cells, which could be causal for increased seizure

susceptibility or the development of SRSs. These explanations may also account for the lack of

effect neuroprotection within other hippocampal subfields has on SRSs.

1. The ‘dormant basket cell’ hypothesis postulates that degeneration of mossy cells results

in reduction of afferent excitatory synaptic input onto insult-resistant inhibitory basket

cells, rendering these cells ‘dormant’ and granule cells hyperexcitable (Sloviter, 1987;

239

Sloviter, 1989; Sloviter et al., 1991; Sloviter et al., 2003). However, the validity of this

theory has been widely challenged on both anatomical and physiological grounds

(reviewed in: Bernard et al., 1998; Ratzliff et al., 2004; Dudek and Sutula, 2007).

2. Alternatively, loss of inhibitory interneurons in the hilus may lead to reduced GABAergic

synaptic input to granule cells that could contribute to abnormal recurrent excitation of

granule cells found in epileptic rats (Kobayashi and Buckmaster, 2003; Ratzliff et al.,

2004; Sun et al., 2007).

A few studies have suggested that epileptogenesis may develop independent of hilar cell loss in

post-status epilepticus rodent models. Numerous studies have confirmed that partial reduction in

SE-induced hilar cell loss by various neuroprotective agents had no effect on the development of

epilepsy (Ebert et al., 2002; Pitkänen et al., 2004; Francois et al., 2005; Pitkänen et al., 2005;

Francois et al., 2006; Jung et al., 2006; Chu et al., 2008) (see Table 9.1). Even though Brandt et

al., (2006) reported complete neuroprotection of the hippocampus and hilus in rats treated with

valproate following electrically-induced SE, this effect was still not sufficient to prevent genesis

of SRSs.

8.7.1.2 Neuroprotection may offer disease-modifying effects

Even though (partial) neuroprotection within the hippocampus did not prevent the development

of chronic epilepsy, it may never the less offer disease-modifying effects. The literature on this,

however, remains controversial. For instance, partial neuroprotection within the hippocampus

has been shown to increase the latency period (Francois et al., 2005) or to reduce the frequency

and severity of SRSs (Bolanos et al., 1998; Pitkänen et al., 2004; Francois et al., 2005; Jung et

al., 2006; Chu et al., 2008) in rats following SE (see Table 8.1). In contrast, Pitkanen et al.,

(2005) and Ebert et al., (2002) showed that partial neuroprotection of the hippocampus increased

the severity of epilepsy; more specifically, the number of surviving hilar cells positively

correlated with the frequency of SRSs. Still, others failed to establish a relationship between

partial (Capella and Lemos, 2002; Brandt et al., 2003b; Narkilahti et al., 2003b; Rigoulot et al.,

2003; Rigoulot et al., 2004; Pitkänen et al., 2004; Francois et al., 2006; Frisch et al., 2007) or

complete (Brandt et al., 2006) neuroprotection of the hippocampus and the severity of SRSs.

A series of studies suggest that neuroprotection within the hippocampus may even play a role in

the prognosis of treatment for epilepsy. These experiments showed that rats developing epilepsy

240

after an episode of SE induced by sustained electrical basolateral amygdala stimulation markedly

differed in their response to phenobarbital (Brandt et al., 2004; Volk et al., 2006; Bethmann et

al., 2008). While approximately 30% of rats did not respond to treatment, SRSs were suppressed

in the other animals, resulting in two subgroups (i.e., responders and nonresponders).

Nonresponders exhibited hippocampal damage, whereas most responders exhibited no overt

brain damage, suggesting a causal relationship between neuronal damage and

pharmacoresistance (Volk et al., 2006; Bethmann et al., 2008). This data is consistent with the

clinical observation that hippocampal sclerosis is associated with poor prognosis of AED

treatment in patients with TLE, but is a good indicator for a positive outcome to surgical

treatment (Schmidt and Löscher, 2005).

8.7.1.3 The effect of neuroprotection on behavioral alterations

The effect of neuroprotection within the hippocampus on SE-induced cognitive and behavioural

morbidity also remains ambiguous. Other studies are in agreement with our findings that partial

neuroprotection within the hippocampus had no effect on SE-induced cognitive and/or

behavioural morbidity (Cilio et al., 2001; Halonen et al., 2001; Narkilahti et al., 2003b; Pitkänen

et al., 2004; Zhou et al., 2007). In contrast to these results, several studies have demonstrated

that partial (Bolanos et al., 1998; Cilio et al., 2001; dos Santos et al., 2005; Frisch et al., 2007;

Cunha et al., 2009; Jun et al., 2009) or almost complete neuroprotection (Rice et al., 1998;

Brandt et al., 2006) of the hippocampus reduced behavioural alterations or improved learning

and memory in rats after SE. The results of these studies are shown in Table 9.1, and are

discussed in sections 6.4.3 (behavioural alterations) or 7.4.6 (impaired learning and memory).

8.7.2 Effect of neuroprotection in extrahippocampal regions

Although limbic seizures have often been attributed to hippocampal pathology, others have

provided evidence that the parahippocampal regions, including the piriform and entorhinal

cortices, may also play an important role. Francois et al., (2005) showed that simultaneous

protection of the hippocampus, parahippocampal cortices, amygdala and thalamus increased the

latency to SRSs; the latency to the first SRS was correlated with neuronal damage in the piriform

and entorhinal cortices, but not in the hippocampus and amygdala. Similarly, André et al.,

(2003) showed that partial neuroprotection (by pregabatrin) in layer II of the piriform cortex and

in layers III and IV of the entorhinal cortex, and without any neuroprotection within the

http://www.ncbi.nlm.nih.gov.myaccess.library.utoronto.ca/pubmed?term=%22Andr%C3%A9%20V%22%5BAuthor%5D

241

hippocampus, caused an increase in the latency to SRSs (see Table 8.1). In a separate study,

André et al., (2007) demonstrated that only simultaneous protection of the hippocampus and the

parahippocampal cortices (plus the amygdala and thalamus by carisbamate) was able to delay or

completely block the occurrence of SRSs, whereas treatments protecting only CA1 and/or CA3

(by caffeine, topiramate, vigabatrin, and amygdala kindling) were not effective. Based on this

set of experiments, the authors suggested that neurodegeneration in the piriform and entorhinal

cortices is a critical factor early in the epileptogenic process, whereas the involvement of the

hippocampus is delayed (Andre et al., 2007; Nehlig, 2007). Still, even with drugs like

carisbamate that offered nearly complete neuroprotection of the hippocampal formation and

parahippcampal cortices, some rats still developed epilepsy (Andre et al., 2007), suggesting that

simply preventing neuronal loss may not be enough to prevent epileptogenesis.

Our findings support the general consensus that neuroprotection, as narrowly defined as

prevention of cell death, is not sufficient to prevent epilepsy (Sankar, 2005). Walker et al.,

(2007) argued that neuroprotection after SE should encompass not only the prevention of

neuronal death, but also the preservation of neuronal and network function. In general, the

majority of patients with MTLE-HS exhibit at least several epileptogenic structures (including

not only the lesional site but also other localized and distant sites) that form complex seizure

networks (Bartolomei et al., 2008b; Bartolomei et al., 2010), and the fact that SRSs can be

induced in rats in the absence of neuronal death, argues against damage in any ‗key‘ or ‗critical‘

brain region as being a prerequisite for epileptogenesis.

No drug has yet been found to prevent or modify the epileptogenic process induced by a brain

insult in humans (Temkin, 2001; Sossa, 2006; Temkin, 2009). Clinical trials conducted have

largely focused on the effect of conventional anti-epileptic drugs (AEDs), including phenytoin,

phenobarbital, carbamazepine, or valproate, in the prevention of epilepsy or disease-

modification. Not surprisingly, AEDs have thus far failed for several proposed reasons (Temkin,

2001; Sossa, 2006; Temkin, 2009): (1) AEDs have been developed for symptomatic suppression

of seizures and not for the prevention or modification of the epileptogenic process, and (2) the

molecular mechanisms underlying epileptogensis and ictogenesis are fundamentally different

(reviewed in: Loscher, 2002; Weaver, 2003). Further clinical trials are necessary to determine

whether new anti-epileptogenic drugs, if any, developed in the laboratory are effective in

humans.


242

Table 8.1 Consequences of neuroprotective drug treatment

Drug Seizure model SE duration

(limited by)

Beginning of drug

treatment

Duration of drug

treatment

1. Atipamezole (α2

receptor antagonist)

Amygdala stimulation Exp 1: 3 h

(diazepam); Exp 2:

not limited

1 week after SE 9 weeks (minipump

implant 100 μg/kg/h)

2. Preconditioning by

amygdala kindling

LDP protocol

lithium/pilocarpine

2 h (diazepam) Preconditioning with fully

kindled amygdala seizures

preceding SE

N/A

3. Carbamazepine

(AED)

Kainate Not limited 1 day after SE 40 mg/kg, i.p., 3

times/day, for 56 days

4. Carbamazepine Pilocarpine (right

dorsal hippocampus)

3 h (thiopental) 1 hr after 3 h SE 120 mg/kg, i.p., for 4

days

5. Carisbamate (RWJ-

333369;

neuromodulator)

LDP protocol

lithium/pilocarpine

2 hr (diazepam) Immediately after SE

onset

60, 90, and 120 mg/kg,

i.p., 2 times/day, for 7

days

6. Carisbamate LDP protocol

lithium/pilocarpine

1 h (diazepam in

controls)

1 h after SE onset 90 and 120 mg/kg, i.p.,

2 times/day, for 6 days

7. Caffeine LDP protocol

lithium/pilocarpine

2 h (diazepam) 14 days prior and 7 days

after SE

0.3 g/L in drinking

water, 14 days prior and

7 days after SE

8. Caffeine LDP protocol

lithium/pilocarpine

2 h (diazepam) 15 days prior and 7 days

after SE

0.3 g/L in drinking

water, 14 days prior and

7 days after SE

9. Celecoxib (COX-2

inhibitor)

LDP protocol

lithium/pilocarpine

1 h (diazepam) 1 day after SE Oral administration, 20

mg/kg, for 14 days

10. Cycloheximide

Pilocarpine

1.5 h (thionembutal) 20 min prior to SE onset 1mg/kg, s.c., for 1 dose

11. z-DEVD-fmk

(caspase-3 inhibitor)

Amygdala stimulation 3 h (diazepam) 3 h after SE onset 6 µg/d/i.c.v., for 1 week

12. Diazepam

(GABAA agonist)

Pilocarpine (right

dorsal hippocampus)

3 h (thiopental) 1 hr after 3 h SE 20 mg/kg, i.p., for 4

days

13. Dizocilpine

(NMDAR antagonist)

Kainate 1.5 h (diazepam) Immediately after

termination of SE

0.1 mg/kg, i.p., for 1

dose

14. Dizocilpine Kainate 1.5 h (diazepam) Immediately after

termination of SE

0.1 mg/kg, i.p., for 1

dose

Abbreviations: h, hour; LDP, N/A, not applicable; low-dose lithium/pilocarpine protocol; i.p., intraperitoneal injection; i.c.v.,

intracerebral injection; min, minutes; P, postnatal day; RLDP, repeated low-dose lithium/pilocarpine protocol; s.c., subcutaneous

injection

243

Table 8.1 (continued)

Neuroprotective effects

Latency to

SRSs

Incidence

of SRSs

Frequency,

severity or

duration of

SRSs

Behavioural

alterations

Impairment

of learning

and memory

References

1. ↓Hilus (p) N.D. N.E. ↓ N.D. N.E. (Pitkänen et al., 2004)

2. ↓ CA1 (c), ↓ CA3 (c),

↓ amygdala (c), ↓

piriform cortex (c), ↓

layer II of entorhinal

cortex (c), ↓ layers III/IV

of entorhinal cortex (p),

↓ hilus (p)

N.E. N.E. N.E. N.D. N.D. (André et al.,

2007)

3. ↓ Hippocampus (p) N.D. N.E. ↓ N.D. N.D. (Capella and

Lemos, 2002)

4. ↓CA1 (p), ↓CA4 (p),

N.E. in CA3

N.D. N.D. N.D N.D. ↓ (Cunha et al.,

2009)

5. ↓ Layer II piriform

cortex (c), ↓ layers III-IV

entorhinal cortex (c), ↓

CA1 (p; dose-related),

N.E. in CA3 and hilus.

Decreased

(52-85 vs 16

days)

↓ N.E. N.D. N.D. (André et al.,

2007)

6. ↓CA1 (p), ↓PC (p),

↓EC (p), ↓amygdala (p),

↓thalamus (p)

Increased –

correlated

with # of

neurons in

PC and EC

N.E. N.D. N.D. N.D. (Francois et al.,

2005)

7. ↓ CA1 (c), ↓ CA3 (p),

↑ layer III of piriform

cortex, N.E. in hilus,

N.E. N.E. N.E. N.D. N.D. (André et al.,

2007)

8. ↓ CA1 (c), ↓hilus (p),

↑layers III-IV PC, N.E.

in EC

N.E. N.E. N.E. N.D. N.D. (Rigoulot et al.,

2003)

9. ↓ CA1 (p), ↓CA3 (c),

↓hilus (p)

N.D ↓ ↓ N.D. N.D. (Jung et al.,

2006)

10. ↓Dorsal hippocampus

(N.S.), ↓(p)

N.D. N.E. N.E. ↓ N.E. (dos Santos et

al., 2005)

11. ↓ CA1 (p), ↓CA3 (p),

↓hilus (p)

N.D. N.E. N.E. N.D. N.E. (Narkilahti et

al., 2003b)

12. ↓CA1 (p), ↓CA3c

(p), ↓hilus (p), ↓layers II-

III PC (p), ↓EC (p),

↓amydala (p)

N.D. N.E. Increased (positively

correlated with #

of hilar neurons)

N.D. N.D. (Pitkänen et al., 2005)

13. ↓layers II-III PC (p),

↓CA1 (c), ↓CA3 (c),

Hilus (p), ↓EC (N.S.),

↓amygdala (N.S.)

N.D. N.D. Increased

(positively

correlated with

# of hilar

neurons)

N.D. N.D. (Ebert et al.,

2002)

14. ↓CA1 (c) N.D. N.E. N.D. ↓ ↓ (Rice et al.,

1998) -

behaviour

(Mello et al.,

1993) –

histopathology

Abbreviations: ↓, beneficial effect of drug treatment; ↑, drug treatment exacerbates neuronal loss, (A) studies that are only

available as abstracts; (c), complete neuroprotection; EC, entorhinal cortex; ND, not determined; NE, drug treatment is not

effective; NS, not specified; NC, nearly complete neuroprotection (>90% of neurons remaining); (p), partial neuroprotection; PC,

piriform cortex




244


Drug Seizure model SE duration

(limited by)

Beginning of drug

treatment

Duration of drug

treatment

15. Dizocilpine Pilocarpine 1 h (diazepam) 20 min prior to

pilocarpine injection

0.1 mg/kg, i.p., for 1 dose

16. Erythropoietin (renal

cytokine)

LDP protocol

lithium/pilocarpine

1 h (diazepam) 1 h after SE onset 5000 IU/kg in PBS, i.p.,

for 7 days

17. Erythropoietin LDP protocol

lithium/pilocarpine

1 h (diazepam) 4 h before SE onset 10 U/g, i.p., for 1 dose

18. Gabapentin (AED) Kainic acid (P35) Not limited 1 day after SE 200 mg/kg, i.p., for 10

days

19. Ketamine (NMDAR

antagonist)

Pilocarpine (right

dorsal hippocampus)

3 h (thiopental) 1 hr after 3 h SE 50 mg/kg, i.p., for 4 days

20. Lamotrigine

(AED)

Perforant path stimulation

(PPS)

2 h after end of

PPS (diazepam)

1 h after SE onset 12.5 mg/kg, i.p., 2


21. Levetiracetam

(AED)

LDP protocol

lithium/pilocarpine

2 hour

(pentobarbital)

24 h after SE onset 50 mg/kg, i.p., 2

times/day, for 2 weeks

22. Phenytoin

(AED)

Pilocarpine (right dorsal

hippocampus)

3 h (thiopental) 1 hr after 3 h SE 60 mg/kg, i.p, for 4 days

23. Pregabalin

(AED)

LDP protocol

lithium/pilocarpine


onset

50 mg/kg, for initial 7

days, 10 mg/kg for

remaining days (until

sacrificed)

24. Pregabatrin

(AED)

LDP protocol

lithium/pilocarpine

2 h (diazepam) 20 min after pilocarpine 50 mg/kg, i.p., for 6 days

(histology), 10 mg/kg, i.p.,

for remaining 55 days

(monitoring SRSs)

25.Retigabine

(potassium channel

opener)

Kainate 1.5 h (diazepam) 0, 1 and 2 h after

termination of SE

3 mg/kg, i.p., for 3 doses

26. Tat-NR2B9c

(synthetic peptide)

RLDP protocol

lithium/pilocarpine

1 h (diazepam) 3 h after SE onset 3 nmol/g, i.p., for 1 dose

27. Topiramate

(AED)

LDP protocol

lithium/pilocarpin


onset

Doses varying 10 – 60

mg/kg, i.p., 2 times/day,

for 7 days

28. Topiramate

LDP protocol

lithium/pilocarpine

1 h (diazepam in

controls)

1 h and 10 h after SE

onset

10 mg/kg or 30 mg/kg, i.p.

2 times/day, for 7 days

29. Topiramate LDP protocol

lithium/pilocarpine

2 h (diazepam) Topiramate at SE onset

and 10 hr later,

diazepam at 2 h and 10

h after SE onset

10 mg/kg, 30 mg/kg, or 60

mg/kg, i.p., for 7 days



injection

245



Latency to SRSs

Incidence of SRSs

Frequency, severity or duration of SRSs

Behavioural alterations

Impairment of learning and memory

References

15. ↓ anterior PC (c),

posterior PC (p), ↓CA1 (p),

↓CA3 (p), ↓thalamus (p),

↓amygdala (p), N.E. in

hilus, substantia nigra pars

reticulata and EC

N.D. N.E. N.E. N.D. N.D. (Brandt et

al., 2003b)

16. ↓CA1(p), ↓CA3 (c),

↓hilus (p)

N.D. N.E. ↓ N.D. N.D. (Chu et al.,

2008)

17. ↓CA1 (p), ↓CA3 (p)

N.D. N.D. N.D. N.D. ↓ (Jun et al.,

2009)

18.↓CA1 (p), ↓CA3 (p) N.D. N.D. N.D. N.E. N.E. (Cilio et al.,

2001)

19. ↓ CA1 (p), ↓ CA3 (p),

N.E. in CA4

N.D. N.D. N.D N.D. ↓ (Cunha et

al., 2009)

20. ↓CA3 (p), ↓hilus (p),

↓PC (p), ↓EC (p), ↓

amygdala (p), NE in CA1

N.D. N.E. N.D. N.D. N.E. (Halonen et

al., 2001)

21.↓ CA1 (P), ↓CA3 (p),

↓hilus (p),

N.D. N.E. N.D. N.D. N.E. (Zhou et al.,

2007)

22. ↓ CA1 (p), ↓ CA3 (p),

N.E. in CA4

N.D. N.D. N.D N.D. ↓ (Cunha et

al., 2009)

23. ↓Layer II PC (p),

↓Layers III-IV EC (p), N.E.

in hippocampus

Increased N.E. N.E. N.D. N.D. (André et al., 2003)

24. ↓Layer II PC (p),

↓Layers III-IV EC (p), N.E.

in hippocampus

Increased N.E. N.E. N.D. N.D. (André et al., 2003)

25.↓layers II-III PC (p),

N.E. in hippocampus, PC,

EC and amygdala

N.D. N.E. N.E. N.D. N.D. (Ebert et al.,

2002)

26. ↓CA1 (p), ↓CA3 (p),

↓CA4(p), N.E. in PC

N.D. N.E. N.D. N.E. (results not

published – see

thesis chapter 6)

N.E. (results not

published – see

thesis chapter 7)

(Dykstra et

al., 2009)

27. ↓CA3 (c), ↓CA1 (p),

N.E. in hilus, EC and PC

N.E. N.E. N.E. N.D. N.D. (André et al., 2003)

28. ↓CA1 (p), ↓CA3 (p)

(only at 30 mg/kg drug

dose), N.E. in hilus, DG,

EC, and PC

N.E. N.E. N.E. N.D. N.D. (Rigoulot et

al., 2004)

29. ↓CA1 (p), ↓hilus (p),

↓layers III-IV EC (p), N.E.

in CA3 and PC

N.E. N.E. N.E. N.D. N.D. (Francois et

al., 2006)


available as abstracts; (c), complete neuroprotection; EC, entorhinal cortex; ND, not determined; NE,not effective; NS, not

specified; (p), partial neuroprotection; PC, piriform cortex

246


Drug Seizure model SE duration (limited

by)

Beginning of drug

treatment

Duration of drug

treatment

30. Topiramate

Pilocarpine 40 min (diazepam only

in controls); 40 min

(topiramate, 2 hr

diazepam in

experimental drug

groups)

Topiramate at 40 min

after SE onset,

diazepam at 2 hr after

SE onset in

experimental drug

groups

20 mg/kg or 100

mg/kg, i.p., for 1 dose

31. Valproate (AED)

Kainate (P35) Not limited in controls 24 h after SE 600 mg/kg, i.p., 2

times/day, for 30

days, tapered to 300

mg/kg, i.p., 2

times/day for final 10

days

32. Valproate

Amygdala

stimulation

4 h (diazepam) 4 hr after SE onset 4 weeks

33. Vigabatrin

(inhibition of GABA

transaminase)

LDP protocol

Lithium/pilocarpine

2 h (diazepam) Immediately after SE

onset

400 mg/kg, i.p., for

initial dose, 250

mg/kg, i.p., 3




injection

247



Latency to SRSs

Incidence of SRSs

Frequency, severity or duration of SRSs

Behavioural alterations

Impairment of learning and memory

References

30.↓CA3 (p) and

↓CA4 (p) at dose of

100 mg/kg TPM, N.E.

with dose of 20 mg/kg

TPM, N.E. in CA1

N.D. N.E. N.E. N.D. ↓ with dose of

20 mg/kg TPM,

N.E. with dose

of 100 mg/kg

TPM

(Frisch et al., 2007) –

improved cognitive

function occurred at

20 mg/kg treatment of

TPM, independent of

neuroprotection

within the

hippocampus

31. ↓CA1 (c), ↓CA3

(p)

N.D. ↓ (during

drug

tapering)

↓ (during

drug tapering)

↓ ↓ (Bolanos et al., 1998)

32. ↓ Hippocampus

and hilus (c –1 out of

9 rats showed

moderate damage in

CA1)

N.D. N.E. N.E. ↓ N.E. (Brandt et al., 2006)

33. ↓ CA3 (c), ↓ CA1

(p), ↑layers II-IV of

entorhinal cortex, N.E.

hilus

N.E. N.E. N.E. N.D. N.D. (André et al., 2003)


available as abstracts; (c), complete neuroprotection; EC, entorhinal cortex; ND, not determined; NE,not effective; NS, not

specified; (p), partial neuroprotection; PC, piriform cortex

248

8.8 Conclusion

This present thesis demonstrates that the lithium/pilocarpine RLDP procedure for the induction

of SE reliably produces neurodegeneration and behavioural alterations in rats, and effectively

models the main features of human MTLE-HS (section 1.1). However, the effectiveness of

using this protocol is strain-dependent as it reduced mortality in Wistar, but not in LEH, rats (see

chapter 3). We showed that the majority of neuronal loss and behavioural alterations were

caused by SE as the IPI, and with the possible exception of neuronal loss in the thalamic

somatosensory nuclei (see section 4.4.2), these processes were unaffected by development of

SRSs at the 3 months recovery time. Stereological analysis of neurons (stained for the neuronal

specific marker [NeuN]) at various times (1 to 3 months) following SE showed regional

variability in the evolution of neuronal loss within the hippocampus, amygdala, thalamus and

piriform cortex, with the majority of neuronal death present by 24 hrs of recovery (see chapter

4). SE resulted in decreased exploratory behavior as assessed in the open field test, increased

aggression to handling, increased hyperreactivity as assessed in the touch respone test, and

anxiolytic effects as measured in the elevated-plus maze test (see chapter 6). Furthermore,

deficits in search strategies use, as well as impaired spatial learning and memory, contributed to

poor performance in the Morris water maze (see chapter 7).

Overall, our results do not support the general hypothesis that genesis of SRSs, cognitive

impairment and behavioural alterations are caused by SE-induced neuronal death. To assess the

relationship between these processes, we first compared the progression of neuronal death and

behavioural alterations in rats after SE. As discussed in section 8.6, our data and comparison of

results from other studies indicate that the majority of neuronal death and behavioural alterations

develop within the first several days after SE. Despite this, a detailed analysis assessing onset of

behavioural changes at even earlier times (i.e., 1, 3, 6 and 12 hours after SE) is required to more

thoroughly assess the temporal relationship between these processes. However, SE animals are

often in poor physical condition immediately following SE, and this precludes assessement of

behavioural alterations in rats less than 1 or 2 days of recovery. Interestingly, Castro et al.,

(2010) demonstrated that when compared to systemic injection of pilocarpine (320 mg/kg), SE in

rats induced by intrahippocampal injection of pilocarpine (total 2.4 mg) recovered almost

immediately following cessation of SE. Because this model also results in hippocampal neuronal

death (Castro et al., 2010; Furtado et al., 2011), it offers an alternative to other SE models in

249

assessing the temporal relationship between seizure-induced neurodegeneration and behavioural

alterations.

As discussed in section 8.7, we next assessed the effect of partial neuroprotection within the

hippocampus (by tat-NR2B9c) on development of SRSs and behavioural alterations in rats

following SE. Our data showed that reducing pyramidal cell loss in the hippocampus had no

effect on the number of rats developing SRSs or on behavioural alterations, and argues against a

causal relationship between neurodegeneration within this region, genesis of SRSs and

behavioural morbidity. As discussed in section 9.3, further studies are necessary to assess the

connection between different patterns of neurogeneration, epileptogenesis and behavioural

alterations caused by SE in rats.

250

Chapter 9

Future directions

9.1 Cell death mechanisms contributing to differential rates of neuronal loss following SE

9.1.1 Previous literature

Previous studies on the pilocarpine model showed that neuronal death occurs by necrosis and/or

apoptosis (Sloviter et al., 1996; Fujikawa et al., 1999; Fujikawa et al., 2000b; Bengzon et al.,

2002; Weise et al., 2005; Henshall, 2007; Henshall and Murphy, 2008; Fujikawa et al., 2010). In

contrast to early neuronal loss, which occurs in the first 24 to 48 hours following a neurological

insult and is predominately necrotic, delayed or secondary neuronal death occurring at later times

has been identified to be mainly apoptotic (Kermer et al., 1999; Snider et al., 1999; Weise et al.,

2005). Different cell death mechanisms have been reported to occur in different brain regions

(Sloviter et al., 1996; Lopez-Meraz et al., 2010), or within the same neuronal population in rats

following SE (Narkilahti et al., 2003a; Weise et al., 2005). Numerous studies have described the

extent and severity of SE-induced neurodegeneration in the pilocarpine and lithium/pilocarpine

models (Turski et al., 1983a; Turski et al., 1983b; Honchar et al., 1983; Fujikawa, 1996; Motte

et al., 1998; Covolan and Mello, 2000; Peredery et al., 2000; Poirier et al., 2000). Still, our

understanding on how SE-induced neuronal death in different brain regions progresses over time

remains unclear. Previous studies have been limited by the semi-quantitative assessment of

neuronal damage and by the limited number of recovery times assessed (see sections 1.5.1.2 and

4.4.1). The effect of SE on neuronal loss is further discussed in section 1.5.

9.1.2 Summary of our findings

In chapter 4, we extended these findings by conducting a detailed, quantitative time course

comparison of SE-induced neuronal death in 19 brain regions within the hippocampus,

amygdala, thalamus and piriform cortex. Neuronal death was assessed by stereological analysis

of neurons (stained for the neuronal specific marker [NeuN]) at ten different intervals after SE

(from 1 hour to 3 months). Our results showed that depending upon the brain region, neuronal

death occurred as early as 1 hour after SE, with the majority of neuronal death in all brain

regions present by 24 hours. While specific regions within the hippocampus (dorsal and ventral

251

CA1) and amygdala (LaDL, LaVM, BLP, BMP) showed additional neuronal loss between 1 and

14 days after SE, the somatosensory thalamic nuclei (VPM, VPL) were the only areas with

additional neuronal death detected after 2 weeks. In general, we demonstrated that different

brain regions exhibit differential rates of neuronal loss following lithium/pilocarpine induced SE.

These findings are further discussed in sections 4.4 and 8.4.

9.1.3 Proposed studies

The cell death mechanisms involved in neuronal death following lithium/pilocarpine-induced SE

remain unclear. While some studies showed that neuronal death in this model is predominately

necrotic ( Fujikawa et al., 1999; Fujikawa et al., 2000b; Fujikawa, 2005; Fujikawa et al., 2010;

Kotariya et al., 2010), others have provided evidence that delayed neuronal loss is apoptotic

(Bengzon et al., 2002; Narkilahti et al., 2003a; Weise et al., 2005; Henshall, 2007; Wang et al.,

2008) (see section 4.4.4). As discussed in section 1.5.1.4, specific signalling cascades

contributing to apoptotic and necrotic morphologies have been reported. For instance, the

expression and activation of calpain was detected in early degenerating necrotic neurons

following SE (Araújo et al., 2008; Wang et al., 2008), whereas caspase-3 was found in delayed

degenerating neurons exhibiting apoptotic features ( Narkilahti et al., 2003a; Weise et al., 2005;

Wang et al., 2008). Other studies, however, have failed to find caspase-3 activation following

prolonged seizures (Fujikawa et al., 2002; Wang et al., 2008). The detailed time-course analysis

of neuronal death we completed in chapter 4 establishes a framework in which further studies

can investigate whether differential rates of neuronal loss reflect regional differences in cell

death mechanisms.

We hypothesize that necrotic and apoptotic cell death mechanisms will be present in regions

exhibiting early (≤ 1 day) and delayed (>1day) neuronal loss, respectively. In view of the fact

that a large proportion of neuronal death occurs by 24 hours after SE (see section 9.1.2), we

expect that necrosis will be the dominate cell death morphology. However, because the dorsal

and ventral CA1 subfields of the hippocampus, the lateral (LaDL and LaVM) and basalateral

amydaloid nuclei (BLP and BMP), and the thalamic somatosensory nuclei showed delayed

neuronal loss (see section 9.1.2), we expect that a proportion of degenerating neurons within

these regions will exhibit apoptotic features. An initial study can focus on hippocampal

subfields that exhibit fast (CA4) and relatively slow (CA1) neuronal loss. Different cell death

252

morphologies can be detected by use of electron and light microscopy. Brain sections can also

be co-stained with Fluoro-jade B (FJB, a marker for degenerating neurons), for NeuN (neurons),

and for specific apoptotic (e.g., caspase-3, caspase-8, p38, ERK) and necrotic (e.g., calpain,

JNK) signalling cascades at various times (1hr – 7 days) following SE. Analysis by confocal

microscopy will permit the identification of degenerating neurons (FJB+/NeuN+) that co-stain

for specific cell-death signalling markers. Although evidence for apoptotic neuronal death has

previously been assessed by the presence of TUNEL staining and DNA laddering, these

morphological changes have also been reported in necrotic neurons (Fujikawa et al., 1999;

Fujikawa et al., 2000b; Fujikawa et al., 2002). Therefore, a combination of experimental

approaches is often required, with confocal microscopy offering the best confirmation on cell

death phenotypes. Several extensive reviews describing apoptotic and necrotic cell death

mechanisms are available (Saraste, 1999; Saraste and Pulkki, 2000; Manning and Zuzel, 2003;

Goldstein and Kroemer, 2007; Vanlangenakker et al., 2008).

9.2 Specific cognitive alterations in rats following SE


As deficits in cognition and memory are commonly observed in human MTLE-HS (Boro and

Haut, 2003; Devinsky, 2003; Motamedi and Meador, 2003; Devinsky, 2004a; Gaitatzis et al.,

2004; Vingerhoets, 2006; Garcia-Morales et al., 2008), causing reduced quality of life for the

patient (Cramer, 2002; Gilliam, 2002; Cramer et al., 2003; Boylan et al., 2004; Johnson et al.,

2004), a major goal in experimental epilepsy studies is to identify and evaluate cognitive and

behavioural dysfunction (Stafstrom, 2006). Previous studies have demonstrated that rats after SE

exhibit elevated escape latencies compared to controls when evaluated in the hidden platform

MWM task (see section 1.6.7.2). Although elevated search times are often interpreted as

impaired spatial learning, others have argued that this deficit may be more attributable to

impairment in behavioural strategies (Schenk and Morris, 1985; Morris, 1989; Whishaw, 1989;

Cain, 1997). This idea is substantiated by observations that rats following SE often exhibit a

thigmotaxic response in the MWM, and that this behaviour occurs in the absence of sensory or

motor impairment (Milgram et al., 1988; Hort et al., 1999; Kubova et al., 2004; McKay and

Persinger, 2004; Groticke et al., 2008; Jun et al., 2009) (see section 1.6.7.2). The effect of SE on

cognitive and behavioural morbidity is further discussed in section 1.6.

253


In chapter 7, we extended previous findings by performing a detailed analysis on the effect of SE

in rats on search strategy use in the hidden platform MWM task. Swim paths were analyzed

according to a categorization developed by Graziano et al., (2003), which is a seven category

qualitative analysis of prototypical behaviour in the MWM: thigmotaxis, circling, random

searching, scanning, self-orienting, approaching target, and ability to directly locate the target.

The search strategies are organized on a continuum of difficulty and efficiency where

thigmotaxis is considered the least efficient way of solving the hidden-platform MWM task,

while direct finding of the hidden platform is the most efficacious (Graziano et al., 2003). These

swim paths were divided into 3 broader categories that included spatial strategies, systematic

non-spatial strategies, and repetitive looping-based strategies (Brody and Holtzman, 2006) (see

section 7.2.3.6).

In general, we found that suboptimal use of search strategies, along with impaired spatial

learning and memory, contributed to poor MWM performance in rats following SE.

Furthermore, we detected between animal differences in the ability to improve performance over

extended training. Our results showed that 22% of rats after SE exhibited no behavioural

impairment, with performance in the MWM and spatial probe tests indistinguishable from

controls. Of the animals that showed MWM impairment compared to controls, 44% acquired

more efficient search strategies, and the increased use of these strategies accounted for their

improved performance. In contrast, 34% of rats showed no improvement in escape latencies or

shift in strategy use.


Previous studies have shown that spatial learning, behavioural strategy learning, and the ability

to switch between behavioural strategies are dissociable from one another (Morris, 1984;

Whishaw and Tomie, 1987; Whishaw and Petrie, 1988; Morris, 1989; Whishaw, 1989; Day and

Schallert, 1996; Day et al., 1999), and that each of these components rely to some extent on

different neural systems (see sections 1.6.7.1, 7.4.4, and 8.5.2.2). Our results showed impaired

spatial memory and search strategy use in rats following SE. Future studies are necessary to

determine whether spatial learning, behavioural strategy learning, and/or the ability to switch

between behavioural strategies are preserved to verify the specificity of cognitive alterations in

254

these animals. Because we were able to separate rats following SE into three different groups

based on MWM performance (see section 9.2.2), our first hypothesis is that these group

differences can be accounted for by selective differences in cognitive alterations. By using

different testing paradigms in the MWM (described below), future studies can specifically assess

the effect of SE on spatial learning, behavioural strategies learning, and the pliability of

switching between different search strategies.

McKay and Persinger (2004) demonstrated that different patterns of brain damage following SE

can differentially affect MWM performance (see section 7.4.5). Because spatial learning and

behavioural strategies learning involve different neuronal networks, our second hypothesis is that

different patterns of neuronal loss are responsible for the group differences we observed in rats

following SE. This hypothesis can be assessed by quantifying neuronal loss in brains regions

that underlie specific cognitive functions, including spatial learning (e.g., dorsal hippocampus,

posterior parietal cortex), behavioural strategies learning (e.g., prefrontal cortex, striatum,

cerebellum, medial thalamus), and the ability to switch between behavioural strategies (e.g.,

striatum, hippocampus, amygdala) (see sections 1.6.7.1 and 8.5.2.2).

9.2.3.1 Spatial learning and memory

Rats treated with anticholinergic drugs (Whishaw and Tomie, 1987; Whishaw, 1989; Day and

Schallert, 1996), following traumatic brain injury (Brody and Holtzman, 2006; Thompson et al.,

2006), or with lesions to specific brain structures (e.g., hippocampus, caudate-putamen)

(Whishaw and Petrie, 1988; Day et al., 1999) tend to perseverate in thigmotaxic responding for

many more trials than controls. Our findings have demonstrated a similar thigmotaxic response

after SE in rats. The authors suggested the animal‘s focus on trying to locate an escape around

the walls of the pool by using repetitive-looping based strategies prevented the ability to acquire

spatial information (Day and Schallert, 1996; Day et al., 1999; Thompson et al., 2006); therefore,

these studies initially trained rats in the MWM with procedures that deterred the animal‘s

thigmotaxic response. In the shrinking platform task, training begins with an escape platform

that occupies nearly the entire pool. The area to which the rats could escape is made smaller by

substituting smaller platforms as training progresses. The use of repetitive looping-based

strategies is discouraged in the first several trials by placing a block between the submerged

platform and the walls of the pool. Although rats following traumatic brain injury still exhibited

255

impaired spatial learning in this task (Thompson et al., 2006), rats treated with anticholinergic

drugs and with hippocampal lesions exhibited similar performance to controls in finding the

submerged platform, and preferentially searched the goal quadrant on the probe trials (Day and

Schallert, 1996; Day et al., 1999).

Our results indicate that rats after SE were capable of acquiring spatial memory by the fact that

they (1) showed an increase in use of spatial strategies during extended MWM training, and (2)

exhibited higher search times when the submerged platform was moved diagonally from its

original location (see section 7.4.4). Because the shrinking platform task discourages the use of

repetitive-looping based strategies, we hypothesize that rats following SE and tested with this

procedure will acquire spatial information and show a preference to the platform quadrant in the

probe tests. This task will confirm whether impaired performance in rats after SE assessed using

the spatial acquisition MWM task is caused by impaired use of spatial information.

9.2.3.2 Acquisition and retention of behavioural-strategies

Acquisition of behavioural strategies can be assessed with a nonspatial training paradigm

(Morris, 1989; Saber and Cain, 2003). In this task, the submerged platform is moved to a

different quadrant of the pool on successive trials, thus preventing the learning of a place

response. We hypothesize that although a majority of rats after SE will acquire more efficient

search strategies in the nonspatial training paradigm, a subset of these animals will not show

improved performance. This is based on the fact that we observed no improvement in

performance with a subset of SE rats when assessed in the spatial acquisition MWM task (see

section 9.2.2). This task will confirm the percentage of rats following SE that exhibit deficits in

acquisition of search strategies.

9.2.3.3 Pliability of search strategy use

A number of different testing paradigms can be used to assess an animal‘s ability to efficiently

alter search strategies. The shift learning paradigm has been used in rats treated with atropine

(Day and Schallert, 1996), with hippocampal lesions (Day et al., 1999), or following traumatic

brain injury (Thompson et al., 2006). In this paradigm, the animal is first trained to learn the

position of a hidden submerged platform using the shrinking platform task (see section 9.3.2.1).

After acquisition of spatial search strategies, the platform is shifted diagonally across from the

256

old location in a probe trial to determine the animal‘s ability to effectively switch search

strategies. Sham animals will efficiently switch from use of spatial strategies to non-spatial,

systematic strategies (Day and Schallert, 1996; Day et al., 1999; Thompson et al., 2006). In

contrast, rats treated with atropine or with hippocampal lesions were demonstrated to initially use

spatial strategies, but then revert to use of repetitive looping-based strategies (Day and Schallert,

1996; Day et al., 1999). Rats subjected to traumatic brain injury showed deficits in spatial

learning (see section 9.3.2.1) and persisted in using repetitive looping-based strategies in the shift

learning paradigm (Thompson et al., 2006).

Day et al., (1999) additionally tested rats with hippocampal lesions on an alternative version of

the shift learning paradigm. In this task, animals were first trained to use spatial strategies using

the shrinking platform task (see section 9.3.2.1). When training was complete, the submerged

platform was randomly shifted around the walls of the pool so that rats were required to switch

to use of repetitive looping-based strategies. Although hippocampal-lesioned rats showed an

initial preference for responding thigmotaxically, once spatial search strategies were acquired in

the shrinking platform task, these animals required more trials compared to controls to switch

back to a thigmotaxic response in the shift-training paradigm (Day et al., 1999). Even with

spatial learning deficits, this procedure can still be used since animals would be required to

switch from use of systematic, non-spatial strategies, to use of repetitive looping-based

strategies.

Because the majority of rats after SE were capable of acquiring spatial strategies but continued to

select less efficient search strategies (see section 9.3.2.1), we hypothesize that these animals will

show deficits in the ability to alter between search strategies. This task will confirm the

percentage of rats following SE that are capable of acquiring search strategies (see section

9.2.3.2), but exhibit behavioural inflexibility.

257

9.3 The causal relationship between neurodegeneration, genesis of SRSs and behavioural alterations


Different patterns of hippocampal neuronal loss are observed in human MTLE (Thom et al.,

2010; Thom et al., 2005; Blümcke et al., 2007; Mueller et al., 2009; de Lanerolle et al., 2003)

and this correlates with the age of an IPI (Thom et al., 2010; Blümcke et al., 2007; Van

Paesschen et al., 1997), or is predictive of response to AED treatment and postsurgical outcome

(Thom et al., 2010; Thom et al., 2005; Blümcke et al., 2007; Mueller et al., 2009; de Lanerolle et

al., 2003) (see section 8.3.1). Extrahippocampal neurodegeneration is observed in patients with

MTLE (Hudson et al., 1993; Du et al., 1993; Wolf et al., 1997; Juhász et al., 1999; Yilmazer-

Hanke et al., 2000; Bernasconi et al., 2003; Natsume et al., 2003; Bernasconi et al., 2005;

Dawodu and Thom, 2005; Thivard et al., 2005); however, futher studies are required to

determine whether different patterns of neuronal loss occur in extrahippocampal regions and if

so, investigate if this is correlated with the severity and distribution of hippocampal neuronal

loss, and the contribution (if any) this damage has on disease outcome. In fact, several studies

reported extrahippocampal neuronal death in the absence of HS (Bernasconi et al., 2001;

Bernasconi et al., 2003).

Animal models of epilepsy have demonstrated a correlation between the pattern of neuronal loss

and severity of the IPI or disease outcome. For instance, several studies showed that adult rats

that were electrically stimulated in the amygdala exhibited different types of self-sustaining

status epilepticus (SSSE). Neuronal loss was found to be more restricted in animals that

displayed ambulatory SSSE (with facial automatisms, neck myoclonus and concomitant

ambulatory behavior) compared with typical SSSE (with facial automatisms, neck and forelimb

myoclonus, rearing and falling, and tonic-clonic seizures) (Tilleli et al., 2005). These findings

indicate that severity of the IPI (i.e., different types of SE) leads to different patterns of neuronal

loss.

Brandt et al., (2003) showed that the type of SSSE not only results in different patterns of

neuronal loss, but also affects the percentage of rats developing epilepsy. In this study, three

different types of SSSE were induced in adult rats by electrical stimulation of the amygdala. In

rats with partial (type I) SSSE, neurodegeneration was restricted to the ipsilateral amygdala,

258

endopiriform nucleus, mediodorsal thalamus and piriform and entorhinal cortices. In rats

exhibiting a partial SSSE with generalized seizures (type II), neurodegeneration was more

marked and less variable compared to rats with type I SSSE, and also extended to include less

severe neuronal loss in the contralateral hemisphere. Variable damage was also detected in the

CA1 and CA3a sectors of the hippocampus. In rats with generalized (type III) SSSE, extensive

bilateral damage was observed and reported to be similar to the pattern of neuronal loss detected

following pilocarpine and kainate-induced SE. Only 33% of rats with type I SSSE developed

SRSs, compared to >90% after type II or type III SSSE. Furthermore, epileptic rats following

type I SSSE exhibited a longer latency to development of epilepsy, and exhibited less severe and

frequent SRSs.

A series of experiments showed rats developing epilepsy after an episode SE markedly differed

in their response to phenobarbital (Brandt et al., 2004; Volk et al., 2006; Bethmann et al., 2008).

While phenobarbital suppressed SRSs in rats with no overt brain damage, rats with hippocampal

pyramidal cell loss did not respond to treatment. These results are further discussed in section

8.7.1.2.

McKay and Persinger (2004) showed that different patterns of neuronal loss (e.g., amygdala,

hippocampus, thalalmus) in rats after SE can differentially affect performance in the MWM.

These results are further discussed in section 7.4.5.


Our findings showed that in adult rats following lithium/pilocarpine-induced SE, partial

neuroprotection (by tat-NR2B9c) within the hippocampus had no effect on the incidence of

SRSs, or on behavioural alterations. In contrast, others demonstrated that neuroprotection of

specific brain regions can affect disease outcome and mitigate behavioural morbidity. These

results are extensively discussed in section 8.7 and compared in Table 8.1. The differential

effects of various neuroprotective strategies on disease outcome may be related to differences in

the pattern of neuroprotection conferred, or in the pattern of neuronal loss acquired by different

models of SE (see table 8.1).

259


A prominent question that arises from our work and previous literature is how different patterns

of seizure-induced neuronal loss affect (1) the development and severity of SRSs, (2) the type of

behavioural alterations displayed, and (3) the effectiveness of different neuroprotective

strategies. Several animal models of epilepsy offer the opportunity for future studies to explore

how restricted versus widespread, or how hippocampal versus extrahippocampal neuronal loss

affect these processes. In general, we hypothesize that different patterns of SE-induced neuronal

loss in adult rats will result in differences in the severity of epilepsy, the specificity of

behavioural deficits displayed, and the effectiveness of different neuroprotective strategies used.

This work can allow us to investigate whether (1) different animal models of epilepsy more

clearly represent different ‗subcategories‘ of neuropathology observed in human MTLE, and (2)

identify specific brain regions or neuronal networks contributing to the severity of epilepsy or

underlying specific behavioural alterations. A comparison of different neuroprotective strategies

effective in selective brain regions can also be used as a tool to assess the causal relationship

between preserved neuronal networks, severity of disease outcome (e.g., severity or frequency of

SRSs) and behavioural changes. For example, as shown in Table 8.1, different neuroprotective

drugs were found to be effective in selective brain regions. Future studies can therefore use a

combination of neuroprotective strategies and different seizure models to characterize the

relationship between different patterns of neuronal loss and disease morbidity.

9.3.3.1 SE models result in different patterns of neuronal loss

In chapter 4, we showed SE induced by the RLDP lithium/pilocarpine procedure results in

widespread neurodegeneration within the hippocampus, thalamus, amygdala and piriform cortex.

This model also results in behavioural and cognitive alterations (see chapters 6 and 7). Castro et

al., (2010) showed that when compared to systemic injection of pilocarpine (320 mg/kg), SE

induced by intrahippocampal injection of pilocarpine (total 2.4 mg, referred to as the H-PILO

model) resulted in significantly less extrahippocampal neuronal death. The H-PILO and

lithium/pilocarpine models offer an opportunity for future studies to assess how differences in

the severity of extrahippocampal neuronal death affect disease outcome and the effectiveness of

different neuroprotective strategies. For instance, even though we showed that partial

neuroprotection in the hippocampus (by tat-NR2B9c) had no effect on seizure development and

260

behavioural alterations in the RLDP lithium/pilocarpine model, it may have a beneficial effect in

the H-PILO model since damage is more restricted to the hippocampus.

As previously described in section 9.3.1, several studies have demonstrated that different types

of SSSE induced by electrical stimulation of the basolateral amygdala also produce differences

in the pattern of neurodegeneration (Brandt et al., 2003; Tilleli et al., 2005). These differences

include (1) neuronal death restricted to the ipsilateral side of seizure onset versus bilateral

neuronal loss, and (2) neuronal death restricted to extrahippocampal regions versus

extrahippocampal and hippocampal neuronal loss. Thus, this SE model also offers the

opportunity to investigate the causal relationship between different patterns of neuronal loss,

development of epilepsy and behavioural morbidity.

261

References

Aarts M, Liu Y, Liu L, Besshoh S, Arundine M, Gurd JW, Wang YT, Salter MW, Tymianski M

(2002) Treatment of ischemic brain damage by perturbing NMDA receptor-PSD-95 protein

interactions. Science 298:846-850.

Aarts MM, Tymianski M (2004) Molecular mechanisms underlying specificity of excitotoxic

signaling in neurons. Curr Mol Med 4:137-147.

Abrahams S, Pickering A, Polkey CE, Morris RG (1997) Spatial memory deficits in patients

with unilateral damage to the right hippocampal formation. Neuropsychologia 35:11-24.

Abrahams S, Morris RG, Polkey CE, Jarosz JM, Cox TC, Graves M, Pickering A (1999)

Hippocampal involvement in spatial and working memory: A structural MRI analysis of patients

with unilateral mesial temporal lobe sclerosis. Brain Cog 41:39-65.

Acharya MM, Hattiangady B, Shetty AK (2008) Progress in neuroprotective strategies for

preventing epilepsy. Progress Neurobiol 84:363-404.

Agam G, Bersudsky Y, Berry GT, Moechars D, Lavi-Avnon Y, Belmaker RH (2009) Knockout

mice in understanding the mechanism of action of lithium. Biochem Soc Trans 37:1121-1125.

Albers GW, Goldstein LB, Hall D, Lesko LM (2001) Aptiganel hydrochloride in acute ischemic

stroke: A randomized controlled trial. JAMA 286:2673-2683.

Al-Hallaq RA, Conrads TP, Veenstra TD, Wenthold RJ (2007) NMDA di-heteromeric receptor

populations and associated proteins in rat hippocampus. J Neurosci 27:8334-8343.

Aliashkevich AF, Yilmazer-Hanke D, Van Roost D, Mundhenk B, Schramm J, Blümcke I (2003)

Cellular pathology of amygdala neurons in human temporal lobe epilepsy. Acta Neuropathol

106:99-106.

André V, Rigoulot MA, Koning E, Ferrandon A, Nehlig A (2003) Long-term pregabalin

treatment protects basal cortices and delays the occurrence of spontaneous seizures in the

lithium/pilocarpine model in the rat. Epilepsia 44:893-903.

André V, Dube C, Francois J, Leroy C, Rigoulot MA, Roch C, Namer IJ, Nehlig A (2007)

Pathogenesis and pharmacology of epilepsy in the lithium/pilocarpine model. Epilepsia 38:41-

47.

Andrioli A, Fabene PF, Spreafico R, Cavalheiro EA, Bentivoglio M (2009) Different patterns of

neuronal activation and neurodegeneration in the thalamus and cortex of epilepsy-resistant

proechimys rats versus wistar rats after pilocarpine-induced protracted seizures. Epilepsia

50(4):832-848

Anisman H, McIntyre DC (2002) Conceptual, spatial and cue learning in the morris water maze

in fast or slow kindling rats: Attention deficit comorbidity. J Neurosci 22:7809-7817.

262

Araújo IM, Gil JM, Carreira BP, Mohapel P, Petersen A, Pinheiro PS, Soulet D, Bahr BA,

Brundin P, Carvalho CM (2008) Calpain activation is involved in early caspase-independent

neurodegeneration in the hippocampus following status epilepticus. J Neurochem 105:666-676.

Arida FA, Scorza CA, Cavalheiro EA (1999) The course of untreated seizures in the pilocarpine

model of epilepsy. Epilepsy Res 34:99-107.

Arnautova EN, Nesmeianova TN (1964) Commission on terminology of the international league

against epilepsy. A proposed international classification of epileptic seizures. Epilepsia 5:297-

306.

Aroniadou-Anderjaska V, Fritsch B, Qashu F, Braga MFM (2008) Pathology and

pathophysiology of the amygdala in epileptogenesis and epilepsy. Epilepsy Res 78:102-116.

Arundine M, Tymianski M (2003) Molecular mechanisms of calcium-dependent

neurodegeneration in excitotoxicity. Cell Calcium 34:325-337.

Avoli M, Louvel J, Pumain R, Köhling R (2005) Cellular and molecular mechanisms of epilepsy

in the human brain. Prog Neurobiol 77:166-200.

Avoli M, D'Antuono M, Louvel J, Köhling R, Biagini G, Pumain R, D'Arcangelo G, Tancredi V

(2002) Network and pharmacological mechanisms leading to epileptiform synchronization in the

limbic system in vitro. Prog Neurobiol 68:167-207.

Azcoitia I, Perez-Martin M, Salazar V, Castillo C, Ariznavarreta C, Garcia-Segura LM,

Tresguerres JAF (2005) Growth hormone prevents neuronal loss in the aged rat hippocampus.

Neurobiol Aging 26:697-703.

Babb TL (1986) Metabolic, morphologic and electrophysiologic profiles of human temporal lobe

foci: An attempt at correlation. Adv Exp Med Biol 293:115-125.

Babb TL, Brown WJ (1986) Neuronal, dendritic and vasular profiles of human temporal lobe

epilepsy correlated with celllular physiology in vivo. Adv Neurol 44:949-966.

Babb TL, Kupfer WR, Pretorius JK, Crandall PH, Levesque MF (1991) Synaptic reorganization

by mossy fibers in human epileptic fascia dentata. Neurosci 42:351-363.

Baldi E, Lorenzini CA, bucherelli C (2003) Task solving by procedural strategies in the morris

water maze. Physiol Behav 78:785-793.

Bannerman DM, Good MA, Butcher SP, Ramsay M, Morris RGM (1995) Distinct components

of spatial learning revealed by prior training and NMDA receptor blockade. Nature 378:182-186.

Bannerman DM, Yee BK, Good MA, Heupel MJ, Iversen SD, Rawlins JN (1999) Double

dissociation of function within the hippocampus: A comparison of dorsal, ventral, and complete

hippocampal cytotoxic lesions. Behav Neurosci 113:1170-1188.

263

Bannerman DM, Rawlins JN, McHugh SB, Deacon RM, Yee BK, Bast T, Zhang WN, Pothuizen

HH, Feldon J (2004) Regional dissociations within the hippocampus--memory and anxiety.

Neurosci Biobehav Rev 28:273-283.

Baram TZ, Eghbal-Ahmadi M, Bender RA (2002) Is neuronal death required for seizure-induced

epileptogenesis in the immature brain? Prog Brain Res 135:365-375.

Baran H, Kepplinger B, Draxler M, Skofitsch G (2004) Choline acetyltransferase, glutamic acid

decarboxylase and somatostatin in the kainic acid model for chronic temporal lobe epilepsy.

Neurosignals 13:290-297.

Bartolomei F, Khalil M, Wendling F, Sontheimer A, Régis J, Ranjeva JP, Guye M, Chauvel P

(2005) Entorhinal cortex involvement in human mesial temporal lobe epilepsy: An

electrophysiologic and volumetric study. Epilepsia 46:677-687.

Bartolomei F, Chauvel P, Wendling F (2008a) Epileptogenicity of brain structures in human

temporal lobe epilepsy: A quantified study from intracerebral EEG. Brain 131:1830.

Bartolomei F, Wendling F, Chauvel P (2008b) The concept of an epileptogenic network in

human partial epilepsies. Neurochirurgie 54:174-184.

Bartolomei F, Cosandier-Rimele D, McGonigal A, Aubert S, Régis J, Gavaret M, Wendling F,

Chauvel P (2010) From mesial temporal lobe to temporoperisylvian seizures: A quantified study

of temporal lobe seizure networks. Epilepsia 51:2147-2158.

Bayley PJ, Frascino JC, Squire LR (2005) Robust habit learning in the absence of awareness and

independent of the medial temporal lobe. Nature 436:550-553.

Bechara A, Damasio H, Damasio AR, Lee GP (1999) Different contributions of the human

amygdala and ventromedial prefrontal cortex to decision-making. J Neurosci 19:5473-5481.

Becker A, Krug M, Schröder H (1997a) Strain differences in pentylenetetrazol-kindling

development and subsequent potentiation effects. Brain Res 763:87-92.

Becker A, Letzel K, Letzel U, Grecksch G (1997b) Kindling of the dorsal and the ventral

hippocampus: Effects on learning performance in rats. Physiol Behav 62:1265-1271.

Becker AJ, Gillardon F, Blumcke I, Langendorfer D, Beck H, Wiestler OD (1999) Differential

regulation of apoptosis-related genes in resistant and vulnerable subfields of the rat epileptic

hippocampus. Mol Brain Res 67:172-176.

Belmaker RH, Bersudskya Y (2007) Lithium–pilocarpine seizures as a model for lithium action

in mania . Neurosci Biobehav Rev 31:843-849.

Ben-Ari Y, Lagowska J, Tremblay E, Le, Gal La Salle, G. (1979) A new model of focal status

epilepticus: Intra-amygdaloid application of kainic acid elicits repetitive secondarily generalized

convulsive seizures. Brain Res 163:176-179.

264

Ben-Ari Y, Tremblay E, Ottersen OP (1980) Injections of kainic acid into the amygdaloid

complex of the rat: An electrographic, clinical and histological study in relation to the pathology

of epilepsy. Neurosci 5:515-528.

Ben-Ari Y, Tremblay E, Riche D, Ghilini g, Naquet R (1981) Electrographic, clinical and

pathological alterations followign systemic administration of kainic acid, bicuculline or

pentetrazole: Metabolic mapping using the dexoyglucose method with special reference to the

pathology of epilepsy. Neurosci 6:1361-1391.

Ben-Ari Y (1985) Limbic seizure and brain damage produced by kainic acid: Mechanisms and

relevance to human temporal lobe epilepsy. Neurosci 14:375-404.

Ben-Ari Y, Dudek FE (2010) Primary and secondary mechanisms of epileptogenesis in the

temporal lobe: There is a before and an after. Epilepsy Curr 10:118-125.

Benedek K, Juhász C, Muzik O, Chugani DC, Chugani HT (2004) Metabolic changes of

subcortical structures in intractable focal epilepsy. Epilepsia 45:1100-1105.

Bengzon J, Mohapel P, Ekdahl CT, Lindvall O (2002) Neuronal apoptosis after brief and

prolonged seizures. Prog Brain Res 135:111-119.

Berg AT, Berkovic SF, Brodie MJ, Buchhalter J, Cross JH, van Emde Boas W, Engel J, French

J, Glauser TA, Mathern GW, Moshé SL, Nordli D, Plouin P, Scheffer IE (2010) Revised

terminology and concepts for organization of seizures and epilepsies: Report of the ILAE

commission on classification and terminology, 2005-2009. Epilepsia 51:676-685.

Bernard C, Esclapez M, Hirsch JC, Ben-Ari Y (1998) Interneurones are not so dormant in

temporal lobe epilepsy: A critical reappraisal of the dormant basket cell hypothesis. Epilepsy Res

32:93-103.

Bernasconi N, Bernasconi A, Caramanos Z, Antel SB, Andermann F, Arnold DL (2003) Mesial

temporal damage in temporal lobe epilepsy: A volumetric MRI study of the hippocampus,

amygdala and parahippocampal region. Brain 126:462-469.

Bernasconi N, Natsume J, Bernasconi A (2005) Progression in temporal lobe epilepsy:

Differential atrophy in mesial temporal structures. Neurology 65(2):223-8 65:223-228.

Berridge MJ, Irvine RF (1989) Inositol phosphates and cell signalling. Nature 341:197-205.

Berridge MJ (2009) Inositol trisphosphate and calcium signalling mechanisms. Biochem

Biophys Acta 1793:933-940.

Bertram EH, Lothman EW, Lenn NJ (1990) The hippocampus in experimental chronic epilepsy:

A morphometric analysis. Ann Neurol 27:43-48.

Bertram EH (1997) Functional anatomy of spontaneous seizures in a rat model of limbic

epilepsy. Epilepsia 38:95-105.

265

Bertram EH, Zhang DX, Mangan P, Fountain N, Rempe D (1998) Functional anatomy of limbic

epilepsy: A proposal for central synchronization of a diffusely hyperexcitable network. Epilepsy

Res 32:194-205.

Bertram EH, Scott C (2000) The pathological substrate of limbic epilepsy: Neuronal loss in the

medial dorsal thalamic nucleus as the consistent change. Epilepsia 41:S3-S8.

Bertram EH, Mangan PS, Zhang D, Scott CA, Williamson JM (2001) The midline thalamus:

Alterations and a potential role in limbic epilepsy. Epilepsia 42:967-978.

Bertram E (2007) The relevance of kindling for human epilepsy. Epilepsia 48:65-74.

Bertram EH, Zhang D, Williamson JM (2008) Multiple roles of midline dorsal thalamic nuclei in

induction and spread of limbic seizures. Epilepsia 49:256-268.

Bertram EH (2009) Temporal lobe epilepsy: Where do the seizures really begin? Epilepsy Behav

14:S32-S37.

Bethmann K, Fritschy JM, Brandt C, Löscher W (2008) Antiepileptic drug resistant rats differ

from drug responsive rats in GABA A receptor subunit expression in a model of temporal lobe

epilepsy. Neurobiol Dis 31:169-187.

Biagini G, Baldelli E, Longo D, Pradelli L, Zini I, Rogawski MA, Avoli M (2006) Endogenous

neurosteroids modulate epileptogenesis in a model of temporal lobe epilepsy. Exp Neurol

201:519-524.

Biagini G, Longo D, Baldelli E, Zoli M, Rogawski MA, Bertazzoni G, Avoli M (2009)

Neurosteroids and epileptogenesis in the pilocarpine model: Evidence for a relationship between

P450scc induction and length of the latent period. Epilepsia 50:53-58.

Binder DK, Steinhäuser C (2006) Functional changes in astroglial cells in epilepsy. Glia 54:358-

368.

Blümcke I, Pauli E, Clusmann H, Schramm J, Becker A, Elger C, Merschhemke M, Meencke

HJ, Lehmann T, von Deimling A, Scheiwe C, Zentner J, Volk B, Romstöck J, Stefan H,

Hildebrandt M (2007) A new clinico-pathological classification system for mesial temporal

sclerosis. Acta Neuropathol 113:235-244.

Blumenfeld H, McNally KA, Vanderhill SD, Paige AL, Chung R, Davis K, Norden AD,

Stokking R, Studholme C, Novotny EJJ, Zubal IG, Spencer SS (2004) Positive and negative

network correlations in temporal lobe epilepsy. Cereb Cortex 14:892-902.

Bolanos AR, Sarkisian M, Yang Y, Hori A, Helmers SL, Mikati M, Tandon P, Stafstrom CE,

Holmes GL (1998) Comparison of valproate and phenobarbital treatment after status epilepticus

in rats. Neurology 51:41-48.

Bonatti E, Kuchukhidze G, Zamarian L, Trinka E, Bodner T, Benke T, Delazer M (2009)

Decision making in ambiguous and risky situations after unilateral temporal lobe epilepsy

surgery. Epilepsy Behav 14:665-673.

266

Bonilha L, Kobayashi E, Mattos JP, Honorato DC, Li LM, Cendes F (2004) Value of extent of

hippocampal resection in the surgical treatment of temporal lobe epilepsy. Arq Neuropsiquiatr

62:15-20.

Bonilha L, Edwards JC, Kinsman SL, Morgan PS, Fridriksson J, Rorden C, Rumboldt Z, Roberts

DR, Eckert MA, Halford JJ (2010a) Extrahippocampal grey matter loss and hippocampal

deafferentation in patients with temporal lobe epilepsy. Epilepsia 51:519-528.

Bonilha L, Elm JJ, Edwards JC, Morgan PS, Hicks C, Lozar C, Rumboldt Z, Roberts DR,

Rorden C, Eckert MA (2010b) How common is brain atrophy in patients with medial temporal

lobe epilepsy? Epilepsia 51:1774-1779.

Boro A, Haut S (2003) Medical comorbidities in the treatment of epilepsy. Epilepsy Behav 4:S2-

S12.

Bouilleret V, Boyet S, Marescaux C, Nehlig A (2000) Mapping of the progressive metabolic

changes occurring during the development of hippocampal sclerosis in a model of mesial

temporal lobe epilepsy. Brain Res 852:255-262.

Boylan LS, Flint LA, Labovitz DL, JAckson SC, Starner K, Devinsky O (2004) Depression but

not seizure frequency predicts quality of life in treatment-resistant epilepsy. Neurology 62:258-

261.

Brandeis R, Brandys Y, Yehuda S (1989) The use of the Morris water maze the study of memory

and learning. Int J Neurosci 48:29-69.

Brandt C, Glien M, Potschka H, Volk H, Löscher W (2003a) Epileptogenesis and

neuropathology after different types of status epilepticus induced by prolonged electrical

stimulation of the basolateral amygdala in rats. Epilepsy Res 55:83-103.

Brandt C, Potschka H, Loscher W, Ebert U (2003b) N-methyl--aspartate receptor blockade after

status epilepticus protects against limbic brain damage but not against epilepsy in the kainate

model of temporal lobe epilepsy. Neurosci 118:727-740.

Brandt C, Volk HA, Löscher W (2004) Striking differences in individual anticonvulsant response

to phenobarbital in rats with spontaneous seizures after status epilepticus. Epilepsia 45:1488-

1497.

Brandt C, Gastens AM, Sun MZ, Hausknecht M, Loscher W (2006) Treatment with valproate

after status epilepticus: Effect on neuronal damage, epileptogenesis, and behavioral alterations in

rats. Neuropharmacology 51:789-804.

Brand M, Labudda K, Markowitsch HJ (2006) Neuropsychological correlates of decision-

making in ambiguous and risky situations. Neural Netw 19:1266-1276.

Brand M, Recknor EC, Grabenhorst F, Bechara A (2007) Decisions under ambiguity and

decisions under risk: Correlations with executive functions and comparisons of two different

gambling tasks with implicit and explicit rules. J Clin Exp Neuropsychol 29:86-99.

267

Brand M, Heinze K, Labudda K, Markowitsch HJ (2008) The role of strategies in deciding

advantageously in ambiguous and risky situations. Cogn Process 9:159-173.

Briellmann RS, Newton MR, Wellard M, Jackson GD (2001) Hippocampal sclerosis following

brief generalized seizures in adulthood. Neurology 57:315-317.

Briellmann RS, Berkovic SF, Syngeniotis A, King MA, Jackson GD (2002) Seizure-associated

hippocampal volume loss: A longitudinal magnetic resonance study of temporal lobe epilepsy.

Ann Neurol 51:641-644.

Broadbent NJ, Squire LR, Clark RE (2004) Spatial memory, recognition memory and the

hippocampus. PNAS 101:14515-14520.

Brody DL, Holtzman DM (2006) Morris water maze search strategy analysis in PDAPP mice

before and after experimental traumatic brain injury. Exp Neurol 197:330-340.

Brown DA (2010) Muscarinic acetylcholine receptors (mAChRs) in the nervous system: Some

functions and mechanisms. J Mol Neurosci 41:340-346.

Browning RA, Nelson DK (1986) Modification of electroshock and pentylenetetrazol seizure

patterns in rats after precollicular transections. Exp Neurol 93:546-556.

Buckmaster PS, Dudek FE (1999) In vivo intracellular analysis of granule cell axon

reorganization in epileptic rats. J Neurophysiol 81:712-721.

Buckmaster PS, Zhang GF, Yamawaki R (2002) Axon sprouting in a model of temporal lobe

epilepsy creates a predominately excitatory feedback circuit. J Neurosci 22:6650-6658.

Burke R, Beukelman DR, Hux K (2004) Accuracy, efficiency and preferences of survivors of

traumatic brain injury when using three organization strategies to retrieve words. Brain Inj

18:497-507.

Butman J, Allegri RF, Thomson A, Fontela E, Abel C, Viaggio B, Drake M, Serrano C, Loñ L

(2007) Behavioral flexibility impairment with negative feedback in refractory temporal lobe

epileptic patients with unilateral amygdala and hippocampal resection. Actas Esp Psiquiatr 35:8-

14.

Cain DP (1997) Prior non-spatial pretraining eliminates sensorimotor disturbances and

impairments in water maze learning caused by diazepam. Psychopharmacology 130:313-319.

Cain DP, Boon F, Corcoran ME (2006) Thalamic and hippocampal mechanisms in spatial

navigation: A dissociation between brain mechanisms for learning how versus learning where to

navigate. Behav Brain Res 170:241-256.

Cammisuli S, Murphy MP, Ikeda-Douglas CJ, Vidya B, Damian Holsinger RM, Head E,

Michalakis M, Racine RJ, Milgram NW (1997) Effects of extended electrical kindling on

exploratory behavior and spatial learning. Behav Brain Res 89:179-190.

268

Cao L, Xu J, Lin Y, Zhao X, Liu X, Chi Z (2009a) Autophagy is upregulated in rats with status

epilepticus and partly inhibited by vitamin E. Biochem Biophys Res Commun 379:949-953.

Cao L, Chen R, Xu J, Lin Y, Wang R, Chi Z (2009b) Vitamin E inhibits activated chaperone-

mediated autophagy in rats with status epilepticus. Neurosci 161:73-77.

Capella HM, Lemos T (2002) Effect on epileptogenesis of carbamazepine treatment during the

silent period of the pilocarpine model of epilepsy. Epilepsia 43:110-111.

Cardoso A, Assunção M, Andrade JP, Pereira PA, Madeira MD, Paula-Barbosa MM, Lukoyanov

NV (2008) Loss of synapses in the entorhinal-dentate gyrus pathway following repeated

induction of electroshock seizures in the rat. J Neurosci Res 86:71-83.

Cardoso A, Carvalho LS, Lukoyanova EA, Lukoyanov NV (2009) Effects of repeated

electroconvulsive shock seizures and pilocarpine-induced status epilepticus on emotional

behavior in the rat. Epilepsy Behav 14:293-299.

Carobrez AP, Bertoglio LJ (2005) Ethological and temporal analyses of anxiety-like behavior:

The elevated plus-maze model 20 years on . Neurosci Biobehav Rev 29:1193-1205.

Carvalho MC, Masson S, Brandao ML, de Souza Silva MA (2008) Anxiolytic-like effects of

substance P administration into the dorsal, but not ventral, hippocampus and its influence on

serotonin. Peptides 29:1191-1299.

Castro OW, Furtado MA, Tilelli CQ, Fernandes A, Pajolla GP, Garcia-Cairasco N (2010)

Comparative neuroanatomical and temporal characterization of fluorojade-positive

neurodegeneration after status epilepticus induced by systemic and intrahippocampal pilocarpine

in Wistar rats. Brain Res 1374:43-55

Cavalheiro EA, Silva DF, Turski WA, Calderazzo-Filho LS, Bortolotto ZA, Turski L (1987) The

susceptibility of rats to pilocarpine-induced seizures is age-dependent. Brain Res 37:43-58.

Cavalheiro EA, Leite JP, Bortolotto ZA, Turski WA, Ikonomidou C, Turski L (1991) Long-term

effects of pilocarpine in rats: Structural damage of the brain triggers kindilng and spontaneous

recurrent seizures. Epilepsia 32:778-782.

Cavalheiro EA, Leite JP, Bortolotto ZA, Turski WA, Ikonomidou C, Turski L (2001) Long-term

effects of pilocarpine in rats: Structural damage of the brain triggers kindling and spontaneous

recurrent seizures. Epilepsia 32:778-82.

Cavalheiro EA, Naffah-Mazzacoratti MG, Mello LE, Leite JP (2006) The pilocarpine model of

seizures. In: Models of seizures and epilepsy (Pitkanen A, Schwartzkroin PA, Moshe SL, eds),

pp433-448. Boston: Elsevier Academic Press.

Cavazos JE, Sutula TP (1990) Progressive neuronal loss induced by kindling: A possible

mechanism for mossy fiber synaptic reorganization and hippocampal sclerosis. Brain Res 527:1-

6.

269

Cavazos JE, Golarai G, Sutula TP (1991) Mossy fiber synaptic reorganization induced by

kindling: Time course of development, progression, and permanence. J Neurosci 11:2795-2803.

Cavazos JE, Das I, Sutula TP (1994) Neuronal loss induced by limbic pathways by kindling:

Evidence for induction of hippocampal slcerosis by repeated brief seizures. J Neurosci 14:3121.

Cavazos JE, Jones SM, Cross DJ (2004) Sprouting and synaptic reorganization in the subiculum

and CA1 region of the hippocampus in acute and chronic models of partial-onset epilepsy.

Neurosci 126:677-688.

Cavazos JE, Cross DJ (2006) The role of synaptic reorganization in mesial temporal lobe

epilepsy. Epilepsy Behav 8:483-493.

Cendes F, Andermann F, Gloor P, Lopes-Cendes I, Andermann E, Melanson D, Jones-Gotman

M, Robitaille Y, Evans A, Peters T (1993) Atrophy of mesial structures in patients with temporal

lobe epilepsy: Cause or consequence of repeated seizures? Ann Neurol 34:795-801.

Cendes F, Andermann F (2002) Do febrile seizures promote temporal lobe epilepsy?

retrospective studies. In: Febrile seizures (In Baram T.Z., Shinnar S, eds), Academic Press, San

Diego.

Chabolla DR (2002) Characteristics of the epilepsies. Mayo Clin Proc 77:981-990.

Chang Q, Gold PE (2003) Switching memory systems during learning: Changes in patterns of

brain acetylcholine and release in the hippocampus and striatum in rats. J Neurosci 23:3001-

3005.

Chard PS, Bleakman D, Christakos S, Fullmer CS, Miller RJ (1993) Calcium buffering

properties of calbindin D28k and parvalbumin in rat sensory neurones. J Physiol 472:341-357.

Chassoux F, Landre E, Rodrigo S, Beuvon F, Turak B, Semah F, Devaux B (2008) Intralesional

recordings and epileptogenic zone in focal polymicrogyria. Epilepsia 49:51-64.

Chauviere L, Rafrafi N, Thinus-Blanc C, Bartolomei F, Esclapex M (2009) Early deficits in

spatial memory and theta rhythm in experimental temporal lobe epilepsy. J Neurosci 29:5402-

5410.

Chen S, Kobayashi M, Honda Y, Kakuta S, Sato F, Kishi K (2007) Preferential neuron loss in

the rat piriform cortex following pilocarpine-induced status epilepticus. Epilepsy Res 4:1-18.

Chin RFM, Neville BGR, Scott RC (2004) A systematic review of the epidemiology of status

epilepticus. Eur J Neurosci 11:800-810.

Choi JY, Kim SJ, Hong SB, Seo DW, Hong SC, Kim BT, Kim SE (2003) Extratemporal

hypometabolism on FDG PET in temporal lobe epilepsy as a predictor of seizure outcome after

temporal lobectomy. Eur J Nucl Med Mol Imaging 30:581-587.

270

Choleris E, Thomas AW, Kavaliers M, Prato FS (2001) A detailed ethological analysis of the

mouse open field test: Effects of diazepam, chlordiazepoxide and an extremely low frequency

pulsed magnetic field. Neurosci Biobehav Rev 25:235-260.

Chu CT (2006) C.T. chu, autophagic stress in neuronal injury and disease. J Neuropathol Exp

Neurol 65:423-432.

Chu K, Jung KH, Lee ST, Kim JH, Kang KM, Kim HK, Lim JS, Park HK, Kim M, Lee SK, Roh

JK (2008) Erythropoietin reduces epileptogenic processes following status epilepticus. Epilepsia

49:1723-1732.

Cilio MR, Bolanos AR, Liu Z, Schmid R, Yang Y, Stafstrom CE, Mikati MA, Holmes GL

(2001) Anticonvulsant action and long-term effects of gabapentin in the immature brain.

Neuropharm 40:139-147.

Cilio MR, Sogawa Y, Cha BH, Liu X, Huang LT, Holmes GL (2003) Long-term effects of status

epilepticus in the immature brain are specific for age and model. Epilepsia 44:518-528.

Clark JD, Rager DR, Calpin JP (1997a) Animal well-being. I. general considerations. Lab Anim

Sci 47:564-570.

Clark JD, Rager DR, Calpin JP (1997c) Animal well-being. II. stress and distress. Lab Anim Sci

47:571-579.

Clark JD, Rager DR, Calpin JP (1997d) Animal well-being. III. an overview of assessment. Lab

Anim Sci 47:580-585.

Clark JD, Rager DR, Calpin JP (1997b) Animal well-being. IV. specific assessment criteria. Lab

Anim Sci 47:586-597.

Clifford DB, Olney JW, Benz AM, Fuller TA, Zorumski CF (1990) Ketamine, phencyclidine,

and MK-801 protect against kainic acid-induced seizure-related brain damage. Epilepsia 31:382-

390.

Clifford DB, Olney JW, Maniotis A, Collins RC, Zorumski CF (1987) The functional anatomy

and pathology of lithium/pilocarpine and high-dose pilocarpine seizures. Neurosci 23:953-968.

Codogno P, Meijer AJ (2005) Autophagy and signaling: Their role in cell survival and cell death.

Cell Death Differ 12:1509-1518.

Cohen MX, Elger CE, Weber B (2008) Amygdala tractography predicts functional connectivity

and learning during feedback-guided decision-making. Neuroimage 39:1396-1407.

Commission on classification and terminology of the international league against epilepsy.

(1989) Epilepsia 30:389-399.

Cornaggia CM, Beghi M, Provenzi M, Beghi E (2006) Correlation between cognition and

behavior in epilepsy. Epilepsia 47:34-39.

271

Coultrap SJ, Nixon KM, Alvestad RM, Valenzuela CF, Browning MD (2005) Differential

expression of NMDA receptor subunits and splice variants among the CA1, CA3 and dentate

gyrus of the adult rat. Curr Opin Neurobiol 135:104-111.

Cousins SL, Papadakis M, Rutter R, Stephenson A (2008) Differential interaction of NMDA

receptor subtypes with the post-synaptic density-95 family of membrane associated guanylate

kinase proteins. J Neurochem 104:903-913.

Cousins SL, Kenny AV, Stephenson FA (2009) Delineation of additional PSD-95 binding

domains within NMDA receptor NR2 subuntis reveals differences between NR2A/PSD-95 and

NR2B/PSD-95 association. Neurosci 158:89-95.

Covolan L, Mello LE (2000) Temporal profile of neuronal injury following pilocarpine or kainic

acid-induced status epilepticus. Epilepsy Res 39:133-152.

Covolan L, Smith RL, Mello LEAM (2000) Ultrastructural identification of dentate granule cell

death from pilocapine-induced seizures. Epilepsy Res 41:9-21.

Covolan L, Mello LE (2006) Assessment of the progressive nature of cell damage in the

pilocarpine model of epilepsy. Braz J Med Biol Res 39:915-924.

Cramer JA (2002) Mood disorders are linked to health-related quality of life in epilepsy.

Epilepsy Behav 3:492.

Cramer JA, Blum D, Reed M, Fanning K (2003) The influence of comorbid depression on

quality of life for people with epilepsy. Epilepsy Behav 4:521.

Croiset G, De Wied D (1992) ACTH: A structure-activity study on pilocarpine-induced epilepsy.

Eur J Pharmacol 229:211-216.

Cui H, Hayashi A, Sun H (2007) PDZ protein interactions underlying NMDA receptor-mediated

excitotoxicity and neuroprotection by PSD-95 inhibitors. J Neurosci 27:9901-9915.

Cull-Candy S, Brickley S, Farrant M (2001) NMDA receptor subunits: Diversity, development

and diseaes. Curr Opin Neurobiol 11:327-335.

Cunha AOS, Mortari MR, Liberato JL, dos Santos WF (2009) Neuroprotective effects of

diazepam, carbamazepine, phenytoin and ketamine after pilocarpine-induced status epilepticus.

Basic Clin Pharmacol Toxicol 104:470-477.

Curia G, Longo D, Biagini G, Jones RSG, Avoli M (2008) The pilocarpine model of temporal

lobe epilepsy. J Neurosci Methods 172:143-157.

D. Blumer, G. Montouris and B. Hermann (1995) Psychiatric morbidity in seizure patients on a

neurodiagnostic monitoring unit, J Neuropsychiatry Clin Neurosci 7:445-456.

Dalby NO, Mody I (2001) The process of epileptogenesis: A pathophysiological approach. Curr

Opin Neurol 14:187-192.

272

D'Ambrosio R (2004) Th erole of glial membrane ion channels in seizures and epileptogenesis.

Pharmacol Ther 103:95-108.

Danober L, Deransart C, Depaulis A, Vergnes M, Marescaux C (1998) Pathophysiological

mechanisms of genetic absence epilepsy in the rat. Prog Neurobiol 55:27-57.

Danzer SC (2008) Postnatal and adult neurogenesis in the development of human disease.

Neuroscientist 14:446-458.

Dashtipour K, Tran PH, Okazaki MM, Nadler JV, Ribak CE (2001) Ultrastructural features and

synaptic connections of hilar ectopic granule cells in the rat dentate gyrus are different from

those of granule cells in the granule cell layer. Brain Res 890:261-271.

Davis M (1992) The role of the amygdala in fear and anxiety. Annu Rev Neurosci 15:353-375.

Davis SM, Lees KR, Albers GW, Diener HC, Markabi S, Karlsson G, Norris J (2000) Selfotel in

acute ischemic stroke: Possible neurotoxic effects of an NMDA antagonist. Stroke 31:347-354.

Dawodu S, Thom M (2005) Quantitative neuropathology of the entorhinal cortex region in

patients with hippocampal sclerosis and temporal lobe epilepsy. Epilepsia 46:23-30.

Day LB, Schallert T (1996) Anticholinergic effects on acquisition of place learning in the morris

water task: Spatial mapping deficit or inability to inhibit nonplace strategies? Behav Neurosci

110:998-1005.

Day LB, Weisand M, Sutherland RJ, Schallert T (1999) The hippocampus is not necessary for a

place response but may be necessary for pliancy. Behav Neurosci 113:914-924.

de Boer HM, Mula M, Sander JW (2008) The global burden and stigma of epilepsy. Epilepsy

Behav 12:540-546.

de Lanerolle NC, Kim JH, Williamson A, Spencer SS, Zaveri HP, Eid T, Spencer DD (2003) A

retrospective analysis of hippocampal pathology in human temporal lobe epilepsy: Evidence for

distinctive patient subcategories. Epilepsia 44:677-687.

de Oliveira DL, Fisher A, Jorge RS, da Silva MC, Leite M, Goncalves CA, Quillfeldt JA, Souza

DO, e Souza TM, Wofchuk S (2008) Effects of early-life LiCl-pilocarpine-induced status

epilepticus on memory and anxiety in adult rats are associated with mossy fiber sprouting and

elevated CSF S100B protein. Epilepsia 49:842-852.

de Rogalski LI, Minokoshi M, Silveira DC, Cha BH, Holmes GL (2001) Recurrent neonatal

seizures: Relationship of pathology to the electroencephalogram and cognition. Brain Res Dev

Brain Res 129:27-38.

DeGiorgio CM, Correale JD, Gott PS, Ginsburg DL, Bracht KA, Smith T, Boutros R, Loskota

WJ, Rabinowicz AL (1995) Serum neuron-specific enolase in human status epilepticus.

Neurology 45:1134-1137.

273

DeGiorgio CM, Gott PS, Rabinowicz AL, Heck CN, Smith TD, Correale JD (1996) Neuron-

specific enolase, a marker of acute neuronal injury, is increased in complex partial status

epilepticus. Epilepsia 37:606-609.

DeGiorgio CM, Heck CN, Rabinowicz AL, Gott PS, Smith T, Correale J (1999) Serum neuron-

specific enolase in the major subtypes of status epilepticus. Neurology 52:746-749.

Degroot A, Treit D (2002) Dorsal and ventral hippocampal cholinergic systems modulate anxiety

in the plus-maze and shock-probe tests. Brain Res 949:60-70.

Delazer M, Zamarian L, Bonatti E, Kuchukhidze G, Koppelstätter F, Bodner T, Benke T, Trinka

E (2010) Decision making under ambiguity and under risk in mesial temporal lobe epilepsy.

Neuropsychologia 48:194-200.

Delazer M, Zamarian L, Bonatti E, Walser N, Kuchukhidze G, Bodner T, Benke T,

Koppelstaetter F, Trinka E (2011) Decision making under ambiguity in temporal lobe epilepsy:

Does the location of the underlying structural abnormality matter? Epilepsy Behav 20:34-37.

DeLorenzo RJ, Towne AR, Pellock JM, Ko D (1992) Status epilepticus in children, adults, and

the elderly. Epilepsia 33:S15-S25.

DeLorenzo RJ, Hauser WA, Towne AR, Boggs JG, Pellock JM, Penberthy L, Garnett L, Fortner

CA, Ko D (1996) A perspective, population-based epidemiologic study of status epilepticus in

richmond, virginia. Neurology 28:10-17.

DeLorenzo RJ, Garnett LK, Towne AR, Waterhouse EJ, Boggs JG, Morton L, Choudhry MA,

Barnes T, Ko D (1999) Comparison of status epilpeticus with prolonged seizure episodes lasting

from 10 to 29 minutes. Epilepsia 40:164-169.

Delorenzo RJ (2006) Epidemiology and clinical presentation of status epilepticus. Adv Neurol

97:199-215.

Deshpande LS, Lou JK, Mian A, Blair RE, Sombati S, DeLorenzo RJ (2007) In vitro status

epilepticus but not spontaneous recurrent seizures cause cell death in cultured hippocampal

neurons. Epilepsy Res 75:171-179.

Deshpande LS, Lou JK, Mian A, Blair RE, Sombati S, Attkisson E, DeLorenzo RJ (2008) Time

course and mechanism of hippocampal neuronal death in an in vitro model of status epilepticus:

Role of NMDA receptor activation and NMDA dependent calcium entry. Eur J Pharmacol 1:73-

83.

Detour J, Schroeder H, Desor D, Nehlig A (2005) A 5-month period of epilepsy impairs spatial

memory, decreases anxiety, but spares object recognition in the lithium/pilocarpine model in

adult rats. Epilepsia 46:499-508.

Devinsky O, Vickrey BG, Cramer J, Perrine K, Hermann B, Meador K, Hays RD (1995)

Development of the quality of life in epilepsy inventory. Epilepsia 36:1089-1104.

274

Devinsky O (2000) The meaning of quality of life to patients with epilepsy. Epilepsy Behav

1:S10-S20.

Devinsky O (2003) Psychiatric comorbidity in patients with epilepsy: Implications for diagnosis

and treatment. Epilepsy Behav 4:S2-S10.

Devinsky O (2004a) Therapy for neurobehavioural disorders in epilepsy. Epilepsia 45:34-40.

Devinsky O (2004b) Diagnosis and treatment of temporal lobe epilepsy. Rev Neurol Dis 1:2-9.

DiMattia BD, Kesner RP (1988) Spatial cognitive maps: Differential role of parietal cortex and

hippocampal formation. Behav Neurosci 102:471-480.

Ding S, Fellin T, Zhu Y, Lee SY, Auberson YP, Meaney DF, Coulter DA, Carmignoto G,

Haydon PG (2007) Enhanced astrocytic Ca2+ signals contribute to neuronal excitotoxicity after

status epilepticus. J Neurosci 27:10674-10684.

Doonan F, Cotter TG (2008) Morphological assessment of apoptosis. Methods 44:200-204.

dos Santos NF, Arida RM, Filho EM, Priel MR, Cavalheiro EA (2000) Epileptogenesis in

immature rats following recurrent status epilepticus. Brain Res Rev 31:269-276.

dos Santos JG, Longo BM, Blanco MM, de Oliveira MGM, Mello LE (2005) Behavioral

changes resulting from the administration of cycloheximide in the pilocarpine model of epilepsy.

Brain Res 1066:37-48.

Drislane FW, Blum AS, Lopez MR, Gautam S, Schomer DL (2009) Duration of refractory status

epilpeticus and outcome: Los of prognostic utility after several hours. Epilepsia 50:1566-1571.

Druga R, Kubova H, Suchomelova L, Haugvicova R (2003) Lithium/pilocarpine status

epilepticus-induced neuropathology of piriform cortex and adjoining structures in rats is age-

dependent. Physiol Res 52:251-264.

Druga R, Mares P, Otahal J, Kubova H (2005) Degenerative neuronal changes in the rat

thalamus induced by status epilepticus at different developmental stages. Epilepsy Res 63:43-65.

Du F, Whetsell WOJ, Abou-Khalil B, Blumenkopf B, Lothman EW, Schwarcz R (1993)

Preferential neuronal loss in layer III of the entorhinal cortex in patients with temporal lobe

epilepsy. Epilepsy Res 16:223-233.

Dudek FE, Hellier JL, Williams PA, Ferraro DJ, Staley KJ (2002) The course of cellular

alterations associated with the development of spontaneous seizures after status epilepticus. Prog


275

Dudek FE, Clark S, Wililams PA, Grabenstatter HL (2006) Kainate-induced status epilepticus: A

chronic model of acquired epilepsy. In: Models of seizures and epilepsy (Pitkanen A,

Schwartzkroin PA, Moshe SL, eds), pp415-432. Boston: Elsevier Academic Press.

Dudek FE, Sutula TP (2007) Epileptogenesis in the dentate gyrus: A critical perspective. Prog

Brain Res 163:755-773.

Duncan JS (1997) Imaging and epilepsy. Brain 120:339-377.

Dyker AG, Edwards KR, Fayad PB, Hormes JT, Lees KR (1999) Safety and tolerability study of

aptiganel hydrochloride in patients with an acute ischemic stroke. Stroke 39:2544-2553.

Dykstra CM, Ratnam M, Gurd JW (2009) Neuroprotection after status epilepticus by targeting

protein interactions with postsynaptic density protein 95. J Neuropathol Exp Neurol 68:823-831.

Ebert U, Brandt C, Loscher W (2002) Delayed sclerosis, neuroprotection, and limbic

epileptogenesis after status epilepticus in the rat. Epilepsia 43:86-95.

Eid T, Williamson A, Lee TW, Petroff OA, de Lanerolle NC (2008) Glutamate and astrocytes -

key players in human mesial temporal lobe epilpesy. Epilepsia 49:42-52.

Engel J, Wolfson L, Brown LL (1978) Anatomical correlates of electrical and behavioral events

related to amygdaloid kindling. Ann Neurol 3:538-544.

Engel J, Bandler R, Griffith NC, Caldecott-Hazard S (1991) Neurobiological evidence for

epilepsy-induced interictal disturbances. Adv Neuro 55:97-111.

Engel J (1993) Update on surgical treatment of the epilepsies. summary of the second

international palm desert conference on the surgical treatment of the epilepsies. Neurology

43:1612-1617.

Engel J (1996) Excitation and inhibition in epilepsy. Can J Neurol Sci 23:167-174.

Engel J (2001) A proposed diagnostic scheme for people with epileptic seizures and with

epilpesy: Report of the ILAE task force on classification and terminology. Epilepsia 42:796-803.

Engel J, Schwartzkroin PA (2006) What should be modeled? In: Models of seizures and epilepsy

(Pitkanen A, Schwartzkroin PA, Moshe SL, eds), pp1-14. Boston: Elsevier Academic Press.

Engel J, Pedley TA, Aicardi J (2008) Basal ganglia species difference: Rodents to primates. In:

Epilepsy: A comprehensive textbook . pp375. Philadelphia, PA: Lippincott Wiliams & Wilkins.

Engin E, Treit D (2007) The role of hippocampus in anxiety: Intracerebral infusion studies.

Behav Pharmacol 18:365-374.

Epp J, Keith JR, Spanswick SC, STone JC, Prusky GT, Sutherland RJ (2008) Retrograde

amnesia for visual memories after hippocampal damage in rats. Learn Mem 15:214-221.

276

Erdogan F, Gölgeli A, Küçük A, Arman F, Karaman Y, Ersoy A (2005) Effects of

pentylenetetrazole-induced status epilepticus on behavior, emotional memory and learning in

immature rats. Epilepsy Behav (Netherlands: Elsevier Science) 6:537-542.

Esclapez M, Hirsch JC, Ben-Ari Y, Bernard C (1999) Newly formed excitatory pathways

provide a substrate for hyperexcitability in experimental temporal lobe epilepsy. J Comp Neurol

408:449-460.

Fabene PF et al (2008) A role for leukocyte-endothelial adhesion mechanisms in epilepsy. Nat

Med (United States) 14:1377-1383.

Falter U, Gower AJ, Gobert J (1992) Resistance of baseline activity in the elevated plus-maze to

exogenous influences. Behav Pharmacol 3:123-128.

Fariello RG, Golden GT, Smith GG, Reyes PF (1989) Potentiation of kainic acid

epileptogenicity and sparing from neuronal damage by an NMDA receptor antagonist. Epilepsy

Res 3:206-213.

Faverjon S, Silveira DC, Fu DD, Cha BH, Akman C, Hu Y, Holmes GL (2002) Beneficial

effects of enriched environment following status epilepticus in immature rats. Neurology

59:1356-1364.

Federico F, Leggio MG, Neri P, Mandolesi L, Petrosini L (2006) NMDA receptor activity in

learning spatial procedural strategies II. the influence of cerebellar lesions. Brain Res Bull

70:356-367.

Felder CC (1995) Muscarinic acetylcholine receptors: Signal transduction through multiple

effectors. FASEB 9:619-625.

Fenton AA, Bures J (1993) Place navigation in rats with unilateral tetrodotoxin inactivation of

the dorsal hippocampus: Place but not procedural learning can be lateralized to one

hippocampus. Behav Neurosci 107:552-564.

Fernandes MJD, Dube C, Boyet S, Marescaux C, Nehlig A (1999) Correlation between

hypermetabolism and neuronal damage during status epilepticus induced by lithium and

pilocarpine in immature and adult rats. J Cereb Blood Flow Metab 19:195-209.

File SE (1993) The interplay of learning and anxiety in the elevated plus maze. Behav Brain Res

58:199-202.

Fisher RS, van Emde Boas W, Blume W, Elger C, Genton P, Lee P, Engel JJ (2005) Epileptic

seizures and epilepsy (ILAE) and the international bureau for epilepsy (IBE). Epilepsia 46:470-

472.

Fix AS, Horn JW, Wightman KA, Johnson CA, Long GG, Storts RW, Farber N, Wozniak DF,

Olney JW (1993) Neuronal vacuolization and necrosis induced by the noncompetitive N-methyl-

D-aspartate (NMDA) antagonist MK(+)801 (dizocilpine maleate): A light and electron

microscopic evaluation of the rat retrosplenial cortex. Exp Neurol 123:204-215.

277

Forder JP, Tymianski M (2009) Postsynaptic mechanisms of excitotoxicity: Involvement of

postsynaptic density proteins, radicals, and oxidant molecules. Neurosci 158:293-300.

Fountain NB (2000) Status epilepticus: Risk factors and complications. Epilepsia 41:23-30.

Franck JE, Pokorny J, Kunkel DD, Schwartzkroin PA (1995) Physiologic and morphologic

characteristics of granule cell circuitry in human epileptic hippocampus. Epilepsia 36:543-558.

Francois J, Ferrandon A, Koning E, Nehlig A (2005) Epileptic outcome is correlated with

protection in temporal cortices: Evidence from neuroprotection studies with a new drug,

RWJ333369, in the lithium/pilocarpine model. Epilepsia 46:62-63.

Francois J, Koning E, Ferrandon A, Nehlig A (2006) The combination of topiramate and

diazepam is partially neuroprotective in the hippocampus but not antiepileptogenic in the

lithium/pilocarpine model of temporal lobe epilepsy. Epilepsy Res 72:147-163.

French JA, Williamson PD, Thadani VM (1993) Characteristics of medial temporal lobe

epilepsy: results of history and physical examination. Ann Neurol 34:774-780.

Friedman LK, Saghyan A, Peinado A, Keesey R (2008) Age- and region-dependent patterns of

Ca2+ accumulations following status epilepticus. Int J Dev Neurosci 26:779-790.

Frisch C, Kudin AP, Elger CE, Kunz WS, Helmstaedter C (2007) Amelioration of water maze

performance deficits by topiramate applied during pilocarpine-induced status epilepticus is

negatively dose-dependent. Epilepsy Res 73:173-180.

Fuerst D, Shah J, Shah A, Watson C (2003) Hippocampal sclerosis is a progressive disorder: A

longitudinal volumetric MRI study. Ann Neurol 53:413-416.

Fujikawa DG, Daniels AH, Kim JS (1994) The competitive NMDA receptor antagonist CGP

40116 protects against status epilepticus-induced neuronal damage. Epilepsy Res 17:207-219.

Fujikawa DG (1995) Neuroprotective effect of ketamine administered after status epilepticus

onset. Epilepsia 36:186-195.

Fujikawa DG (1996) The temporal evolution of neuronal damage from pilocarpine-induced

status epilepticus. Brain Res 725:11-22.

Fujikawa DG, Shinmei SS, Cai B (1999) Lithium/pilocarpine-induced status epilepticus

produces necrotic neurons with internucleosomal DNA fragmentation in adult rats. Eur J

Neurosci 11:1605-1614.

Fujikawa DG, Itabashi HH, Wu A, Shinmei SS (2000a) Status epilepticus-induced neuronal loss

in humans without systemic complications or epilepsy. Epilepsia 41:981-991.

Fujikawa DG, Shinmei SS, Cai B (2000b) Kainic acid-induced seizures produce necrotic, not

apoptotic, neurons with internucleosomal DNA cleavage: Implications for programmed cell

death mechanisms. Neurosci 98:41-53.

278

Fujikawa DG, Ke X, Trinidad RB, Shinmei SS, Wu A (2002) Caspase-3 is not activated in

seizure-induced neuronal necrosis with internucleosomal DNA cleavage. J Neurochem 83:229-

240.

Fujikawa DG (2005) Prolonged seizures and cellular injury: Understanding the connection.

Epilepsy Behav 7:3-11.

Fujikawa DG, Zhao S, Ke X, Shinmei SS, Allen SG (2010) Mild as well as severe insults

produce necrotic, not apoptotic, cells: Evidence from 60-min seizures. Neurosci Lett 469:333-

337.

Furtado MA, Braga GK, Oliveira JA, Del Vecchio F, Garcia-Cairasco N (2002) Behavioral,

morphologic, and electroencephalographic evaluation of seizures induced by intrahippocampal

microinjection of pilocarpine. Epilepsia 43:S37-S39.

Furtado MA, Castro OW, Del Vecchio F, de Oliveira JA, Garcia-Cairasco N (2011) Study of

spontaneous recurrent seizures and morphological alterations after status epilepticus induced by

intrahippocampal injection of pilocarpine. Epilepsy Behav 20(2):257-266

Gaitatzis A, Trimble MR, Sander JW (2004) The psychiatric comorbidity of epilespy. Acta

Neurol Scand 110:207-220.

Garcia-Morales I, de la Pena Mayor, P., Kanner AM (2008) Psychiatric comorbidities in

epilepsy: Identification and treatment. Neurologist 14:S15-S25.

Gardella D, Hatton WJ, Rind HB, Rosen GD, von Bartheld CS (2003) Differential tissue

shrinkage and compression in the z-axis: Implications for optical disector counting in vibratome-

, plastic- and cryosections. J Neurosci Methods 124:45.

Gaskin S, Gamliel A, Tardif M, Cole E, Mumby DG (2009) Incidental (unreinforced) and

reinforced spatial learning in rats with ventral and dorsal lesions of the hippocampus. Behav


Geschwind N (1983) Interictal behavioral changes in epilepsy. Epilepsia 24:S23-S30.

Gilliam F (2002) Optimizing epilepsy management: Seizure control, reduction, tolerability, and

co-morbidities. introduction. Neurology 58:S9-S20.

Gleissner U, Helmstaedter C, Elger CE (1998) Right hippocampal contribution to visual

memory: A presurgical and postsurgical study in patients with temporal lobe epilepsy. J Neurol

Neurosurg Psychiatry 65:665-669.

Glien M, Brandt C, Potschka H, Voigt H, Ebert U, Löscher W (2001) Repeated low-dose

treatment of rats with pilocarpine: Low mortality but high proportion of rats developing epilepsy.

Epilepsy Res 46:111.

Glikmann-Johnston Y, Saling MM, Chen J, Cooper KA, Beare RJ, Reutens DC (2008) Structural

and functional correlates of unilateral mesial temporal lobe spatial memory impairment. Brain

131:3006-3018.

279

Goddard GV, McIntyre DC, Leech CK (1969) A permanent change in brain function resulting

from daily electrical stimulation. Exp Neurol 25:295-330.

Goffin K, Nissinen J, Van Laere K, Pitkanen A (2007) Cyclicity of spontaneous recurrent

seizures in pilocarpine model of temporal lobe epilepsy in rats. Exp Neurol 205:501-505.

Golden GT, Smith GG, Ferrar TN, Reyes PF, Kulp JK, Fariello RG (1991) Strain differences in

convulsive response to the excitotoxin kainic acid. Neuroreport 2:141-144.

Golden GT, Smith GG, Ferraro TN, Reyes PF (1995) Rat strain and age differences in kainic

acid induced seizures. Epilepsy Res 20:151-159.

Goldstein P, Kroemer G (2007) Death by necrosis: Towards a molecular definition Trends

Biochem Sci 32:37-43.

Goodkin HP, Yeh JL, Kapur J (2005) Status epilepticus increases the intracellular accumulation

of GABAA receptors. J Neurosci 25:5511-5520.

Goodkin HP, Kapur J (2009) The impact of diazepam's discovery on the treatment and

understanding of status epilepticus. Epilepsia 50:2011-2018.

Gorter JA, Goncalves Pereira PM, van Vliet EA, Aronica, E. Lopes da Silva, F.H., Lucassen P

(2004) Neuronal cell death in a rat model of mesial temporal lobe epilepsy is induced by the

initial status epilepticus and not by later repeated spontaneous seizures. Epilepsia 44:647-658.

Götz-Trabert K, Hauck C, Wagner K, Fauser S, Schulze-Bonhage A (2008) Spread of ictal

activity in focal epilepsy. Epilepsia 49:1594-1601.

Graziano A, Petrosini L, Bartoletti A (2003) Automatic recognition of explorative strategies in

the morris water maze. J Neurosci Methods 130:33-44.

Griebel G (1995) 5-hydroxytryptamine-interacting drugs in animal models of anxiety disorders:

more than 30 years of research. Pharmacol Ther 65:319-395.

Groticke I, Hoffmann K, Loscher W (2008) Behavioral alterations in a mouse model of temporal

lobe epilepsy induced by intrahippocampal injection of kainate. Exp Neurol 213:71-83.

Guénot M (2004) Surgical treatment of epilepsy: Outcome of various surgical procedures in

adults and children. Rev Neurol (Paris) 160:S241-S250.

Guerreiro CA, Jones-Gotman M, Andermann F, Bastos A, Cendes F (2001) Severe amnesia in

epilepsy: Causes, anatomopsychological considerations, and treatment. Epilepsy Behav 2:224-

246.

Gupta RC, Dettbarn W (2003) Prevention of kainic acid seizures-induced changes in levels of

nitric oxide and high-energy phosphates by 7-nitroindazole in rat brain regions. Brain Res

981:184-192.

280

Gupta R, Koscik TR, Bechara A, Tranel D (2011) The amygdala and decision-making.

Neurophsychologia 49(4):1315-1324

Gutbrod K, Krouzel C, Hofer H, Müri R, Perrig W, Ptak R (2006) Decision-making in amnesia:

Do advantageous decisions require conscious knowledge of previous behavioural choices?.

Neuropsychologia 44:1315-1324.

Haberly LB PJ (1978) Association and commissural fiber systems of the olfactory cortex of the

rat. J Comp Neurol 178:711-740.

Haim E, Belmaker RH (2001) The effects of inositol treatment in animal models of psychiatric

disorders . J Affect Disord 62:113-121.

Hallera J, Kruk MR (2006) Normal and abnormal aggression: Human disorders and novel

laboratory models. Neurosci Biobehav Rev 30:292-303.

Halonen T, Nissinen J, Pitkänen A (2001) Effect of lamotrigine treatment on status epilepticus-

induced neuronal damage and memory impairment in rat. Epilepsy Res 46:205-223.

Hamilton SE, Loose MD, Qi M, Levey AI, Hille B, McKnight GS, Idzerda RL, Nathanson NM

(1997) Disruption of the m1 receptor gene ablates muscarinic receptor-dependent M current

regulation and seizure activity in mice. Proc Natl Acad Sci USA 94:13316.

Handforth A, Ackermann RF (1988) Functional [14C]2-deoxyglucose mapping of progressive

states of status epilepticus induced by amygdala stimulation in rat. Brain Res 460:94-102.

Handforth A, Ackermann RF (1992) Hierarchy of seizure states in the electrogenic limbic status

epilepticus model: Behavioral and electrographic observations of initial states and temporal

progression. Epilepsia 33:589-600.

Handforth A, Ackermann RF (1995) Mapping of limbic seizure progressions utilizing the

electrogenic status epilepticus model and the 14C-2-deoxyglucose method. Brain Res Brain Res

Rev 20:1-23.

Handforth A, Treiman DM (1995) Functional mapping of the early stages of status epilepticus: A

C14-2-deoxyglucose study in the lithium pilocarpine model in rat. Neurosci 64:1057-1073.

Harding AJ, Halliday GM, Cullen K (1994) Practical considerations for the use of the optical

dissector in estimating neuronal number. J Neurosci Methods 51:83-89.

Hardingham GE (2009) Coupling of the NMDA receptor to neuroprotective and

neurodestructive events. Biochem Soc Trans 37:1147-1160.

Harwood SM, Yaqoob MM, Allen DA (2005) Caspase and calpain function in cell death:

Bridging the gap between apoptosis and necrosis. Ann Clin Biochem 42:415-431.

Hattiangady B, Shetty AK (2008) Implications of decreased hippocampal neurogenesis in

chronic temporal lobe epilepsy. Epilepsia 49:26-41.

281

Hattiangady B, Rao MS, Shetty AK (2004) Chronic temporal lobe epilpesy is associated with

severely declined dentate neurogenesis in the adult hippocampus. Neurobiol Dis 17:473-490.

Hatton WJ, Von Bartheld CS (1999) Analysis of cell death in the trochlear nucleus of the chick

embryo: Calibration of the optical disector counting method reveals systematic bias. J Comp

Neurol 409:169.

Hawley WR, Grissom EM, Dohanich GP (2011) The relationships between trait anxiety, place

recognition memory, and learning strategy. Behav Brain Res 216:525-530.

Heinrichs SC, Seyfried TN (2006) Behavioral seizures correlates in animal models of epilepsy:

A road map for assay selection, data interpretation, and the search for causal mechanisms.


Hellier JL, Patrylo PR, Buckmaster PS, Dudek FE (1998) Recurrent spontaneous motor seizures

after repeated low-dose systemic treatment with kainate: Assessment of a rat model of temporal

lobe epilepsy. Epilepsy Res 31:73-84.

Helmstaedter C (2007) Cognitive outcome of status epilepticus in adults. Epilepsia 48:85-90.

Henry TR, Mazziotta JC, Engel JJ, Christenson PD, Zhang JX, Phelps ME, Kuhl DE (1990)

Quantifying interictal metabolic activity in human temporal lobe epilepsy. J Cereb Blood Flow

Metab 10:748-757.

Henshall DC (2007) Apoptosis signalling pathways in seizure-induced neuronal death and

epilepsy. Biochem Soc Trans 35:421-423.

Henshall DC, Murphy BM (2008) Modulators of neuronal cell death in epilepsy. Curr Opin

Pharmacol 8:75-81.

Hermann BP, Seidenberg M, Schoenfeld J, Davies K (1997) Neuropsychological characteristics

of the syndrome of mesial temporal lobe epilepsy. Arch Neurol 54:369-376.

Herzog AG (1999) Psychoneuroendocrine aspects of temporolimbic epilepsy: Part I. brain,

reproductive steroids, and emotions. Psychosomatics 40:95-101.

Hesdorffer DC, Logroscino G, Cascino G, Annegers JF, Hauser WA (1998) Risk of unprovoked

seizure after acute symptomatic seizure: Effect of status epilepticus. Ann Neurol 44:908-812.

Hodges H (1996) Maze procedures: The radial-arm and water maze compared. Brain Res Cogn


Hogg S (1996) A review of the validity and variability of the elevated plus-maze as an animal

model of anxiety. Pharmacol Biochem Behav 54:21-30.

Holmes GL, Sarkisian M, Ben-Ari Y, Chevassus-Au-Louis N (1999) Mossy fiber sprouting after

recurrent seizures during early development in rats. J Comp Neurol 404:537-553.

282

Holtkamp M, Schuchmann S, Gottschalk S, Meierkord H (2004) Recurrent seizures do not cause

hippocampal damage. J Neurol 251:458-463.

Honchar MP, Olney JW, Sherman WR (1983) Systemic cholinergic agents induce seizures and

brain damage in lithium-treated rats. Science 220:323-325.

Hort F, Broiek G, Mares P, Langmeier M, Komirek V (1999) Cognitive functions after

pilocarpine-induced status epilepticus: changes during silent period precede appearance of

spontaneous recurrent seizures. Epilepsia 40:177-183.

Hort J, Brozek G, Komárek V, Langmeier M, Mareš P (2000) Interstrain differences in cognitive

functions in rats in relation to status epilepticus. Behav Brain Res 112:77-83.

Horvath ZA, Czopf J, Buzsaki G (1997) MK-801-induced neuronal damage in rats. Brain Res

753:181-195.

Houser CR, Miyashiro JE, Swartz BE, Walsh GO, Rich JR, Delgado-Escueta AV (1990) Altered

patterns of dynorphin immunoreactivity suggest mossy fiber reorganization in human

hippocampal epilepsy. J Neurosci 10:267-282.

Hudson LP, Munoz DG, Miller L, McLachlan RS, Girvin JP, Blume WT (1993) Amygdaloid

sclerosis in temporal lobe epilepsy. Ann Neurol 33:622-631.

Hung PL, Lai MC, Yang SN, Wang CL, Liou CW, Wu CL, Wang TJ, Huang LT (2002)

Aminophylline exacerbates status epilepticus-induced neuronal damages in immagure rats: A

morphological, motor and behavioural study. Epilepsy Res 49:218-225.

Huo JZ, Dykstra CM, Gurd JW (2006) Increase in tyrosine phosphorylation of the NMDA

receptor following the induction of status epilepticus. Neurosci Lett 401:266-270.

Ikonomidou C, Turski L (2002) Why did NMDA receptor antagonists fail clinical trials for

stroke and traumatic brain injury? Lancet Neurol 34:235-244.

Inglish J, Morris RGM (2004) Reversible hippocampal inactivation partially dissociates how and

where to search in the water maze. Behav Neurosci 118:1022-1032.

Ingvar M (1986) Cerebral blood flow and metabolic rate during seizures. relationship to epileptic

brain damage. Ann N Y Acad Sci 462:194-206.

Ingvar M, Folbergrova J, Siesjo BK (1987) Metabolic alterations underlying the development of

hypermetabolic necrosis in the substantia nigra in status epilepticus. J Cereb Blood Flow Metab

7:103-108.

Jabs R, Seifert G, Steinhäuser C (2008) Astrocytic function and its alteration in the epileptic

brain. Epilepsia 49:3-12.

Jan MM, Sadler M, Rahey SR (2010) Electroencephalographic features of temporal lobe

epilepsy. Can J Neurol Sci 37:439-448.

283

Jing G, Zhao-Fu C, Xue-Wu L, Pei-Yan S, Rong W (2007) Mitochondrial dysfunction and

ultrastructural damage in the hippocampus of pilocarpine-induced epileptic rat. Neurosci Lett

411:152-157.

Jing M, Shingo T, Yasuhara T, Kondo A, Morimoto T, Wang F, Baba T, Yuan WJ, Tajiri N,

Uozumi T, Murakami M, Tanabe M, Miyoshi Y, Zhao S, Date I (2009) The combined therapy of

intrahippocampal transplantation of adult neural stem cells and intraventricular erythropoietin-

infusion ameliorates spontaneous recurrent seizures by suppression of abnormal mossy fiber

sprouting. Brain Res 1295:203-217.

Johnson EK, Jones JE, Seidenberg M, Hermann BP (2004) The releative impact of anxiety,

depression, and clinical features on health-related quality of life in epilepsy. Epilepsia 45:544-

550.

Jones NC, Salzberg MR, Kumar G, Couper A, Morris MJ, O'Brien TJ (2008) Elevated anxiety

and depressive-like behavior in a rat model of genetic generalized epilepsy suggesting common

causation. Exp Neurol 209:254-260.

Jope RS, Gu X (1991) Seizures increase acetylcholine and choline concentrations in rat brain

regions. Neurochem Res 16:1219-1226.

Jope RS, Williams MB (1994) Lithium and brain signal transduction systems. Biochem Pharm

47:429-441.

Juhász C, Nagy F, Watson C, da Silva EA, Muzik O, Chugani DC, Shah J, Chugani HT (1999)

Glucose and [11C]flumazenil positron emission tomography abnormalities of thalamic nuclei in

temporal lobe epilepsy. Neurology 53:2037-2045.

Jun Y, Jiang TX, Yuan GHY, B.S., Jun Z, Xiao JM, Jian CX, Heng X, Xiao XZ, Xin XX (2009)

Erythropoietin pre-treatment prevents cognitive impairments following status epilepticus in rats.

Brain Res 1282:57-66.

Jung KH, Chu K, Lee ST, Kim J, Sinn DI, Kim JM, Park DK, Lee JJ, Kim SU, Kim M, Lee SK,

Roh JK (2006) Cyclooxygenase-2 inhibitor, celecoxib, inhibits the altered hippocampal

neurogenesis with attenuation of spontaneous recurrent seizures following pilocarpine-induced

status epilepticus. Neurobiol Dis 23:237-246.

Jung KH, Chu K, Lee ST, Kim JH, Kang KM, Song EC, Kim SJ, Park HK, Kim M, Lee SK, Roh

JK (2009) Region-specific plasticity in the epileptic rat brain: A hippocampal and

extrahippocampal analysis. Epilepsia 50:537-549.

Jutila L, Immonen A, Partanen K, Partanen J, Mervaala E, Ylinen A, Alafuzoff I, Paljärvi L,

Karkola K, Vapalahti M, Pitkänen A (2002) Neurobiology of epileptogenesis in the temporal

lobe. Adv Tech Stand Neurosurg 27:5-22.

Kalvianinen R, Salmenpera T (2002) Do recurrent seizures cause neuronal damage? Prog Brain

Res 135:279-295.

284

Kapur J (2006) Is mesial temporal sclerosis a necessary component of temporal lobe epilepsy?

Epilepsy Curr 6:208-209.

Kelly OP, McIntosh J, McIntyre DC, Merali Z, Anisman H (2003) Anxiety in rats selective bred

for fast and slow kindling rates: Situation-specific outcomes. Stress 6:289-295.

Kennedy MR, Carney E, Peters SM (2003) Predictions of recall and study strategy decisions

after diffuse brain injury. Brain Inj 17:1043-1064.

Kermer P, Klocker N, Bahr M (1999) Neuronal death after brain injury. models, mechanisms,

and therapeutic strategies in vivo. Cell Tissue Res 298:383-395.

Khan GM, Smolders I, Lindekens H, Manil J, Ebinger G, Michotte Y (1999) Effects of diazepam

on extracellular brain neurotransmitters in pilocarpine-induced seizures in rats. Eur J Pharmacol

373:153-161.

King D, Spencer S (1995) Invasive electroencephalography in mesial temporal lobe epilepsy. J

Clin Neurophysiol 12:32-45.

Kirsch I, Lynn SJ, Vigorito M, Miller RR (2004) The role of cognition in classical and operant

conditioning. J Clin Psychol 60:369-392.

Kjelstrup KG, Tuvnes FA, Steffenach HA, Murison R, Moser EI, Moser MB (2002) Reduced

fear expression after lesions of the ventral hippocampus. Proc Natl Acad Sci USA 99:10825-

10830.

Klitgaard H, Matagne A, Vanneste-Goemaere J, Margineanu DG (2002) Pilocarpine-induced

epileptogenesis in the rat: Impact of initial duration of status epilepticus on electrophysiological

and neuropathological alterations. Epilepsy Res 51:93-107.

Knake S, Hamer HM, Rosenow F (2009) Status epilepticus: A critical review. Epilepsy Behav

15:10-14.

Kobayashi M, Buckmaster PS (2003) Reduced inhibition of dentate granule cells in a model of

temporal lobe epilepsy. J Neurosci 23:2440-2452.

Kobayashi E, Grova C, Tyvaert L, Dubeau F, Gotman J (2009) Structures involved at the time of

temporal lobe spikes revealed by interindividual group analysis of EEG/fMRI data. Epilepsia

50:2549-2556.

Koch UR, Musshoff U, Pannek HW, Ebner A, Wolf P, Speckmann EJ, Köhling R (2005)

Intrinsic excitability, synaptic potentials, and short-term plasticity in human epileptic neocortex.

J Neurosci Res 80:715-726.

Kondratyev A, Gale K (2004) Latency to onset of status epilepticus determines molecular

mechanisms of seizure-induced cell death. Mol Brain Res 121:86-94.

Kornau H, Schenker LT, Kennedy MB (1995) Domain interaction between NMDA receptor

subunits and the postsynaptic density protein PSD-95. Science 269:1737-1740.

285

Kotariya NT, Bikashvili TZ, Zhvaniya MG, Chkhikvishvili TG (2010) Ultrastructure of

hippocampal field CA1 in rats after status epilepticus induced by systemic administration of

kinaic acid. Neurosci Behav Physiol 40:127-130.

Krsek P, Mikuleck A, Druga R, Hlink Z, Kubov H, Mares P (2001) An animal model of

nonconvulsive status epilepticus: A contribution to clinical controversies. Epilepsia 42:171-180.

Krystal JH, Karper LP, Seibyl JP, Freeman GK, Delaney R, Bremner JD, Heninger GR, Bowers

MB, Charney DS (1994) Subanesthetic effects of the noncompetitive NMDA antagonist,

ketamine, in humans. psychotomimetic, perceptual, cognitive, and neuroendocrine responses.

Arch Gen Psychiatry 51:199-214.

Kubova H, Mares P, Suchomelova L, Brozek G, Druga R, Pitkanen A (2004) Status epilepticus

in immature rats leads to behavioural and cognitive impairment and epileptogenesis. Eur J

Neurosci 19:3255-3265.

Kuruba R, Hattiangady B, Shetty AK Hippocampal neurogenesis and neural stem cells in

temporal lobe epilepsy. Epilepsy Behav 14:65-73.

Kuzmiski JB, MacVicar BA (2001) Cyclic nucleotide-gated channels contribute to the

cholinergic plateau potential in hippocampal CA1 pyramidal neurons. J Neurosci 21:8707-8714.

Labudda K, Frigge K, Horstmann S, Aengenendt J, Woermann FG, Ebner A, Markowitsch HJ,

Brand M (2009) Decision making in patients with temporal lobe epilepsy. Neuropsychologia

47:50-58.

Lahti AC, Koffe lB, LaPorte D, Tamminga CA (1995) Subanesthetic doses of ketamine stimulate

psychosis in schizophrenia. Neuropsychopharmacology 13:9-19.

Lanke J, Månsson L, Bjerkemo M, Kjellstrand P (1993) Spatial memory and stereotypic

behaviour of animals in radial arm mazes. Brain Res 605:221-228.

Lau A, Tymianski M (2010) Glutamate receptors, neurotoxicity and neurodegeneration. Pflugers

Arch 460(2):525-542

Lee HK, Choi SS, Han KJ, Han EJ, Suh HW (2003) Cycloheximide inhibits neurotoxic

responses induced by kainic acid in mice. Brain Res Bull 61:99-107.

Leggio MG, Federico F, Neri P, Graziano A, Mandolesi L, Petrosini L (2006) NMDA receptor

activity in learning spatial procedural strategies I. the influence of hippocampal lesions. Brain

Res Bull 70:347-355.

Leggio MG, Neri P, Graziano A, Mandolesi L, Molinari M, Petrosini L (1999) Cerebellar

contribution to spatial event processing: Characterization of procedural learning. Exp Brain Res

127:1-11.

Leite JP, Bortolotto ZA, Cavalheiro EA (1990) Spontaneous seizures in rats: An experimental

model of partial epilepsy. Neurosci Biobehav Rev 14:511-517.

286

Leite JP, Cavalheiro EA (1995) Effects of conventional antiepileptic drugs in a model of

spontaneous recurrent seizures in rats. Epilepsy Res 20:93-104.

Leite JP, Garcia-Cairasco N, Cavalheiro EA (2002) New insights from the use of pilocarpine and

kainate models. Epilepsy Res 50:93-103.

Lemos T, Cavalheiro EA (1995) Suppression of pilocarpine-induced status epilepticus and the

late development of epilepsy in rats. Exp Brain Res 102:423-428.

Letty S, Lerner-Natoli M, Rondouin G (1995) Differential impairments of spatial memory and

social behavior in two models of limbic epilepsy. Epilepsia 36:973-982.

Levine B, Klionsky DJ (2004) Development by self-digestion: Molecular mechanisms and

biological functions of autophagy. Dev Cell 6:463-477.

Lewis DV (1999) Febriel convulsions and mesial temporal sclerosis. Current Opin Neurol

12:197-201.

Li XG, Somogyi P, Ylinen A, Buzsaki G (1994) The hippocampal CA3 network: An in-vivo

intracellular labeling study. J Comp Neurol 339:181-208.

Lieb JP, Hoque K, Skomer CE, Song XW (1987) Inter-hemispheric propagation of human mesial

temporal lobe seizures: A coherence/phase analysis. Electroencephalogr Clin Neurophysiol

67:101-119.

Lieb JP, Dasheiff RM, Engel JJ (1991) Role of the frontal lobes in the propagation of mesial

temporal lobe seizures. Epilepsia 32:822-837.

Liu Z, Nagao T, Desjardins GC, Gloor P, Avoli M (1994) Quantitative evaluation of neuronal

loss in the dorsal hippocampus in rats with long-term pilocarpine seizures. Epilepsy Res 17:247.

Liu RS, Lemieux L, Sander JW, Sisodiya SM, Duncan JS (2002) Seizure-associated

hippocampal volume loss: A longitudinal magnetic resonance study of temporal lobe epilepsy.

Ann Neurol 52:861.

Loddenkemper T, Kotagal P (2005) Lateralizing signs during seizures in focal epilepsy. Epilepsy

Behav 7:1-17.

Longo BM, Mello LE (1997) Blockade of pilocarpine- or kainate-induced mossy fiber sprouting

by cycloheximide does not prevent subsequent epileptogenesis in rats. Neurosci Lett 226:163-

166.

Lopez-Meraz ML, Niquet J, Wasterlain CG (2010) Distinct caspase pathways mediate necrosis

and apoptosis in subpopulations of hippocampal neurons after status epilepticus. Epilepsia 51:56-

60.

Lorente De Nó R (1934) Studies on the structure of the cerebral cortex. II. continuation of the

study of the ammonic system. Journal Für Psychologie Und Neurologie 46:113-177.

287

Lorenzana AL, Chancer Z, Schauwecker PE (2007) A quantitative train locus on chromosome 18

is a critical determinant of excitotoxic cell death susceptibility. Eur J Neurosci 25:1998-2008.

Löscher W, Ebert U (1996) The role of the piriform cortex in kindling. Prog Neurobiol 50:427-

481.

Löscher W (2002) Animal models of epilepsy for the development of antiepileptogenic and

disease-modifying drugs. A comparison of the pharmacology of kindling and post-status

epilepticus models of temporal lobe epilepsy. Epilepsy Res 50:105-123.

Löscher W (2007) Mechanisms of drug resistance in status epilepticus. Epilepsia 48:S74-S77.

Löscher W, Brandt C (2010) Prevention or modification of epileptogenesis after brain insults:

Experimental approaches and translational research. Pharmacol Rev 62:668-700.

Lothman EW, Collins RC (1981) Kainic acid induced limbic seizures: Metabolic, behavioural,

electroencephalographic and neuropathological correlates. Brain Res 218:318.

Lothman EW, Bertram EH, Stringer JL (1991) Functional anatomy of hippocampal seizures.

Prog Neurobiol 37:1-82.

Lowenstein DH (1999) Status epilepticus: An overview of the clinical problem. Epilepsia 40:S3-

S8.

Lowenstein DH, Bleck T, Macdonald RL (1999) It's time to revise the definition of status


Lowenstein DH (2001) Structural reorganization fo hippocampal networks caused by seizure

activity. Int Rev Neurobiol 45:209-236.

Luongo R, Oliveira DA, Lebrun I, Sandoval MR (2009) Diazepam and pentobarbital protect

against scorpion venom toxin-induced epilepsy. Brain Res Bull 79:296-302.

Lynch DR, Guttmann RP (2002) Excitotoxicity: Perspectives based on N-methyl-D-aspartate

receptor subtypes. J Pharmacol Exp Ther 300:717-723.

Macdonald RL, Kapur J (1999) Acute cellular alterations in the hippocampus after status

epiletpicus. Epilepsia 40:S9-S20.

Maillard L, Vignal JP, Gavaret M, Guye M, Biraben A, McGonigal A, Chauvel P, Bartolomei F

(2004) Semiologic and electrophysiologic correlations in temporal lobe seizure subtypes.

Epilepsia 45:1590-1599.

Majak K, Pikkarainen M, Kemppaninen S, Jolkkonen E, Pitkänen A (2002) Projections from the

amygdaloid complex to the claustrum and the endopiriform nucleus: A phaseolus vulgaris

leucoagglutinin study in the rat. J Comp Neurol 451:236-249.

Majak K, Pitkanen A (2004) Do seizures cxause irreversible cognitive damage? evidence from

animal studies. Epilepsy Behav 5:S35-S44.

288

Majak K, Rönkkö S, Kemppaninen S, Pitkänen A (2004) Projections from the amygdaloid

complex to the piriform cortex: A PHA-L study in the rat. J Comp Neurol 476:414-428.

Majores M, Schoch S, Lie A, Becker AJ (2007) Molecular neuropathology of temporal lobe

epilepsy: Complementary approaches in animal models and human disease tissue. Epilepsia

48:4-12.

Malhotra AK, Pinals DA, Weingartner H, Sirocco K, Missar CD, Pickar D, Breier A (1996)

NMDA receptor function and human cognition: The effects of ketamine in healthy volunteers.

Neuropsychopharmacology 14:301-307.

Mangan PS, Scott CA, Williamson JM, Bertram EH (2000) Aberrant neuronal physiology in the

basal nucleus of the amygdala in a model of chronic limbic epilepsy. Neurosci 101:377-391.

Manning F, Zuzel K (2003) Comparison of types of cell death: Apoptosis and necrosis. J Biol

Educ 37:141-145.

Marcangelo MJ, Ovsiew F (2007) Psychiatric aspects of epilepsy. Psychiatr Clin North Am

30:781-802.

Marescaux C, Vergnes M, Depaulis A (1992) Genetic absence epilepsy in rats from strasbourg--

a review. J Neural Transm Suppl (Wien) 35:37-69.

Margerison JH, Corsellis JA (1966) Epilepsy and the temporal lobes. A clinical,

electroencephalographic and neuropathological study of the brain in epilepsy, with particular

reference to the temporal lobes. Brain 89:499-530.

Martin ED, Pozo MA (2006) Animal models for the development of new neuropharmacological

therapeutics in the status epilepticus. Curr Neuropharm 4:33-40.

Maslansky JA, Powelt R, Deimengiant C, Patelt J (1994) Assessment of the muscarinic receptor

subtypes involved in pilocarpine-induced seizures in mice. Neurosci Lett 168:225-228.

Masukawa LM, Wang H, O'Connor MJ, Uruno K (1996) Prolonged field potentials evoked by 1

hz stimulation in the dentate gyrus of temporal lobe epileptic human brain slices. Brain Res

721:132-139.

Mathern GW, Babb TL, Leite JP, Pretorius K, Yeoman KM, Kuhlman PA (1996) The

pathogenic and progressive features of chronic human hippocampal epilepsy. Epilepsy Res

26:151-161.

Mathern GW, Adelson PD, Cahan LD, Leite JP (2002) Hippocampal neuron damage in human

epilepsy: Meyer's hypothesis revisited. Prog Brain Res 135:237-251.

Matsuura M, Oana Y, Kato M, Kawana A, Kan R, Kubota H, Nakano T, Hara T, Horikawa N

(2003) A multicenter study on the prevalence of psychiatric disorders among new referrals for

epilepsy in Japan. Epilepsia 44:114.

289

McDonald AJ, Pearson JC (1989) Coexistence of GABA and peptide immunoreactivity in non-

pyramidal neurons of the basolateral amygdala. Neurosci Lett 100:53-58.

McDonald RJ, White NM (1993) A triple dissociation of memory systems: Hippocampus,

amydgala and dorsal striatum. Behav Neurosci 107:3-22.

McDonald RJ, White NM (1994) Parallel information processing in the water maze: Evidence

for independent memory systems involving dorsal striatum and hippocampus. Behav Neurol Bio

61:260-270.

McDonald AJ, Mascagni F (2002) Immunohistochemical characterization of somatostatin

containing interneurons in the rat basolateral amygdala. Brain Res 943:237-244.

McDonough JH, Jr McLeod CG, Jr Nipwoda MT (1987) Direct microinjection of soman or VX

into the amygdala produces repetitive limbic convulsions and neuropathology. Brain Res

435:123-137.

McIntyre DC, Kelly ME (1990) Pyriform cortex involvement in limbic kindling. Soc Neurosci

Abstr 16:1107.

McIntyre DC, Kelly ME, Dufresne C (1999) FAST and SLOW amygdala kindling rat strains:

Comparison of amygdala, hippocampal, piriform and perirhinal cortex kindling. Epilepsy Res

35:197-209.

McIntyre DC, Poulter MO, Gilby K (2002) Kindling: Some old and some new. Epilepsy Res

50:79-82.

McIntyre CK, Marriott LK, Gold PE (2003) Patterns of brain acetylcholine release predict

individual differences in preferred learning strategies in rats. Neurobiol Learn Mem 79:177-183.

McIntyre DC, Gilby KL (2007) Genetically seizure-prone or seizure-resistant phenotypes and

their associated behavioral comorbidities. Epilepsia 48:30-32.

McKay BE, Persinger MA (2004) Normal spatial and contextual learning for ketamine-treated

rats in the pilocarpine epilepsy model. Pharmacol Biochem Behav 78:111-119.

McKhann GM, Schoenfeld-McNeill J, Born DE, Haglund MM, Ojemann GA (2000)

Intraoperative hippocampal electrocorticography to predict the extent of hippocampal resection

in temporal lobe epilepsy surgery. J Neurosurg 93:44-52.

McKhann GM, Wenzel HJ, Robbins CA, Sosunov AA, Schwartzkroin PA (2003) Mouse strain

differences in kainic acid sensitivity, seizure behavior, mortality and hippocampal pathology.

Neurosci 122:551-561.

McLin JP, Steward O (2006) Comparison of seizure phenotype and neurodegeneration induced

by systemic kainic acid in inbred, outbred and hybrid mouse strains. Eur J Neurosci 24:2191-

2292.

290

McNamara JO, Huang YZ, Leonard S (2006) Molecular signaling mechanisms underlying

epileptogenesis. Sci STKE 356:1-17.

Meldrum BS, Brierley JB (1973) Prolonged epileptic seizures in primates: Ischemic cell change

adn its relation to ictal physiological events. Arch Neurol 28:10-17.

Meldrum BS, Horton RW, Brierley JB (1974) Epileptic brain damage in adolescent baboons

following seizures induced by allylglycine. Brain 97:407-418.

Meldrum BS (1999) The revised operational definition of generalised tonic-clonic (TC) status

epilepticus in adults. Epilepsia 40:123-124.

Meldrum BS (2002) Concept of activity-induced cell death in epilepsy: Historical and

comtemporary perspectives. Progress Brain Res 135:1-11.

Mello LE, Cavalheiro EA, Babb TL, Kupfer WR, Pretorius JK, Tan AM, Finch DM (1993)

Circuit mechanisms of seizures in the pilocarpine model of chronic epilepsy: Cell loss and mossy

fiber sprouting. Epiepsia 34:985-995.

Melloa LE, Mendez-Oterobs R (1996) Expression of 9-O-acetylated gangliosides in the rat

hippocampus. Neurosci Lett 213:17-2-.

Menard J, Treit D (2001) The anxiolytic effects of intra-hippocampal midazolam are antagonized

by intra-septal L-glutamate. Brain Res 888:163-166.

Mendes de Freitas R, Aguiar LM, Vasconcelos SM, Sousa FC, Viana GS, Fonteles MM (2005)

Modifications in muscarinic, dopaminergic and serotonergic receptors concentrations in the

hippocampus and striatum of epileptic rats. Life Sci 78:253-258.

Meurs A, Clinckers R, Ebinger G, Michotte Y, Smolders I (2008) Seizure activity and changes in

hippocampal extracellular glutamate, GABA, dopamine and serotonin. Epilepsy Res 78:50-59.

Midzyanovskaya LS, Shatskova AB, Sarkisova KY, van Luijtelaar G, Tuomisto L, Kuznetsova

GD (2005) Convulsive and nonconvulsive epilepsy in rats: Effects on behavioral response to

novelty stress. Epilepsy Behav 5:543-551.

Mikati MA, Tarif S, Lteif L, Jawad MA (2001) Time sequence and types of memory deficits

after experimental status epilepticus. Epilepsy Res 43:97-101.

Milgram NW, Isen DA, Mandel D, Palantzas H, Pepkowski MJ (1988) Deficits in spontaneous

behavior and cognitive function following systematic administration of kainic acid.

Neurotoxicology 9:611-624.

Mingo NS, Cottrell G, Zhang L, Wallace MC, Burnham WM, Eubanks JH (1997) Kainic acid-

induced generalized seizures alter the regional hippocampal expression of the rat m1 and m3

muscarinic acetylcholine receptor genes. Epilepsy Res 29:71-79.

291

Mintzer S, Cendes F, Soss J, Andermann F, Engel JJ, Dubeau F, Olivier A, Fried I (2004)

Unilateral hippocampal sclerosis with contralateral temporal scalp ictal onset. Epilepsia 45:792-

802.

Miranda R, Marriott LK, Gold PE (2006) Patterns of brain acetylcholine release predict

individual differences in preferred learning strategies in rats. Behav Neurosci 120:641-650.

Mohajeri MH, Saini K, Li H, Crameri A, Lipp H, Wolfer DP, Nitsch RM (2003) Intact spatial

memory in mice with seizure-induced partial loss of hippocampal pyramidal neurons. Neurobio

of Disease 12:174-181.

Montecot C, Rondi-Reig L, Springhetti V, Seylaz J, Pinard E (1998) Inhibition of neuronal (type

1) nitric oxide synthase prevents hyperaemia and hippocampal lesions resulting from kainate-

induced seizures. Neurosci 84:791-800.

Moran NF, Lemieux L, Kitchen ND, Fish DR, Shorvon SD (2001) Extrahippocampal temporal

lobe atrophy in temporal lobe epilepsy and mesial temporal sclerosis. Brain 124:167-175.

Moreira CM, Masson S, Carvalho MC, Brandao ML (2007) Exploratory behaviour of rats in the

elevated plus-maze is differentially sensitive to inactivation of the basolateral and central

amygdaloid nuclei. Brain Res Bull 71:466-473.

Morimoto K, Fahnestock M, Racine RJ (2004) Kindling and status epilepticus models of

epilepsy: Rewiring teh brain. Prog Neurobiol 73:1-60.

Morris R (1984) Developments of a water-maze procedure for studying spatial learning in the

rat. J Neurosci Methods 11:47-60.

Morris RG (1989) Synaptic plasticity and learning: Selective impairment of learning rats and

blockade of long-term potentiation in vivo by the N-methyl-D-aspartate receptor antagonist AP5.

J Neurosci 9:3040-3057.

Morris RGM, Frey U (1997) Hippocampal synaptic plasticity: Role in spatial learning or the

automatic recording of attended experience? Philos Trans R Soc Lond B 352:1489-1503.

Motamedi G, Meador K (2003) Epilepsy and cognition. Epilepsy Behav 4:S25-S38.

Motte J, Gernandes MJ, Baram TZ, Nehlig A (1998) Spatial and temporal evolution of neuronal

activation, stress and injury in lithium/pilocarpine seizures in adult rats. Brain Res 793:(1-2):61-

72.

Moussa RC, Ikeda-Douglas CJ, Thakur V, Milgram NW, Gurd JW (2001) Seizure activity

results in increased tyrosine phosphorylation of the N-methyl-d-aspartate receptor in the

hippocampus. Mol Brain Res 95:36-47.

Mueller SG, Laxer KD, Barakos J, Cheong I, Garcia P, Weiner MW (2009) Subfield atrophy

pattern in temporal lobe epilepsy with and without mesial sclerosis detected by high-resolution

MRI at 4 tesla: Preliminary results. Epilepsia 50:1474-1483.

292

Muir KW (2006) Glutamate-based therapeutic approaches: Clinical trials with NMDA

antagonists. Curr Opin Pharmacol 6:53-60.

Mullen RJ, Buck CR, Smith AM (1992) NeuN, a neuronal specific nuclear protein in vertebrates.

Development 116:201-211.

Müller C, Grötickea I, Bankstahla M, Löscher W (2009) Behavioral and cognitive alterations,

spontaneous seizures, and neuropathology developing after a pilocarpine-induced status

epilepticus in C57BL/6 mice. Exp Neurol 219:284-297.

Murashima YL, Yoshii M, Suzuki J (2000) Role of nitric oxide in the epileptogenesis of EL

mice. Epilepsia 41:195-199.

Murthy KS (2008) Inhibitory phosphorylation of soluble guanylyl cyclase by muscarinic m2

receptors via Gβ-dependent activation of c-src kinase. J Pharmacol Exp Ther 325:183-189.

Nadler JV (2003) The recurrent mossy fiber pathway of the epilpetic brain. Neurochem Res

28:1649-1658.

Naegele JR (2007) Neuroprotective strategies to avert seizure-induced neurodegeneration in


Nagao T, Alonso A, Avoli M (1996) Epileptiform activity induced by pilocarpine in the rat

hippocampal-entorhinal slice preparation. Neurosci 72:399-408.

Nakanishi S, Masu M, Bessho Y, Nakajima Y, Hayashi Y, Shigemoto R (1994) Molecular

diversity of glutamate receptors and their physiological functions. EXS 71:71-80.

Nandhagopal R (2006) Generalized convulsive status epilepticus: An overview. Postgrad Med J

82:723-732.

Napolitano CE, Orriols MA (2010) Graduated and sequential propagation in mesial temporal

epilepsy: Analysis with scalp ictal EEG. J Clin Neurophysiool 27:285-291.

Narkilahti S, Pirttilä TJ, Lukasiuk K, Tuunanen J, Pitkänen A (2003a) Expression and activation

of caspase-3 following status epilepticus in the rat. Eur J Neurosci 18:1486-1496.

Narkilahti S, Nissinen J, Pitkanen A (2003b) Administration of caspase 3 inhibitor during and

after status epilepticus in rat: Effect on neuronal damage and epileptogenesis.

Neuropharmacology 44:1068-1088.

Natsume J, Bernasconi N, Andermann F, Bernasconi A (2003) MRI volumetry of the thalamus

in temporal, extratemporal and idiopathic generalized epilepsy. Neurology 41:1964-1968.

Nayel MH, Awad IA, Luders H (1991) Extent of mesiobasal resection determines outcome after

temporal lobectomy for intractable complex partial seizures. Neurosurgery 29:55-60.

Nehlig A (2007) What is animal experimentation telling us about new drug treatments of status

epilepticus? Epilepsia 48:78-81.

293

Nevander G, Ingvar M, Auer R, Siesjo BK (1985a) Status epilepticus in well-oxygenated rats

causes neuronal necrosis. Ann Neurol 18:281-290.

Newberg AB, Alavi A, Berlin J, Mozley PD, O'Connor M, Sperling M (2000) Ipsilateral and

contralateral thalamic hypometabolism as a predictor of outcome after temporal lobectomy for

seizures. J Nucl Med 41:1964-1968.

Niessen HG, Angenstein F, Vielhaber S, Frisch C, Kudin A, Elger CE, Heinze HJ, Scheich H,

Kunz WS (2005) Volumetric magnetic resonance imaging of functionally relevant structural

alterations in chronic epilepsy after pilocarpine-induced status epilepticus in rats. Epilepsia

46:1021-1026.

Niimura M, Moussa R, Bissoon N, Ikeda-Douglas C, Milgram NW, Gurd JW (2005) Changes in

phosphorylation of the NMDA receptor in the rat hippocampus induced by status epilepticus. J

Neurochem 92:1377-1385.

Niquet J, Liu H, Wasterlain CG (2005) Programmed neuronal necrosis and status epilepticus.

Epilepsia 46:S43-S48.

Nissinen J, Halonen T, Koivisto E, Pitkänen A (2000) A new model of chronic temporal lobe

epilepsy induced by electrical stimulation of the amygdala in rat. Epilepsy Res 38:177-205.

Nissinen J, Lukasiuk K, Pitanen A (2001) Is mossy fiber sprouting present at the time of the first

spontaneosu seizures in rat experimental temporal lobe epilepsy? Hippocampus 11:299-310.

Noe KH, Manno EM (2005) Mechanisms underlying status epilepticus. Drugs Today 41:257-

266.

Okazaki MM, Evenson DA, Nadler JV (1995) Hippocampal mossy fiber sprouting and synapse

formation after status epilepticus in rats: Visualization after retrograde transport of biocytin. J

Comp Neurol 352:515-534.

Olney JW, Collins RC, Sloviter RS (1986) Excitotoxic mechanisms of epileptic brain damage.

Adv Neurol 44:857-877.

Olney JW, Labruyere J, Price MT (1989) Pathological changes induced in cerebrocortical

neurons by phencyclidine and related drugs. Science 244:1360-1362.

Olney JW, Labruyere J, Wang G (1991) NMDA antagonist neurotoxicity: Mechanism and

prevention. Science 254:1515-1518.

Ono J, Vieth RF, Walson PD (1990) Electrocorticographical observation of seizures induced by

pentylenetetrazol (PTZ) injection in rats. Funct Neurol 5:345-352.

Ormandy GC, Song L, Jope RS (1991) Analysis of the convulsant-potentiating effects of lithium

in rats. Exp Neurol 111:356-361.

Osborne NN, Tobin AB, Ghazi H (1988) Role of inositol trisphosphate as a second messenger in

signal transduction processes: An essay. Neurochem Res 13:177-191.

294

Packard MG, McGaugh JL (1996) Inactivation of hippocampus or caudate nucleus with

lidocaine differentially affects expression of place and response learning. Neurobiol Learn Mem

65:65-72.

Packard MG (2009) Anxiety, cognition, and habit: A multiple memory systems perspective.

Brain Res 1293:121-128.

Pail M, Brázdil M, Marecek R, Mikl M (2010) An optimized voxel-based morphometric study of

grey matter changes in patients with left-sided and right-sided mesial temporal lobe epilepsy and

hippocampal sclerosis (MTLE/HS). Epilepsia 51:511-518.

Pandis C, Sotiriou E, Kouvaras E, Asprodini E, Papatheodoropoulos C, Angelatou F (2006)

Differential expression of NMDA and AMPA receptor subunits in the rat dorsal and ventral

hippocampus. Neurosci 140:163-175.

Pang Z, Bondada V, Sengoku T, Siman R, Geddes JW (2003) Calpain facilitates the neuron

death induced by 3-nitropropionic acid and contributes to the necrotic morphology. J

Neuropathol Exp Neurol 62:633-643.

Papatheodoropoulos C (2007) NMDA receptor-dependent high-frequency network oscillations

(100-300 hz) in rat hippocampal slices. Neurosci Lett 414:197-202.

Parent JM, Yu TW, Leibowitz RT, Geschwind DH, Sloviter RS, Lowenstein DH (1997) Dentate

granule cell neurogenesis is increased by seizures and contributes to aberrant network

reorganization in the adult rat hippocampus. J Neurosci 17:3727-3738.

Parent JM (2007) Adult neurogenesis in the intact and epileptic dentate gyrus. Prog Brain Res

163:529-540.

Parent JM, Murphy GG (2008) Mechanisms and functional significance of aberrant seizure-

induced hippocampal neurogenesis. Epilepsia 49:19-25.

Peredery O, Persinger MA, Parker G, Mastrosov L (2000) Temporal changes in neuronal dropout

following inductions of lithium/pilocarpine seizures in the rat. Brain Res 881:9-17.

Perini GI, Tosin C, Carraro C, Bernasconi G, Canevini MP, Canger R, Pellegrini A, Testa G

(1996) Interictal mood and personality disorders in temporal lobe epilepsy and juvenile

myoclonic epilepsy. J Neurol Neurosurg Psychiatry 61:601-605.

Persinger MA, Bureau YR, Kostakos M, Peredery O, Falter H (1993) Behaviors of rats with

insidious, multifocal brain damage induced by seizures following single peripheral injections of

lithium and pilocarpine. Physiol Behav 53:849-866.

Persinger MA, Dupont MJ (2004) Emergence of spontaneous seizures during the year following

lithium/pilocarpine-induced epilepsy and neuronal loss within the right temporal cortices.


Peterson CJ, Vinayak S, Pazos A, Gale K (1992) A rodent model of focally evoked self-

sustaining status epilepticus. Eur J Pharmacol 221:151-155.

295

Pitkänen A, Tuunanen J, Kalviainen R, Partanen KS, T. (1998) Amygdala damage in

experimental and human temporal lobe epilepsy. Epilepsy Res 32:233-252.

Pitkänen M, Pikkarainen N, Nurminen A, Ylinen A (2000) Reciprocal connections between the

amygdala and the hippocampal formation, perirhinal cortex, and postrhinal cortex in rat. A

review. Ann NY Acad Sci 911:369-391.

Pitkänen A, Nissinen J, Nairismägi J, Lukasiuk K, Gröhn OH, Miettinen R, Kauppinen R (2002)

Progression of neuronal damage after status epilepticus and during spontaneous seizures in a rat

model of temporal lobe epilepsy. Prog Brain Res 135:67-83.

Pitkänen A, Kubova H (2004) Antiepileptic drugs in neuroprotection. Expert Opin Pharmacother

5:777-798.

Pitkänen A, Narkilahti S, Bezvenyuk Z, Haapalinna A, Nissinen J (2004) Atipamezole, an

alpha(2)-adrenoceptor antagonist, has disease modifying effects on epileptogenesis in rats.

Epilepsy Res 61:119-140.

Pitkänen A, Kharatishvili I, Narkilahti S, Lukasiuk K, Nissinen J (2005) Administration of

diazepam during status epilepticus reduces development and severity of epilepsy in rat. Epilepsy

Res 63:27-42.

Pitkänen A, McIntosh TK (2006) Animal models of post-traumatic epilepsy. J Neurotrauma

23:241-261.

Pitkänen A, Lukasiuk K (2009) Molecular and cellular basis of epileptogenesis in symptomatic

epilepsy. Epilepsy Behav 14:16-25.

Poirier JL, Čapek R, De Koninck Y (2000) Differential progression of dark neuron and fluoro-

jade labelling in the rat hippocampus following pilocarpine-induced status epilepticus. Neurosci

97:59-68.

Post RM (2004) Neurobiology of seizures and behavioral abnormalities. Epilepsia 45:4-14.

Priel MR, dos Santos NF, Cavalheiro EA (1996) Developmental aspects of the pilocarpine model

of epilepsy. Epilepsy Res 26:121.

Priel MR, Albuquerque EX (2002) Short-term effects of pilocarpine on rat hippocampal neurons

in culture. Epilepsia 43:40-46.

Prut L, Belzung C (2003) The open field as a paradigm to measure the effects of drugs on

anxiety-like behaviors. Eur J Pharmacol 463:3-33.

Racine RJ (1972) Modification of seizure activity by electrical stimulation. II. motor seizure.

Electroencephalogr Clin Neurophysiol 32:281-294.

Racine RJ, Steingart M, McIntyre DC (1999) Development of kindling-prone and kindling-

resistant rats: Selective breeding and electrophysiological studies. Epilepsy Res 35:183-195.

296

Rajasekaran K, Jayakumar R, Venkatachalam K (2003) Increased neuronal nitric oxide synthase

(nNOS) activity triggers picrotoxin-induced seizures in rats and evidence for participation of

nNOS mechanism in the action of antiepileptic drugs. Brain Res 979:85-97.

Ramos A (2008) Animal models of anxiety: Do I need multiple tests? Trends Pharmacol Sci

29:493-498.

Ratzliff AH, Howard AL, Santhakumar V, Osapay I, Soltesz I (2004) Rapid deletion of mossy

cells does not result in a hyperexcitable dentate gyrus: Implications for epileptogenesis. J

Neurosci 24:2259-2269.

Rice AC, DeLorenzo RJ (1999) N-methyl-D-aspartate receptor activation regulates refratoriness

of status epilepticus to diazepam. Neurosci 93:117-123.

Rice AC, Floyd CL, Lyeth DG, Hamm RJ, DeLorenzo RJ (1998a) Status epilepticus causes

long-term NMDA receptor-dependent behavioral changes and cognitive deficits. Epilepsia

39:1148-1157.

Rice AC, Floyd CL, Lyeth DG, Hamm RJ, DeLorenzo RJ (1998b) Status epilepticus causes

long-term NMDA receptor-dependent behavioral changes and cognitive deficits. Epilepsia

39:1148-1157.

Ridet JL, Malhotra SK, Privat A, Gage FH (1997) Reactive astrocytes: Cellular and molecular

cutes to biological function. Trends Neurosci 20:570-577.

Rigoulot MA, Leroy C, Koning E, Ferrandon A, Nehlig A (2003) Prolonged low-dose caffeine

exposure protects against hippocampal damage but not against the occurrence of epilepsy in the

lithium/pilocarpine model. Epilepsia 44:529-535.

Rigoulot MA, Koning E, Ferrandon A, Nehlig A (2004) Neuroprotective properties of

topiramate in the lithium/pilocarpine model of epilpesy. J Pharmacol Exp Ther 308:787-795.

Roch C, Leroy C, Nehlig A, Namer IJ (2002) Magnetic resonance imaging in the study of the

lithium/pilocarpine model of temporal lobe epilepsy in adult rats. Epilepsia 43:325-335.

Rodgers RJ (1997) Animal models of anxiety: Where next? Behav Pharmacol 8:477-504.

Rodgers RJ, Dalvi A (1997) Anxiety, defence and the elevated plus-maze. Neurosci Biobehav

Rev 21:801-810.

Rodgers RJ, Cao BJ, Dalvi A, Holmes A (1997) Animal models of anxiety: An ethological

perspective. Braz J Med Biol Res 30:289-304.

Rosen JB, Schulkin J (1998) From normal fear to pathological anxiety. Psychol Rev 105:325-

350.

Rosenblum K, Futter M, Jones M, Hulme EC, Bliss TV (2000) ERKI/II regulation by the

muscarinic acetylcholine receptors in neurons. J Neurosci 20:977-985.

297

Rosenow F, Hamer HM, Knake S (2007) The epidemiology of convulsive and nonconvulsive

status epilepticus. Epilepsia 48:82-84.

Rowland LM, Astur RS, Jung RE, Bustillo JR, Lauriello J, Yeo RA (2005) Selective cognitive

impairments associated with NMDA receptor blockade in humans. Neuropsychopharmacology

30:633-639.

Runke D, McIntyre DC (2008) Assessment of anxiety-like behaviors in female rats bred for

differences in kindling susceptibility and amygdala excitability. Brain Res 1240:143-152.

Ryvlin P, Cinotti L, Froment JC, Le Bars D, Landais P, Chaze M, Galy G, Lavenne F, Serra JP,

Mauguière F (1991) Metabolic patterns associated with non-specific magnetic resonance

imaging abnormalities in temporal lobe epilepsy. Brain 114:2363-2383.

Saber AJ, Cain DP (2003) Combined beta-adrenergic and cholinergic antagonism produces

behavioral and cognitive impairments in the water maze: Implications for alzheimer disease and

pharmacotherapy with beta-adrenergic antagonists. Neuropsychopharmacology 28:1247-1256.

Salmond CH, Menon DK, Chatfield DA, Pickard JD, Sahakian BJ (2005) Deficits in decision-

making in head injury survivors. Neurotrauma 22:613-622.

Sanberg PR, Fibiger HC (1979) Body weight, feeding, and drinking behaviors in rats with kainic

acid-induced lesions of striatal neurons—With a note on body weight symptomatology in

huntington's disease. Exp Neurol 66:444-466.

Sankar R (2005) Neuroprotection in epilepsy: The holy grail of antiepileptogenic therapy.

Epilepsy Rev 7:S1-S2.

Santos NF, Marques RH, Correia L, Sinigaglia-Coimbra R, Calderazzo L, Sanabria ERG,

Cavalheiro EA (2000) Multiple pilocarpine-induced status epilepticus in developing rats: A long-

term behavioral and electrophysiological study. Epilepsia 41:S57-S63.

Saraste A (1999) Morphologic criteria and detection of apoptosis. Herz 24:189-195.

Saraste A, Pulkki K (2000) Morphologic and biochemical hallmarks of apoptosis. Cardiovasc

Res 45:528-537.

Sardo P, Ferraro G (2007) Modulatory effects of nitric oxide-active drugs on the anticonvulsant

activity of lamotrigine in an experimental model of partial complex epilepsy in the rat. BMC

Neurosci 8:47-57.

Sarkisova KY, Midzianovskaia IS, Kulikov MA (2003) Depressive-like behavioral alterations

and c-fos expression in the dopaminergic brain regiosn in WAG/Rij rats with genetic absence

epilepsy. Behav Brain Res 144:211-226.

Sattler R, Charlton MP, Hafner M, Tymianski M (1998) Distinct influx pathways, not calcium

load, determine neuronal vulnerability to calcium neurotoxicity. J Neurochem 71:2349-2364.

298

Sattler R, Xiong Z, Lu WY, Hafner M, MacDonald JF, Tymianski M (1999) Specific coupling of

NMDA receptor activation to nitric oxide neurotoxicity by PSD-95 protein. Science 284:1845-

1848.

Saucier D, Cain DP (1995) Spatial learning without NMDA receptor-dependent long-term

potentiation. Nature 378:132-133.

Sayin U, Sutula TP, Stafstrom CE (2004) Seizures in the developing brain caues adverse long-

term effects on spatial learning and anxiety. Epilepsia 45:1539-1548.

Scarr E (2009) Muscarinic receptors in psychiatric disorders – can we mimic ‗health‘?.

Neurosignals 17:298-310.

Scharfman HE, Goodman JH, Sollas AL (2000) Granule-like neurons at the hilar/CA3 border

after status epilepticus and their synchrony with area CA3 pyramidal cells: Functional

implications of seizure-induced neurogenesis. J Neurosci 20:6144-6158.

Scharfman HE, Sollas AE, Berger RE, Goodman JH, Pierce JP (2003) Perforant path activation

of ectopic granule cells that are born after pilocarpine-induced seizures. Neurosci 121:1017-

1029.

Schauwecker PE, Williams RW, Santos JB (2004) Genetic control of sensibility to hippocampal

cell death induced by kainic acid: A quantitative train loci analysis. J Comp Neurol 477:96-107.

Schenk F, Morris RG (1985) Dissociation between components of spatial memory in rats after

recovery from the effects of retrohippocampal lesions. Exp Brain Res 58:11-28.

Schmidt D, Löscher W (2005) Drug resistance in epilepsy: Putative neurobiologic and clinical

mechanisms.. Epilepsia 46:858-877.

Schmued LC, Hopkins KJ (2000) Fluoro-jade B: A high affinity fluorescent marker for the

localization of neuronal degeneration. Brain Res 874:123-130.

Scholtes FB, Renier WO, Meinardi H (1994) Generalized convulsive status epilepticus: Causes,

therapy and outcome in 346 patients. Epilepsia 25:1104-1112.

Schramm J (2008) Temporal lobe epilepsy surgery and the quest for optimal extent of resection:

A review. Epilepsia 49:1296-1307.

Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF (1999) In vivo protein transduction: Delivery

of a biologically active protein into the mouse. Science 285:1569-1572.

Schwob JE, Fuller T, Price JL, Olney JW (1980) Widespread patterns of neuronal damage

followign systemic or intracerebral injections of kainic acid: A histological study. Neurosci

5:991-1014.

Scorza FA, Arida RM, Naffah-Mazzacoratti Mda G, Scerni DA, Calderazzo L, Cavalheiro EA

(2009) The pilocarpine model of epilepsy: What have we learned? An Acad Bras Cienc 81:345-

365.

299

Scott RC, King MD, Gadian DG, Neville BG, Connelly A (2003) Hippocampal abnormalities

after prolonged febrile convulsion: A longitudinal MRI study. Brain 126:2551-2557.

Scott RC, Gadian DG, King MD, Chong WK, Cox TC, Neville BG, Connelly A (2002)

Magnetic resonance imaging findings within 5 days of status epilepticus in childhood. Brain

125:1951-1959.

Segal M (1988) Synaptic activation of the cholinergic receptor in rat hippocampus. Brain Res

452:79-82.

Seifert G, Carmignoto G, Steinhäuser C (2010) Astrocyte dysfunction in epilepsy. Brain Res Rev

63:212-221.

Seino M (2006) Classification criteria of epileptic seizures and syndromes. Epilepsy Res

70S:S27-S33.

Sharma AK, Reams RY, Jordan WH, Miller MA, Thacker HL, Snyder PW (2007) Mesial

temporal lobe epilepsy: pathogenesis, induced rodent models and lesions. Toxicol Pathol 35:984-

999.

Sharma AK, Jordan WH, Reams RY, Hall DG, Snyder PW (2008) Temporal profile of clinical

signs and histopathologic changes in an F-344 rat model of kainic acid-induced mesial temporal

lobe epilepsy. Toxicol Pathol 36:932-943.

Shetty AK, Turner DA (1997) Fetal hippocampal cells grafted to kainate-lesioned CA3 region of

adult hippocampus suppress aberrant supragranular sprouting of host mossy fibers. Exp Neurol

143:231-245.

Shetty AK (2002) Entorhinal axons exhibit sprouting in CA1 subfield of the adult hippocampus

in a rat model of temporal lobe epilepsy. Hippocampus 12:534-542.

Shetty AK, Zaman V, Hattiangady B (2005) Repair of the injured adult hippocampus through

graft-mediated modulation of the plasticity of the dentate gyrus in a rat model of temporal lobe

epilepsy. J Neurosci 25:8401.

Shi L, Argenta AE, Winseck AK, Brunso-Bechtold JK (2004) Stereological quantification of

GAD-67-immunoreactive neurons and boutons in the hippocampus of middle-aged and old

fischer 344 x brown norway rats. J Comp Neurol 478:282-291.

Shorvon S (1994) The outcome of tonic-clonic status epilepticus. Curr Opin Neurol 7:93-95.

Siddiqui AH, Joseph SA (2005) CA3 axonal sprouting in kainate-induced chronic epilepsy.

Brain Res 1066:146.

Singleton RH, Povlishock JT (2004) Identification and characterization of heterogeneous

neuronal injury an ddeath in regions of diffuse brain injury: Evidence for multiple independent

injury phenotypes J Neurosci 24:3542-3553.

300

Sloviter RS (1987) Decreased hippocampal inhibition and a selective loss of interneurons in

experimental epilepsy. Science 235:73-76.

Sloviter RS (1989) Calcium-binding protein (calbindin-D28k) and parvalbumin

immunocytochemistry: Localization in the rat hippocampus with specific reference to the

selective vulnerability of hippocampal neurons to seizure activity. J Comp Neurol 280:183-196.

Sloviter RS, Sollas AL, Barbaro NM, Laxer KD (1991) Calcium-binding protein (calbindin-

D28k) and parvalbumin immunocytochemistry in the normal and epileptic human hippocampus.

J Comp Neurol 308:381-396.

Sloviter RS (1994) The functional organization of the hippocampal dentate gyrus and its

relevance to the pathogenesis of temporal lobe epilepsy. Ann Neurol 35:640-654.

Sloviter RS, Dean E, Sollas AL, Goodman JH (1996) Apoptosis and necrosis induced in

different hippocampal neuron populations by repetitive perforant path stimulation in the rat. J

Comp Neurol 366:516-533.

Sloviter RS (1999) Status epilepticus-induced neuronal injury and network reorganization.

Epilepsia 40:S34-S39.

Sloviter RS, Zappone CA, Harvey BD, Bumanglag AV, Bender RA, Frotscher M (2003)

―Dormant basket cell‖ hypothesis revisited: Relative vulnerabilities of dentate gyrus mossy cells

and inhibitory interneurons after hippocampal status epilepticus in the rat. J Comp Neurol

459:44.

Smith BN, Dudek FE (2002) Network interactions mediated by new excitatory connections

between CA1 pyramidal cells in rats with kainate-induced epilepsy. J Neurophys 87:1655-1658.

Smolders I, Khan GM, Manil J, Ebinger G, Michotte Y (1997) NMDA receptor-mediated

pilocarpine-induced seizures: Characterization in freely moving rats by microdialysis. B J

Pharmacol 121:1171-1179.

Snider BJ, Gottron FJ, Choi DW (1999) Apoptosis and necrosis in cerebrovascular disease. Ann

N Y Acad Sci 893:243-253.

Sogawa Y, Monokoshi M, Silveira DC, Cha BH, Cilio MR, McCabe BK, Liu X, Hu Y, Holmes

GL (2001) Timing of cognitive deficits following neonatal seizures: Relationship to histological

changes in the hippocampus. Brain Res Dev Brain Res 131:73-83.

Sokol DK, Demyer WE, Edwards-Brown M, Sanders S, Garg B (2003) From swelling to

sclerosis: Acute change in mesial hippocampus after prolonged febrile seizure. Seizure 12:237-

240.

Sossa M (2006) A new frontier in epilepsy: Novel antiepileptogenic drugs. J Pharmacol Sci

100:487-494.

Spencer SS, Spencer DD (1994) Entorhinal-hippocampal interactions in medial temporal lobe


301

Spencer SS, Kim J, deLanerolle N, Spencer DD (1999) Differential neuronal and glial relations

with parameters of ictal discharge in mesial temporal lobe epilepsy. Epilepsia 40:708-712.

Sperk G, Wieser R, Widmann R, Singer EA (1986) Kainic acid induced seizures: Changes in

somatostatin, substance P and neurotensin. Neurosci 17:1117-1126.

Sperling MR, Gur RC, Alavi A, Gur RE, Resnick S, O'Connor MJ, Reivich M (1990)

Subcortical metabolic alterations in partial epilepsy. Epilepsia 31:145-155.

Stafstrom CE, Chronopoulos A, Thurber S, Thompson JL, Holmes GL (1993) Age-dependent

cognitive and behavioral deficits after kainic acid seizures. Epilepsia 34:420-432.

Stafstrom CE (2002) Assessing the behavioral and cognitive effects of seizures on the

developing brain. Prog Brain Res 135:377-390.

Stafstrom CE (2006) Behavioral and cogntiive testing procedures in animal models of epilesy.

In: Models of seizures and epilepsy (Pitkanen A, Schwartzkroin PA, Moshe SL, eds), pp613-628.

Boston: Elsevier Academic Press.

Sun C, Mtchedlishvili Z, Bertram EH, Erisir A, Kapur J (2007) Selective loss of dentate hilar

interneurons contributes to reduced synaptic inhibition of granule cells in an electrical

stimulation-based animal model of temporal lobe epilepsy. J Comp Neurol 500:876-893.

Sun HS, Doucette TA, Liu YT, Fang Y, Teves L, Aarts M, Ryan CL, Bernard PB, Lau A, Forder

JP, Salter MW, Wang YT, Tasker RA, Tymianski M (2008) Effectiveness of PSD95 inhibitors in

permanent and transient focal ischemia in the rat. Stroke 39:2544-2553.

Sun QJ, Duan RS, Wang AH, Shang W, Zhang T, Zhang XQ, Chi ZF (2009) Alterations of

NR2B and PSD-95 experssion in hippocampus of kainic acid-exposed rats with behavioural

deficits. Behav Brain Res 201:292-299.

Sutherland RJ, Whishaw IQ, Regehr JC (1982) Cholinergic receptor blockade impairs spatial

localization by ues of distal cues in the rat. J Comp Physiol Psychol 96:563-573.

Sutula T (2002) Seizure-induced axonal sprouting: Assessing connectiosn between injury, local

circuits and epileptognesis. Epilepsy Curr 2:86-91.

Sutula T, Cascino G, Cavazos J, Parada I, Ramirez L (1989) Mossy fiber synaptic reorganization

in the epileptic human temporal lobe. Ann Neurol 26:321-330.

Sutula T, Zhang P, Lynch M, Sayin U, Golarai G, Rod R (1998) Synaptic and axonal remodeling

of mossy fibers in the hilus and supragranular region of the dentate gyrus in kainate-treated rats.

J Comp Neurol 390:578-594.

Sutula TP, Ockuly J (2006) Kindling, spontaneous seizures, and the consequences of epilepsy:

More than a model. In: Models of seizures and epilepsy. pp395-406.Elsevier Inc.

302

Sutula TP, Dudek FE (2007) Unmasking recurrent excitation generated by mossy fiber sprouting

in the epileptic dentate gyrus: An emergent property of a complex system. Prog Brain Res

163:541-563.

Swanson TH (1995) The pathophysiology of human mesial temporal lobe epilepsy. J Clin

Neurophysiol 12:2-22.

Swinkels WA, Kuyk J, van Dyck R, Spinhoven P (2005) Psychiatric comorbidity in epilepsy.


Szyndler J, Wierzba-Bobrowica T, Skorzewska A, Maciejak P, Walkowiak J, Lechowicz W,

Turzynska D, Bidzinski A, Plaznik A (2005) Behavioral, biochemical and histological studies in

a model of pilocarpine-induced spontaneous recurrent seizures. Pharmacol Biochem Behav

81:15-23.

Tauck DL, Nadler JV (1985) Evidence of functional mossy fiber sprouting in hippocampal

formation of kainic acid-treated rats. J Neurosci 5:1016-1022.

Tellez-Zenteno JF, Pondal-Sordo M, Matijevic S, Wiebe S (2004) National and regional

prevalence of self-reported epilepsy in canada. Epilepsia 45:1623-1629.

Téllez-Zenteno JF, Dhar R, Wiebe S (2005) Long-term seizure outcomes following epilepsy

surgery: A systematic review and meta-analysis. Brain 128:1198.

Temkin NR (2001) Antiepileptogenesis and seizure prevention trials with antiepileptic drugs:

Meta-analysis of controlled trials. Epilepsia 42:515-524.

Temkin NR (2009) Preventing and treating posttraumatic seizures: The human experience.

Epilepsia 50:10-13.

Thivard L, Lehéricy S, Krainik A, Adam C, Dormont D, Chiras J, Baulac M, Dupont S (2005)

Diffusion tensor imaging in medial temporal lobe epilepsy with hippocampal sclerosis.

Neuroimage 28:682-690.

Thom M, Zhou J, Martinian L, Sisodiya S (2005) Quantitative post-mortem study of the

hippocampus in chronic epilepsy: Seizures do not inevitably cause neuronal loss. Brain

128:1344-1357.

Thom M, Martinian L, Catarino C, Yogarajah M, Keopp MJ, Caboclo L, Sisodiya SM (2009)

Bilateral reorgnaization of the dentate gryus in hippocampal sclerosis: A postmortem study.

Neurology 73:1033-1040.

Thom M, Liagkouras I, Elliot KJ, Martinian L, Harkness W, McEvoy A, Caboclo LO, Sisodiya

SM (2010) Reliability of patterns of hippocampal sclerosis as predictors of postsurgical outcome.

Epilepsia 51:1801-1808.

303

Thompson HJ, LeBold DG, Marklund N, Morales DM, Hagner AP, McIntosh TK (2006)

Cognitive evaluation of traumatically brain-injured rats using serial testing in the morris water

maze. Restor Neurol Neurosci 24:109-114.

Tilelli CQ, Vecchio FD, Fernandes A, Garcia-Cairasco N (2005) Different types of status

epilepticus lead to different levels of brain damage in rats. Epilepsy Behav 7:401-410.

Titlic M, Basic S, Hajnsek S, Lusic I (2009) Comorbidity psychiatric disorders in epilepsy: A

review of literature. Bratisl Lek Listy 110:105-109.

Tokuhara D, Sakuma S, Hattori H, Matsuoka O, Yamano T (2007) Kainic acid dose affects

delayed cell death mechanism after status epilepticus. Brain Dev 29:2-8.

Toplak ME, Sorge GB, Benoit A, West RF, Stanovich KE (2010) Decision-making and

cognitive abilities: A review of associations between iowa gambling task performance, executive

functions, and intelligence. Clin Psychol Rev 30:562-581.

Towne AR, Pellock JM, Ko D, DeLorenzo RJ (1994) Determinants of mormtality in status


Treiman DM (2010) Management of refractory complex partial seizures: Current state of the art.

Neuropsychiatr Dis Treat 24:297-308.

Treit D, Menard J, Royan C (1993) Anxiogenic stimuli in the elevated plus-maze. Pharmacol

Biochem Behav 44:463-469.

Turner DA, Wheal HV (1991) Excitatory synaptic potentials in kainic acid-denervated rat CA1

pyramidal neurons. J Neurosci 11:2794.

Turski WA, Czuczwar SJ, Kleinrok Z, Turski L (1983a) Cholinomimetics produce seizures and

brain damage in rats. EXS 39:1408-1411.

Turski WA, Cavalheiro EA, Schwarz M, Czuczwar SJ, Kleinrok Z, Turski L (1983b) Limbic

seizures produced by pilocarpine in rats: Behavioural, electroencephalographic and

neuropathological study. Behav Brain Res 9:315-335.

Turski WA, Cavalheiro EA, Bortolotto ZA, Mello LM, Schwarz M, Turski L (1984) Seizures

produced by pilocarpine in mice: A behavioral, electroencephalographic and morphological

analysis. Brain Res 321:237-253.

Turski WA, Cavalheiro EA, Calderazzo-Filho LS, Kleinrok Z, Czuczwar SJ, Turski L (1985)

Injections of picrotoxin and bicuculline into the amygdaloid complex of the rat: An

electroencephalographic, behavioural and morphological analysis. Neurosci 14:37-53.

Turski L, Ikonomidou C, Turski WA, Bortolotto ZA, Cavalheiro EA (1989) Review -

cholinergic mechanisms and epileptogenesis - the seizures induced by pilocarpine - a novel

experimental-model of intractable epilepsy. Synapse 3:154-171.

304

Tuunanen J, Halonen T, Pitkänen A (1996) Status epilepticus causes selective regional damage

and loss of GABAergic neurons in the rat amygdaloid complex. Eur J Neurosci 8:2711-2725.

Tuunanen J, Halonen T, Pitkänen A (1997) Decrease in somatostatin-immunoreactive neurons in

the rat amygdaloid complex in a kindling model of temporal lobe epilepsy. Epilepsy Res 26:315-

327.

Tuunanen J, Lukasiuk K, Halonen T, Pitkänen A (1999) Status epilepticus-induced neuronal

damage in the rat amygdaloid complex: Distribution, time-course and mechanisms. Neurosci

94:473-495.

Tymianski M, Charlton MP, Carlen PL, Tator CH (1993) Source specificity of early calcium

neurotoxicity in cultured embryonic spinal neurons. J Neurosci 13:2085-2104.

van Der Linden S, Panzica F, de Curtis M (1999) Carbachol induces fast oscillations in the

medial but not in the lateral entorhinal cortex of the isolated guinea pig brain. J Neurophysiol

82:2441-2450.

van Paesschen W, Revesz T, Duncan JS, King MD, Connelly A (1997) Quantitative

neuropathology and quantitative magnetic resonance imaging of the hippocampus in temporal

lobe epilepsy. Ann Neurol 42:756-766.

van Vliet EA, da Costa Araujo S, Redeker S, van Schaik R, Aronica E, Gorter JA (2007) Blood-

brain barrier leakage may lead to progression of temporal lobe epilepsy. Brain 130:521-534.

Vanlangenakker N, Vanden-Berghe T, Krysko DV, Festjens N, Vandenabeele P (2008)

Molecular mechanisms and pathophysiology of necrotic cell death. Curr Mol Med 8:207-220.

Vazquez B, Devinsky O (2003) Epilepsy and anxiety. Epilepsy Behav 4:S20-S25.

Veliskova J, Miller AM, Nunes ML, Brown LL (2005) Regional neural activity of the substantia

nigra during peri-ictal generalized seizure stages. Neurobiol Dis 20:752-759.

Veliskova J (2006) Behavioral chracterization of seizures in rats. In: Models of seizures and

epilepsy (Pitkanen A, Schwartzkroin PA, Moshe SL, eds), pp601-612. Boston: Elsevier

Academic Press.

Vingerhoets G (2006) Cognitive effects of seizures. Seizure 15:221-226.

Vinton AB, Carne R, Hicks RJ, Desmond PM, Kilpatrick C, Kaye AH, O'Brien TJ (2007) The

extent of resection of FDG-PET hypometabolism relates to outcome of temporal lobectomy.

Brain 130:548-560.

Volk HA, Arabadzisz D, Fritschy JM, Brandt C, Bethmann K, Löscher W (2006) Antiepileptic

drug-resistant rats differ from drug-responsive rats in hippocampal neurodegeneration and

GABA(A) receptor ligand binding in a model of temporal lobe epilepsy. Neurobiol Dis 21:633-

646.

305

Vorhees CV, Williams MT (2006) Morris water maze: Procedures for assessing spatial and

related forms of learning and memory. Nature Protocols 1:848-858.

Vosler PS, Brennan CS, Chen J (2008) Calpain-mediated signaling mechanisms in neuronal

injury and neurodegeneration. Mol Neurobiol 38:78-100.

Walker M (2007) Neuroprotection in epilepsy. Epilepsia 48:66-68.

Wall PM, Messier C (2001) Methodological and conceptual issues in the use of the elevated plus

maze as a psychological measurement instrument of animal anxiety-like behavior. Neurosci

Biobehav Rev 25:275-286.

Walsh RN, Cummins RA (1976) The open field test: A critical review. Psychol Bull 83:482-504.

Walter C, Murphy BL, Pun RY, Spieles-Engemann AL, Danzer SC (2007) Pilocarpine-induced

seizures cause selective time-dependent changes to adult-generated hippocampal dentate granule

cells. J Neurosci 27:7541-7652.

Wang CA, Lai MC, Lui CC, Yang SN, Tiao MM, Hsieh CS, Lin HH, Huang LT (2007) An

enriched environment improves cognitive performance after early-life status epilepticus

accompanied by an increase in phosphorylation of extracellular signal-regulated kinase 2.


Wang S, Wang S, Shan P, Song Z, Dai T, Wang R, Chi Z (2008) Mu-calpain mediates

hippocampal neuron death in rats after lithium/pilocarpine-induced status epilepticus. Brain Res

Bull 76:90-96.

Wang Y, Qun Zh (2010 (ahead of print)) Molecular and cellular mechanisms of excitotoxic

neuronal death. Apoptosis .

Weaver DF (2003) Epileptogenesis, ictogenesis and the design of future antiepileptic drugs. Can

J Neurol Sci 30:4-7.

Weise J, Engelhorn T, Dorfler A, Aker S, Bahr M, Hufnagel A (2005) Expression time course

and spatial distribution of activated caspase-3 after experimental status epilepticus: Contribution

of delayed neuronal cell death to seizure-induced neuronal injury. Neurobiology of Disease

18:582-590.

Wendling F, Chauvel P, Biraben A, Bartolomei F (2010) From intracerebral EEG signals to

brain connectivity: Identification of epileptogenic networks in partial epilepsy. Front Syst

Neurosci 25:154.

Wess J, Eglen RM, Gautam D (2007) Muscarinic acetylcholine receptors:Mutant mice provide

new insights for drug development. Nat Rev Drug Discov 6:721-733.

West MJ, Gunderson HJG (1990) Unbiased stereological estimation of the number of neurons in

the human hippocampus. J Comp Neurol 296:1-22.

306

Whishaw IQ, Mittleman G, Bunch ST, Dunnet SB (1987) Impairments in the acquisition,

retention and selection of spatial navigation strategies after medial caudate-putamen lesions in

rats. Behav Brain Res 24:125-138.

Whishaw IQ, Tomie JA (1987) Cholinergic receptor blockade produces impairments in a

sensorimotor subsystem for place navigation in the rat: Evidence from sensory, motor, and

acquisition tests in a swimming pool. Behav Neurosci 101:603-616.

Whishaw IQ, Petrie BF (1988) Cholinergic blockade in the rat impaires strategy selection but not

learning and retention of nonspatial visual discrimination problems in a swimming pool. Behav

Neurosci 102:662-667.

Whishaw IQ (1989) Dissociating performance and learning deficits on spatial navigation tasks in

rats subjected to cholinergic muscarinic blockade. Brain Res Bull 23:347-358.

Whitman MC, Greer CA (2009) Adult neurogenesis and the olfactory system. Prog Neurobiol

89:162-175.

Wieser HG (2004) ILAE commission report: Mesial temporal lobe epilepsy with hippocampal

sclerosis. Epilepsia 45:695-714.

Williamson A, Patrylo PR (2007) Physiological studies of human dentate granule cells. Prog

Brain Res 163:183-198.

Wittner L, Huberfeld G, Clémenceau S, Eross L, Dezamis E, Entz L, Ulbert I, Baulac M, Freund

TF, Maglóczky Z, Miles R (2009) The epileptic human hippocampal cornu ammonis 2 region

generates spontaneous interictal-like activity in vitro. Brain 132:3032-3046.

Wolf HK, Buslei R, Schmidt-Kastner R, Schmidt-Kastner PK, Pietsch T, Wiestler OD, Blümcke

I (1996) NeuN: A useful neuronal marker for diagnostic histopathology. J Histochem Cytochem

44:1167-1171.

Wolf HK, Aliashkevich AF, Blümcke I, Wiestler OD, Zentner J (1997) Neuronal loss and gliosis

of the amygdaloid nucleus in temporal lobe epilepsy. A quantitative analysis of 70 surgical

specimens. Acta Neuropathol 93:606-610.

Wu CL, Huang LT, Liou CW, Wang TJ, Tung YR, Hsu HY, Lai MC (2001)

Lithium/pilocarpine-induced status epilepticus in immature rats result in long-term deficits in

spatial learning and hippocampal cell loss. Neurosci Lett 312:113-117.

Wuarin JP, Dudek FE (2001) Excitatory synaptic input to granule cells increases with time after

kainate treatment. J Neurophysiol 85:1067-1077.

Wyneken U, Smalla KH, Marengo JJ, Soto D, de la Cerda A, Tischmeyer W, Grimm R,

Boeckers TM, Wolf G, Orrego F, Gundelfinger ED (2001) Kainate-induced seizures alter protein

composition and N-methyl-d-aspartate receptor function of rat forebrain postsynaptic densities.

Neurosci 102:65-74.

307

Xu B, McIntyre DC, Fahnestock M, Racine RJ (2004) Strain differences affect the induction of

status epilepticus and seizure-induced morphological changes. Eur J Neurosci 20(2):403-418

Yakovlev AG, Faden AI (2004) Mechanisms of neural cell death: Implications for development

of neuroprotective treatment strategies. NeuroRx 1:5-16.

Yamano M, Akamatsu N, Tsuji S, Kobayakawa M, Kawamura M (2011) Decision-making in

temporal lobe epilepsy examined with the iowa gambling task. Epilepsy Res 1:38.

Yang J, Huang Y, Yu X, Sun H, Li Y, Deng Y (2007) Erythropoietin preconditioning suppresses

neuronal death following status epilepticus in rats. Acta Neurobiol Exp 67:141-148.

Yilmazer-Hanke DM, Wolf HK, Schramm J, Elger CE, Wiestler OD, Blümcke I (2000)

Subregional pathology of the amygdala complex and entorhinal region in surgical specimens

from patients with pharmacoresistant temporal lobe epilepsy. J Neuropathol Exp Neurol 59:907-

920.

Zhang X, Cui SS, Wallace AE, Hannesson DK, Schmued LC, Saucier DM, Honer WG,

Corcoran ME (2002) Relations between brain pathology and temporal lobe epilepsy. J Neurosci

22:6052-6061.

Zhou JL, Zhao Q, Holmes GL (2007) Effect of levetiracetam on visual-spatial memory following

status epilepticus. Epilepsy Res 73:65-74.

308

Appendices

Appendix I: Literature comparison

309

310

311

312

313

314

315

316

317

318

319

320

321

322

Appendix II: Temporal reduction in neuron densities within regions of the hippocampus, thalamus, amygdala and piriform cortex

323

324

325

326

Appendix III: Convolution analyses

3.1 Assessment of performance based on shift in strategy use

To determine the extent by which a shift in strategy contributed to improved performance

during acquisition learning, a convolution analysis was performed (Brody and Holtzman, 2006).

The convolution analysis removes the changes in performance of each strategy from the

assessment, and permits only the shift in strategy as the determinant of predicted performance.

In the calculations, the average escape latency across all 14 days for each strategy was multiplied

by the frequency with which each strategy was used on t hat day. For example, the average

escape latency across all 14 days of acquisition testing when RLDP SE rats used the direct find

strategy was 5.5 s, approaching: 8.7 s, orienting: 15.6 s, searching: 37.8 s, random searching:

54.4 s, circling: 49 s, and thigmotaxis: 56.9 s. On day 1, RLDP SE rats used the direct find

strategy 1.4% of the time, approaching: 5.7%, orienting: 15.6%, scanning: 24.3%, random

searching: 33.6%, circling: 7.1%, and thigmotaxis: 17.9%. Thus, the predicted performance for

day 1 using the convolution analysis was:

5.5 x 0.014 + 8.7 x 0.057 + 15.6 x 0.100 + 37.8 x 0.243 + 54.4 x 0.336 + 49.0 x 0.0714 + 56.9 +

0.179 = 43.3 s.

The actual performance was 47.8 ± 2.3 s. The same analysis was applied to the search strategy

use of the RLDP SE rats on day 14, and yielded a predicted performance of:

5.5 x 0.045 + 8.7 x 0.293 + 15.6 x 0.150 + 37.8 x 0.248 + 54.4 x 0.158 + 49.0 x 0.030 + 56.9 +

0.075 = 28.9 s.

The actual performance was 27.6 ± 3.3 s. Consequently, the convolution analysis predicted a

33% improvement in RLDP SE rats from day 1 to day 14. This value is close to the 40%

improvement of the actual performance derived experimentally in RLDP SE rats.

327

3.2 Assessment of performance based on improved efficacy within each strategy

The convolution analysis was also used to assess the extent of which improved performance in

each search strategies contributed to the overall improvement in performance during acquisition

learning (Brody and Holtzman, 2006). In this assessment, the frequency with which each

strategy was used across all 14 days was multiplied by the average escape latency within each

strategy for the specific day. For example, across all 14 days of acquisition learning, RLDP SE

rats used direct find 2.8% of the time, approaching: 18.6%, orienting: 15.7%, scanning: 22.1%,

random searching: 20.7%, circling: 7.3% and thigmotaxis: 12.7%. On day 1, the average latency

used in RLDP SE rats with direct find was 2.7 s, approaching: 7.3 s, orienting: 18.4 s, scanning:

42.9 s, random searching, 57.4 s, circling: 56.7 s and thigmotaxis: 59.3 s. As a result, the

predicted performance for Day 1 using convolution analysis was:

0.028 x 2.7 + 0.186 x 7.3 + 0.157 x 18.4 + 0.221 x 42.9 + 0.207 x 57.4 + 0.073 x 56.7 + 0.127 x

59.3 = 37.3 s.

The actual performance was 47.8 ± 2.3 s. The same analysis was applied to the search strategy

use of the RLDP SE rats on day 14, and yielded a predicted performance of:

0.028 x 3.1 + 0.186 x 6.8 + 0.157 x 15.6 + 0.221 x 34.9 + 0.207 x 51.2 + 0.073 x 49.9 + 0.127 x

56.0 = 32.9 s.

The actual performance was 27.6 ± 3.3 s. In these calculations, the effect of the shift in strategy

is removed from the analysis, and only the change in the efficiency for each strategy is used to

predict performance. This analysis predicted a 12% improvement in RLDP SE rats from day 1

to day 14. This value is not approximate to the 40% improvement of the actual performance

observed in RLDP SE rats.

Documents

THE DEVELOPMENT OF NEURODEGENERATION AND … · THE DEVELOPMENT OF NEURODEGENERATION AND BEHAVIOURAL ALTERATIONS AFTER LITHIUM/PILOCARPINE-INDUCED STATUS EPILEPTICUS IN RATS Crystal