discovery.ucl.ac.uk · University of Lo ndo n Abstract A Patch-Clamp Study Of The Single Channel Properties Of N ative N -M eth y l-d -A spar tate (NM DA) Receptors In N eurons From

A Pa t c h -Clam p St u d y O f T h e Sin g l e C h a n n e l

P ro perties O f N ative N -M e t h y l -d -A spa r t a t e

(N M D A ) R eceptors In N e u r o n s F r o m T h e R at

C e n t r a l N ervo us System

by

Juan Carlos Pina-Crespo

A thesis submitted before the

University of London

in partial fulfillment o f the requirements fo r the degree o f

Doctor of Philosophy

Department of Pharmacology

University College London

1998

ProQuest Number: U642098

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U n iver sity o f Lo n d o n

A b str a ct

A Pa t c h -Clam p St u d y O f T h e Sin g l e C h a n n e l Pro per ties

O f N a tiv e N - M e t h y l -d -A s p a r t a t e (N M D A ) Re c e pto r s In

N e u r o n s F r o m T h e R a t C e n t r a l N e r v o u s System

by Juan Carlos Pina-Crespo

Native subtypes of NM D A receptors with distinct developmental and anatomical distribution

are present in the brain. Knowledge of their function and pharmacology would greatly help

in understanding NM DA receptor involvement in physiological and pathological processes in

the central nervous system. The single-channel properties of native NM DA receptors were

studied in outside-out patches excised from neuronal cell bodies in hippocampal and striatal

slices from 0-day old rats. Steady state channel activations produced by glutamate (50 - 100

nM) or NM DA (1 - 5 pM) and glycine (3 - 10 pM) were studied at -60 mV in an extracellular

solution containing 1 mM Ca and no added Mg^ ,

In patches from striatal neurons (n=9), glutamate and NM DA produced a single pattern of

channel activity characterized by the presence of two distinct unitary currents of 54 pS (83 ± 1

%) and 44 pS (17 ± 1 %), Frequent and symmetric direct transitions between 44 and 54 pS

currents suggested a common channel population obeying microscopic reversibility. In the

presence of a saturating glycine concentration , spermine (100 pM) produced an overall

increase in receptor activity; a property described only in recombinant N M D A receptors

containing N R la and NR2B subunits. The functional and pharmacological properties of PO

striatum NM DA receptors were consistent with these being a NR2B-like N M D A receptor

population.

In dentate gyrus neurons, glutamate and NM DA produced two patterns of single-channel

activity. One similar to that of PO striatum NM DA receptors and characterized by only high

conductance (42 and 51 pS) unitary currents and a second pattern showing, in addition to high

conductance (42 and 49 pS) currents, low conductance (17 and 42 pS) single-channel currents.

Direct transitions between 42 and 51 pS currents were symmetric while those between 17 and

42 pS were asymmetric. N o direct transitions linked 17 and 49 pS or 17, 42 and 49 pS currents,

Ifenprodil produced a selective reduction of single-channel activity mediated by high

conductance NM DA channels in both patches with only high conductance and patches with

high and low conductance single-channel activity. Based on their different functional and

pharmacological properties and striking similarities with N R la/N R 2B and N R la/N R 2D

recombinant NM DA receptors, it was concluded that high and low conductance single

channel activity were the result of the activation of two functionally and pharmacologically

different NM DA receptor-channel populations: high conductance ifenprodil-sensitive NR2B-

like NM DA receptors and low conductance ifenprodil-insensitive NR2D-like NM DA

receptors.

TABLE OF CONTENTS

Page

Title page ............................................................................................................................................ 1

Abstract .............................................................................................................................................. 2

Table of c o n ten ts .......................................................................................................................... 4

List o f Figures.............................................................................................................................. 12

List OF Ta b l e s .............................................................................................................................. 15

A c k n o w l e d g m e n t s ................................................................................................................... 19

List o f abréviations...................................................................................................................... 20

Chapter 1: In t r o d u c t io n 22

1.1 Historical background of research into glutamate role in the brain................... 23

1.2 Classification of glutamate receptors........................................................................ 27

1.1.1 Metabotropic glutamate receptors............................................................ 28

1.2.2 lonotropic glutamate receptors.................................................................. 28

1.2.2.1 AMP A and Kainate receptors (Non-NMDA receptors) 29

1.2.2.2 NM DA receptors........................................................................... 30

1.2.2.3 Delta ( Ô ) subunits (orphan receptors)........................................ 31

1.3 NM DA receptor-mediated synaptic transmission.................................................. 31

1.4 Molecular biology of NM DA receptors.................................................................. 32

1.4.1 N R l gene fam ily............................................................................................ 32

1.4.2 NR2 gene fam ily............................................................................................ 34

1.4.2.1 NR2A subunit................................................................................ 34

1.4.2.2 NR2B subunit.................................................................................. 34

1.4.2.3 NR2C subunit................................................................................ 35

1.4.2.4 NR2D subunit................................................................................ 35

1.4.3 NR3 gene family ( % subunits)..................................................................... 35

1.5 Expression of NM DA receptor subunits in hippocampus and striatum of 0-day-old rats................................................................................................................... 36

1.5.1 N R l subunit.................................................................................................. 36

1.5.2 NR2A subunit................................................................................................. 36

1.5.3 NR2B subunit................................................................................................. 36

1.5.4 NR2C subunit................................................................................................. 37

1.5.5 NR2D subunit................................................................................................. 37

1.5.6 NR3A or x-1 subunit................................................................................... 37

1.6 Subunit composition of NM DA receptors............................................................. 37

1.6.1 Factors regulating NM DA receptor subunit composition...................... 39

1.6.1.1 Preferential assembly between N R l and NR2 subunits 39

1.6.1.2 Differential expression of N R l and NR2 subunits.................. 39

1.7 Subunit stoichiometry of NM DA receptors........................................................... 40

1.8 Properties of recombinant NM DA receptors...................................................... 40

1.8.1 Functional properties................................................................................... 40

1.8.1.1 Macroscopic currents..................................................................... 40

1.8.1.1.1 NR1/NR2A receptors................................................... 41

1.8.1.1.2 NR1/NR2B receptors................................................... 41

1.8.1.1.3 NR1/NR2C receptors................................................... 41

1.8.1.1.4 NR1/NR2D receptors................................................. 41

1.8.1.2 Single-channel currents................................................................... 41

1.8.1.2.1 High and low conductance NM DA channels 42

1.8.1.2.2 Shut tim es....................................................................... 42

1.8.1.2.3 Open tim es..................................................................... 42

1.8.1.2.4 Open times conditional on amplitude....................... 42

1.8.2 Pharmacological properties........................................................................... 44

1.8.2.1 Effects of spermine......................................................................... 44

1.8.2.1.1 Inhibitory effects.......................................................... 45

A. Reduction in receptor affinity for agonists 45

B. Voltage-dependent inhibition............................... 45

1.9.2.1.2 Stimulatory effects.......................................................... 45

A. Glycine independent stimulation....................... 45

B. Glycine dependent stimulation........................... 46

1.8.2.2 Effects of ifenprodil....................................................................... 46

1.9 Experiments conducted in this study........................................................................ 46

Chapter 2: Materials a n d Methods 47

2.1 Solutions........................................................................................................................ 47

2.1.1 Shcing solution................................................................................................. 47

2.1.2 External solution for recording.................................................................... 47

2.1.3 Internal solution for recording...................................................................... 47

2.2 Drugs and chemicals....................................................................................................... 47

2.3 Drug application........................................................................................................... 48

2.4 Brain sHce preparation................................................................................................... 48

2.5 Cell visualization and identification............................................................................. 49

2.6 Patch pipette fabrication.............................................................................................. 49

2.7 Patching procedure......................................................................................................... 50

2.8 Data acquisition and analysis....................................................................................... 51

2.8.1 Detection and recording of single channels currents................................ 51

2.8.2 Record digitization......................................................................................... 51

2.8.3 Detection and measurement of events in the digitized record................ 51

2.9 Display and analysis of single channel data.............................................................. 52

2.9.1 Stabihty plots.................................................................................................. 52

2.9.1.1 Stability plots for ampUtudes.......................................................... 52

2.9.1.2 Stability plots for open times, shut times and P pen...................... 52

2.9.2 Distribution of fitted amphtudes................................................................. 53

2.9.3 Distribution of open times and shut times.................................................. 53

2.9.4 Distribution of open times conditional on amplitude.............................. 53

2.9.5 Bursts............................................................................................................... 54

2.9.5.1 Distribution of burst lengths........................................................ 54

2.9.5.2 Distribution of the total time per burst.................................... 55

2.9.5.3 Burst Ppp ......................................................................................... 55

2.9.6 Clusters........................................................................................................... 55

2.9.7 Super-clusters.................................................................................................. 55

2.9.8 Alignment of activations.............................................................................. 55

2.9.9 Current-voltage relationship p lots............................................................. 56

2.9.10 Direct transitions between conductance levels......................................... 56

Chapter 3: Single-Ch a n n e l Properties o f N M D A Receptors in Striatal 57

N euro ns from 0 D ay-Old (PO) Rats

3.1 Summary......................................................................................................................... 57

3.2 Introduction.................................................................................................................. 58

3.3 Results............................................................................................................................. 59

3.3.1 Presence of functional NM DA receptors in PO-striatal neurons 59

3.3.2 Properties of single channel currents.......................................................... 59

3.3.2.1 Stabihty plots for amphtudes........................................................ 59

3.3.2.2 Distribution of single channel current amplitudes.................... 59

3.3.2.3 Single channel conductance.......................................................... 62

3.3.2.4 Direct transitions between conductance states........................... 62

3.3.3 Properties of single-channel activations..................................................... 66

3.3.3.1 Stabihty plots for open times, shut times and P p n.................. 66

3.3.3.2 Distribution of shut tim es............................................................ 66

3.3.3.3 Distribution of all individual open times.................................... 70

3.3.3.4 Distribution of open times conditional on ampUtude.............. 70

3.3.3.4.1 Open times to the main level (54 pS)......................... 73

3.3.3.4.2 Open times to the sublevel (44 pS)........................... 73

3.3.3.5 Bursts............................................................................................... 73

3.3.3.5.1 Distribution of burst lengths...................................... 73

3.3.3.5.2 Distribution of total time per burst........................... 76

3.3.3.5.3 Burst P pen....................................................................... 76

3.3.3.6 Clusters............................................................................................. 76

3.3.3.6.1 Distribution of cluster lengths.................................... 76

3.3.3.6.2 Distribution of total time per cluster......................... 76

3.3.3.6.3 Cluster Popen..................................................................... 79

3.3.3.7 Super clusters.................................................................................... 80

3.3.3.7.1 Distribution of super cluster lengths........................... 80

3.3.3.7.2 Distribution of total time per super cluster................ 80

3.3.3.7.3 Super cluster P pg .......................................................... 80

3.3.3.8 Ahgnment of activations................................................................. 80

3.3.3.8.1 Ahgnment of clusters................................................... 80

3.3.3.8.2 Alignment of super-clusters........................................... 80

3.4 Discussion..................................................................................................................... 86

3.4.1 Single channel conductance.......................................................................... 86

3.4.2 Shut times......................................................................................................... 87

3.4.3 Main level openings....................................................................................... 88

3.4.4 Sublevel openings............................................................................................ 89

3.4.5 Bursts, clusters and super-clusters................................................................. 89

Chapter 4: Effects o f Spermine o n the Single-Ch a n n e l Properties o f 90

N M D A Receptors from PO-Striatal N eurons

4.1 Summary........................................................................................................................ 90

4.2 Introduction................................................................................................................. 91

4.3 Results............................................................................................................................ 91

4.3.1 Spermine affects NM DA receptor-channels in PO-striatal neurons. . . . 91

4.3.2 Effects on steady state single-channel behaviour....................................... 93

4.3.3 Effects on single-channel current amphtudes........................................... 93

4.3.4 Effects on shut tim es..................................................................................... 93

4.3.5 Effects on open periods................................................................................ 97

4.3.6 Effects on bursts............................................................................................. 99

4.3.7 Effects on clusters........................................................................................... 100

4.3.8 Effects on the decay time-course of ahgned clusters.................................. 101

4.4 Discussion....................................................................................................................... 103

Chapter 5: Single-Ch a n n e l Properties o f N M D A Receptors in 105

H ippocampal Granule Cells from o D ay-Old (PO) Rats

5.1 Summary........................................................................................................................ 105

5.2 Introduction................................................................................................................. 107

5.3 Results............................................................................................................................ 107

5.3.1 Presence of functional NM DA receptors in PO-hippocampal granulecells................................................................................................................... 107

5.3.2 Properties of single-channel currents........................................................... 107

5.3.2.1 Stabihty of single-channel current amphtudes........................ 107

5.3.2.2 Distribution of single-channel current amphtudes..................... 110

5.3.2.3 Conductance of single channel currents.............................. I l l

5.3.2.4 Direct transitions between conductance states......................... I l l

5.3.3 Properties of single-channel activations.................................................... 114

5.3.3.1 Stabihty plot analysis of shut times, open times and 117

5.3.3.2 Distribution of shut tim es.......................................................... 121

5.3.3.2.1 Patches with only high conductance openings 123

5.3.3.2.2 Patches with high & low conductance openings.. . . 123

5.3.3.2.3 Distribution of shut times bordered on each side by50 pS currents................................................................... 125

5.3.3.3 Distribution of all individual open tim es............................. 128

5.3.3.3.1 Distribution of open times conditional on currentamphtude....................................................................... 130

A. 17 pS currents.......................................................... 130

B. 33 pS currents......................................................... 130

C. 42 pS currents......................................................... 133

D. 50 pS currents...................................................... 133

5.3.3.4 Bursts............................................................................................... 133

5.3.3.4.1 Distributions of burst lengths...................................... 135

5.3.3.4.2 Distribution of the total open time per burst 135

5.3.3.4.3 Burst Popen....................................................................... 135

5.3.3.5 Bursts of 50 pS currents.................................................................. 135

5.3.3.6 Clusters............................................................................................. 139

5.3.3.6.1 Distributions of cluster lengths.................................. 139

5.3.3.6.2 Distribution of the total open time per cluster . . . . 142

5.3.3.6.3 Cluster Popen................................................................... 142

5.3.3.7 Clusters of 50 pS currents.............................................................. 142

5.3.3.8 Super clusters.................................................................................. 145

5.3.3.8.1 Distributions of super cluster lengths......................... 145

5.3.3.8.2 Distribution of the total open time per super cluster 145

5.3.3.8.3 Super cluster Popen.......................................................... 148

5.4 Discussion.................................................................................................................... 148

5.4.1 NM DA receptors mediate two patterns of single channel activity inPO hippocampal granule cells....................................................................... 148

5.4.1.1 High conductance pattern of single-channel activity................ 148

5.4.1.2 High and low conductance pattern of single-channel activity.. 150

5.4.2 Presence of direct transitions between conductance levels.................... 150

5.4.3 Properties of single channel activations..................................................... 151

5.4.1.1 Shut times........................................................................................ 151

5.4.1.2 Open times......................................................................................... 153

5.4.1.2.1 17 pS currents.............................................................. 153

5.4.1.2.2 33 pS currents.............................................................. 153

5.4.1.2.3 42 pS currents.............................................................. 153

5.4.1.2.4 50...pS currents.............................................................. 155

5.4.1.3 Bursts................................................................................................. 156

5.4.1.4 Clusters............................................................................................... 158

5.4.1.5 Super-clusters.................................................................................... 160

Chapter 6: Effects of Ifenprodil o n the Single-Ch a n n e l Properties of

N M D A R e c e p to r s f r o m PO H ip p ocam p al G r a n u le C e l ls 163

6.1 Summary......................................................................................................................... 163

6.2 Introduction..................................................................................................................... 164

6.3 Results............................. 164

6.3.1 Effects of ifenprodil on single-channel activity........................................ 164

6.3.2 Effects on single-channel current amphtudes............................................. 165

6.3.3 Effects on shut times.................................................................................... 168

6.3.3.1 In patches with only high conductance currents...................... 168

6.3.3.2 In patches with high and low conductance currents................... 168

6.3.4 Effects on individual open tim es................................................................ 168

6.3.4.1 In patches with only high conductance currents...................... 173

6.3.4.2 In patches with high and low conductance currents................... 173

6.3.5 Effects on open times conditional on amphtude...................................... 173

6.3.5.1 Effects on 17 and 33 pS currents................................................... 175

6.3.5.2 Effects on 42 pS currents................................................................. 175

6.3.5.3 Effects on 50 pS currents................................................................. 179

6.3.6 Effects on bursts........................................................................................... 179

6.3.6.1 Effects on burst lengths................................................................ 179

6.3.6.2 Effects on total open time per burst.......................................... 181

6.3.6.3 Effects on burst ..................................................................... 182

6.3.7 Effects on clusters........................................................................................ 182

6.3.7.1 Effects on cluster lengths................................................................. 182

6.3.7.2 Effects on total open time per cluster.......................................... 184

6.3.7.3 Effects on cluster ..................................................................... 185

6.3.1.7 Effects on the decay time-course of ahgned clusters............... 185

6.4 Discussion....................................................................................................................... 185

6.4.1 Effects on single-channel current amphtudes............................................ 186

6.4.2 Effects on shut times.................................................................................... 186

6.4.3 Effects on individual open tim es................................................................ 186

6.4.4 Effects on bursts........................................................................................... 187

6.4.5 Effects on clusters........................................................................................ 187

Chapter 7: General D iscussion 188

7.1 NM DA receptors in PO striatum................................................................................. 188

7.1.1 Can PO-striatum NM DA receptors be considered a homogeneous population of NR la/NR 2B native NM DA receptors?............................. 188

7.1.2 Future experiments........................................................................................... 190

10

7.2 NM DA receptors in PO dentate gyrus................................................ 191

7.2.1 Is the high and low conductance pattern of single-channel activity the result of the activation of two different NM DA channels or a single NM DA channel species with two different single-channel behaviour?. . 191

7.2.2 Is it possible from the data obtained so far to have an idea what subunits are taking part in the assembly of these two functionally and pharmacologically different NM DA receptor populations?....................... 192

7.2.3 Future experiments........................................................................................... 193

7.2 Physiological role of NM DA receptors in the immature brain........................... 193

7.3 Concluding remarks............................................................... 195

References..................................................................................................................................... 196

11

L i s t o f F i g u r e s

N um ber Page

C h a p t e r 3

3.1 Functional NM DA receptor-channels in PO striatal neurons.......................................... 60

3.2 Single-channel current amphtude of NM DA receptors in PO striatal neurons 61

3.3 Single-channel conductance of NM DA receptors in PO striatal neurons................... 63

3.4 Direct transitions between consecutive open-channel current levels.......................... 64

3.5 Stability plot analysis of shut times, open times and Popen during activation ofNM DA receptors in PO striatal neurons............................................................................ 68

3.6 Distribution of the duration of all shut times and individual open times..................... 69

3.7 Distribution of the duration of individual open times conditional on amphtude. . . 72

3.8 Distribution of burst lengths and total open time per burst........................................... 75

3.9 Distribution of cluster lengths and total open time per cluster..................................... 78

3.10 Distribution of super-cluster lengths and total open time per super-cluster................. 81

3.11 Alignment of clusters of single NMDA-channel activations.......................................... 83

3.12 Alignment of super-clusters of single-channel currents..................................................... 84

C h a p t e r 4

4.1 Effects of spermine on NM DA receptor single-channel currents from PO striatalneurons................................................................................................................................ 92

4.2 Stabihty plot analysis of the effect of spermine on amplitudes, shut times, opentimes and P pen....................................................................................................................... 94

4.3 Effects of spermine on the amphtude of NM DA receptor single-channel currentsfrom PO striatal neurons...................................................................................................... 95

4.4 Distribution of shut times and open periods under control conditions (A,B) andafter 100 pM spermine (C,D)............................................................................................. 96

4.5 Distribution of burst lengths and total open time per burst under controlconditions (A,B) and after 100 pM spermine (C,D)........................................................ 98

12

4.6 Alignment of clusters of openings from single NM D A-channel activations undercontrol conditions (A,B) and after 100 |iM spermine (C,D).......................................... 102

C h a p t e r s

5.1 Types of single-channel activity mediated by NM DA receptors in PO-hippocampalgranule cells............................................................................................................................ 108

5.2 Single-channel current amplitudes of NM DA receptors in PO-hippocampal granulecells.......................................................................................................................................... 109

5.3 Conductance of NM DA receptor single-channel currents in PO-hippocampalgranule cells............................................................................................................................ 112

5.4 Direct transitions between consecutive open-channel current levels............................ 115

5.5 Stabihty plot analysis of shut times, open times and .............................................. 118

5.6 Sectioning of stabihty plot for amphtudes in patches containing high and lowconductance single channel activity.................................................................................. 119

5.7 Stabihty plot analysis of shut times, open times and P pen for pooled sections of data containing mainly high amphtude (Aj, B , Cj, DJ and mainly low amphtude(A2 , B2 , C2 , D 2) single channel currents.............................................................................. 120

5.8 Distribution of the duration of shut times........................................................................ 122

5.9 Distribution of the duration of shut times bordered on each side by 50 pS currentsin patches with high and low conductance (A) and only high conductance (B) single-channel currents........................................................................................................ 126

5.10 Distribution of the duration of ah individual apparent openings.................................. 129

5.11 Distribution of the duration of individual open times conditional on amphtude ina patch with high and low conductance single-channel activity.................................... 131

5.12 Distribution of burst lengths and total open time per burst in the presence ofglutamate and N M D A ........................................................................................................ 136

5.13 Distribution of burst lengths and total open time per burst for bursts of 50 pScurrents................................................................................................................................... 138

5.14 Distribution of cluster lengths and total open time per cluster in the presence ofglutamate and N M D A ........................................................................................................ 141

5.15 Distribution of cluster lengths and total open time per cluster for clusters of 50 pScurrents................................................................................................................................... 144

5.16 Distribution of super-cluster lengths and total open time per super-cluster in thepresence of glutamate and N M D A.................................................................................... 147

13

C h a p t e r 6

6.1 Effects of ifenprodil on single-channel activity mediated by NM DA receptors inPO-hippocampal granule cells............................................................................................. 166

6.2 Effects of ifenprodil on the distribution of single-channel current amphtudes inpatches with high and low conductance (A,C) and only high conductance (B,D) single-channel activity mediated by NM DA receptors................................................... 167

6.3 Distribution of the duration of shut times under control conditions (A,B) and after1 pM ifenprodil (C, D) in patches containing high and low conductance (A,C) and only high conductance (B,D) NM DA receptor single-channel activity....................... 169

6.4 Distribution of individual open times under control conditions (A,B) and after 1pM ifenprodil (C, D) in patches containing high and low conductance (A,C) and only high conductance (B,D) NM DA receptor single-channel activity....................... 170

6.5 Distribution of the duration of individual openings to the 17 and 33 pS conductance levels under control conditions (A,B) and after 1 pM ifenprodil (C,D ) ........................................................................................................................................... 174

6.6 Distribution of the duration of individual open times for the 42 pS conductancelevel under control conditions (A,B) and after 1 pM ifenprodil (C, D)....................... 176

6.7 Distribution of the duration of individual open times for the 50 pS conductancelevel under control conditions (A,B) and after 1 pM ifenprodil (C, D)....................... 177

6.8 Distribution of burst lengths and total open time per burst under controlconditions (A,B) and after 1 pM ifenprodil (C, D ).......................................................... 180

6.9 Distribution of clusters lengths and total open time per cluster under controlconditions (A,B) and after 1 pM ifenprodil (C, D ).......................................................... 183

14

L i s t o f T a b l e s

N u m be r Page

C h a p t e r 1

1.1 Classification of ionotropic glutamate receptors............................................................... 29

1.2 Classification of N R l splice variants................................................................................... 33

1.3 Single channel conductance of N R 1/N R 2 recombinant NM DA receptors 43

1.4 Distribution of shut times for N R 1/N R 2 recombinant NM DA receptors 43

1.5 Open times including all amphtude levels in NR 1/N R2 recombinant NM DAreceptors................................................................................................................................ 44

1.6 Open times conditional on unitary current amphtude in N R 1/N R 2 recombinantN M D A receptors................................................................................................................. 44

C h a p t e r 3

3.1 Distribution of single channel current amphtudes........................................................... 59

3.2 Analysis of direct transitions between ah conductance levels detected in POstriatum NM DA receptors and comparison with those of high conductance N R 1/N R 2A and NR1/NR2B recombinant NM DA receptors................................. 65

3.3 Direct transitions at different negative membrane potentials...................................... 6

3.4 Distribution of shut times..................................................................................................

3.5 Distribution of individual open times to ah amplitude levels.........................................

3.6 Distribution of individual open times to the main level (54 pS).....................................

3.7 Distribution of individual open times to the sublevel (44 pS).......................................

3.8 Distribution of burst lengths..............................................................................................

3.9 Distribution of total open time per burst..........................................................................

3.10 Burst

3.11 Distribution of cluster lengths......................

3.12 Distribution of total open time per cluster.

3.13 Cluster

15

3.14 Distribution of super-cluster lengths.................................................................................... 82

3.15 Distribution of total open time per super-cluster.............................................................. 82

3.16 Super-cluster ................................................................................................................... 82

3.17 Decay time-course of aligned clusters................................................................................. 85

3.18 Decay time-course of aligned super-clusters...................................................................... 85

3.19 Comparison between shut times from PO striatum and recombinant NM DAreceptors................................................................................................................................. 87

3.20 Comparison between main level openings from PO striatum and recombinant 88NM DA receptors.................................................................................................................

3.21 Comparison between sublevel openings from PO striatum and recombinantNM DA receptors................................................................................................................. 89

C h a p t e r 4

4.1 Distribution of single-channel current amphtudes........................................................... 93

4.2 Distribution of shut times..................................................................................................... 97

4.3 Distribution of open periods................................................................................................. 97

4.4 Distribution of burst lengths................................................................................................. 99

4.5 Distribution of total open time per burst......................................................................... 99

4.6 Mean charge transfer per burst............................................................................................ 100

4.7 Distribution of cluster lengths............................................................................................ 100

4.8 Distribution of total open time per cluster.......................................................................... 101

4.9 Mean charge transfer per cluster....................................................................................... 101

4.10 Decay time-course of ahgned clusters................................................................................. 103

C h a p t e r 5

5.1 Distribution of single-channel current amphtudes................................................ I l l

5.2 Frequencies of direct transitions between shut and open levels.......................... 113

5.3 Frequencies of direct transitions between open-channel current levels............. 116

5.4 Distribution of shut times for glutamate and NM DA..................................................... 124

5.5 Distribution of shut times bordered on each side by 50 pS currents............................ 127

16

5.6 Distribution of individual open times to all amplitude levels.............................. 128

5.7 Distribution of open times for 17 pS...currents................................................................ 132

5.8 Distribution of open times for 33 pS currents................................................................ 132



5.11 Distribution of burst lengths.............................................................................................. 137

5.12 Distribution of total open time per burst........................................................................... 137

5.13 Burst ............................................................................................................................... 137

5.14 Distribution of burst lengths for bursts of 50 pS currents................................... 140

5.15 Distribution of total open time per burst for bursts of 50 pS currents................ 140

5.16 Burst for bursts of 50 pS currents............................................................................. 140

5.17 Distribution of cluster lengths............................................................................................ 143

5.18 Distribution of total open time per cluster........................................................................ 143

5.19 Cluster ........................................................................................................................... 143

5.20 Distribution of cluster lengths for clusters of 50 pS currents...................................... 146

5.21 Distribution of total open time per cluster for clusters of 50 pS currents............... 146

5.22 Cluster for clusters of 50 pS currents....................................................................... 146

5.23 Distribution of super-cluster lengths................................................................................ 149

5.24 Distribution of total open time per super-cluster........................................................... 149

5.25 Super-cluster P„p .......................................................................................................... 149

5.26 Direct transitions involving three consecutive conductance levels........................ 152

5.27 Comparison of shut times between PO- dentate gyrus, PO-striatum andrecombinant NM DA receptors................................................................................ 154

5.28 Individual open times for 17 pS currents in PO-dentate gyrus and N R 1/N R 2Drecombinant NM DA receptors................................................................................ 154

5.29 Comparison of open times for 40 pS currents from PO-dentate gyrus, PO-striatum and recombinant NM DA receptors......................................................... 155

5.30 Comparison of open times for 50 pS currents from PO-dentate gyrus, PO- 156striatum and recombinant NM DA receptors.................................................................

17

5.31 Comparison of burst lengths between dentate gyrus and striatum............................. 157

5.32 Comparison of total open time per burst between dentate gyrus and striatum. . . 157

5.33 Comparison of mean burst Popen between dentate gyrus and striatum...................... 158

5.34 Comparison of cluster lengths between dentate gyrus and striatum........................ 159

5.35 Comparison of total open time per cluster between dentate gyrus and striatum. . 159

5.36 Comparison of mean cluster P ^ between dentate gyrus and striatum........ 160

5.37 Comparison of super-cluster lengths between dentate gyrus and striatum......... 161

5.38 Comparison of total open time per super-cluster between dentate gyrus andstriatum............................................................................................................................ 161

5.39 Comparison of mean super-cluster P ^ between dentate gyrus and striatum. . . . 162

C h a p t e r 6

6.1 Distribution of single channel current amplitudes......................................................... 165

6.2 Distribution of shut times.................................................................................................. 171

6.3 Distribution of individual open times for currents to all amplitude levels................ 172

6.4 Distribution of individual open times for the 17 and 33 pS levels............................. 175

6.5 Distribution of individual open times to the 42 pS level............................................. 178

6.6 Distribution of individual open times to the 50 pS level............................................. 178

6.7 Distribution of burst lengths............................................................................................. 181

6.8 Distribution of total open time per burst......................................................................... 181

6.9 Burst P^^.......................................................................................................................... 182

6.10 Distribution of clusters lengths......................................................................................... 184

6.12 Distribution of total open time per cluster....................................................................... 184

6.13 Cluster Pppen...................................................................................................................... 184

6.14 Decay time-course of aligned clusters......................................................................... 184

18

ACKNOWLEDGMENTS

I would like to thank my wife Yasbel, for all her love, support and patience during all these

years. I would Hke to thank my sons Juan Carlos and Julian David, for making me the happiest

man on earth.

I would like to thank my parents Maro and Rosa and my aunt Rodolfa, for being always

supportive and positive about my choices. I would like to thank my brothers Hildemaro, Jesus,

Pedro and sisters Rosa, Carmen AHcia and Gloria for their continuous support and

encouragement.

I would also hke to thank Raul and Carmen for all their care and support.

I would hke to thank Nelson Dalo for his unconditional support, encouragement, enthusiasm

and friendship. For being a continuous source of inspiration and for infecting me with the love

for science.

I would like to thank Lilian Spencer for her friendship and support.

I would like to express my thanks to Anis Contractor for his continuous support and

encouragement. I would also like to thank Adrian Wong, Beth Rycroft and Fiona Halliday,

fellow members of the lab, for making the environment here much more fun and enjoyable.

I would like to thank David Colquhoun for his example of commitment to science. Don

Jenkinson for giving me the opportunity of being part of the great UCL family. PhiUipe Behe,

David Wyllie, Peter Stern and Sunjew Kamboj for many stimulating conversations, for

providing me with helpful advise, ideas and data.

I would like to thank CONICIT, Inter-American Bank of Development and Universidad

Centroccidental ‘Lisandro Alvarado’ for providing me with the time and financial support.

Finally, I would Hke to express my thanks to my supervisor Alasdair J. Gibb for his guidance,

support, encouragement and friendship. For his good sense of humor, his abiHty to laugh and

joke when things were going wrong, cells were dead, chambers were broken, solutions were acid

and things seemed to be going nowhere. For being always kind, considerate, helpful and

supportive.

19

LIST OF ABBREVIATIONS

- ^ c r i f

AIDS:

AMPA:

C M :

Ca " + :

cAMP:

cDNA:

CONSAM:

DA:

DN Q X :

EGTA:

EKDIST:

EPSC:

GABA:

G A D :

G luR :

G l u R l / G l u R - A :

G l u R 2 / G l u R - B :

G l u R 3 / G l u R - C :

G l u R 4 / G l u R - D :

GluR5;

G l u R 6

G luR 7:

H E R 293 cel ls:

HEPES:

IC 5 0 :

K + :

KA:

K A l :

KA2:

LTD:

Critical amplitude

Acquired immunodeficiency syndrome

a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

Cornu Ammon region 1

Calcium ions

Cyc l ic adenosine m o n o p h o s p h a te

com p lem en tary d e o x y r ib o n u c le i c acid

Continuous sampling program

D o m o i c acid

6,7-Dinitroquinoxaline-2,3-dione

Ethylene Glycol-bis(|3-aminoethyl Ether) N ,N ,N ’,N ’-Tetraacetic acid

Single channel distribution program

Excitatory post-synaptic current

y-aminobutyr ic acid

Glutamic acid descarboxilase

Glutamate receptor

Glutamate receptor subunit 1 or A

Glutamate receptor subunit 2 or B

Glutamate receptor subunit 3 or C

Glutamate receptor subunit 4 or D

Glutamate receptor subunit 5



H um an em b ryon ic k id n e y 293 cells

N-[2-Hydroxyethyl]piperazine-N’-[2-ethanesulfonic acid]

Inh ib i tory concentra t ion 50

Potassium ions

Kainate

Kainate receptor subunit 1

Kainate receptor subunit 2

Long term depression

20

LTP:

M g' + :

m G l u R l

m G l u R 2

m G l u R 3

m G l u R 4

n iG lu R S

m G l u R 6

inG luR Z

m G l u R S

m G l u R s :

M K801:

m R N A :

N a + :

NM DA:

NM DAR:

NRl:

NR2A:

NR2B:

NR2C:

NR2D:

PO:

P •^ Open*

pS:

PSD-95:

SAP-102:

SCAN:

G if

Xen U 1 :

Zn'":

Long term potentiation

Magnes ium ions

M etabotropic glutamate receptor 1

M etabotrop ic glutamate receptor 2







M etabotrop ic glutamate receptors

D i z o c i l p in e , 5-Methyl - lO, 1 l -d ihydr o -5H -d ibe nz o [a ,d ]

cyc lohe pte n-5 ,10 - im ine .

Messenger r ibonuc le ic acid

Sodium ions

N-M ethy l -d -A spart ic acid

N-M ethy l -d -A sp art ate receptor

N -M ethy l -d -A sp art ate receptor subunit 1

N -M ethy l -d -A spart ate receptor subunit 2A

N -M ethy l -d -A sp art ate receptor subunit 2B

N -M ethy l -d -A spart ate receptor subunit 2C

N -M ethy l -d -A sp art ate receptor subunit 2D

Postnatal day 0, day of birth

Probabi l i t y of the channel be ing open

picos iem en

Posynapt ic dens i ty protein 95

Synapt ic associated protein 102

Single channel analysis program

Critical shut t im e or crit ical gap length

X e nop us glutamate receptor subunit 1

Zinc ions

21

CHAPTER 1

Introduction

The functional and pharmacological characterisation of native subtypes of NM DA

receptors is a fundamental step in the understanding of NM DA receptor involvement in a

variety of physiological and pathological processes in the central nervous system (CoUingridge

& Watkins, 1994; Monaghan & Wenthold, 1997). Through the identification and localisation

of NM DA receptor subtypes to specific neuronal populations it should be possible to improve

our understanding of how NM DA receptor-mediated signalling is regulated in different brain

regions and how subtype-selective drugs could be more efficiently used to target specific brain

areas affected by particular neuropathological conditions, increasing therapeutic efficacy and

reducing at the same time the potential side effects of drugs.

Recent evidence from studies using recombinant receptors has shown that the functional

(reviewed by Stern & Colquhoun, 1998) and pharmacological (reviewed by Lynch et a i, 1997)

properties of NM DA receptors are determined by their subunit composition. In the brain,

native subtypes of NM DA receptors are generated by differences in the pattern of expression of

NM DA receptor subunits, changes in the level of expression of particular subunits during brain

development and differential assembly of NM DA receptor subunits within individual neurons

(reviewed by Feldmeyer & CuU-Candy, 1996; Monaghan et a i, 1997; Watanabe, 1997). A single

neuron or neuronal population can potentially express different associations of NM DA receptor

subunits producing functionally and pharmacologically different NM DA receptor subtypes

which may serve different physiological functions. By analysing the functional and

pharmacological properties of these receptors it should be possible to identify which NM DA

receptor subtypes are present in the mammaHan central nervous system and how they

participate in the wide variety of processes in which NM DA receptor-mediated signalling have

been shown to be involved.

The aims of this introductory chapter will be to present, first of all, the rationale for

this study and the methods used; secondly, to describe the historical background of research

into glutamate receptor-mediated signalling in the brain, and thirdly, to discuss some of the

evidence that may help to explain the molecular and structural basis of the functional and

pharmacological diversity of NM DA receptors in the brain, especially in the hippocampus

and striatum.

Usually, the functional and pharmacological properties of native NM DA receptors have

been studied by recording macroscopic responses generated by activation of all the receptors

present in the cell membrane (reviewed by Mayer et a i, 1994). In contrast, the combination of

22

single-channel recording techniques (Hamill et a l, 1981), which allow the study of the

microscopic behaviour of single receptor macromolecules in isolation, and patch-clamping in

brain slices (Edwards et a l, 1989), which allow specific neuronal cell types to be studied,

represents a more powerful approach for a relatively more accurate functional and

pharmacological identification of native species of NM DA receptors. A particular subtype of

NM DA receptor can be identified by its distinctive single channel properties. Single-channel

recording allows the study of permeation and activation properties such as magnitude and

number of conductance states, duration and number of open and shut states and probabihty of

being open (P^^). It also allows a detailed study of the effects of agonists, antagonists and channel

blockers on specific single channel properties.

By recording and analysing individual single-channel receptor activations arising from

native NM DA receptors obtained from neurons in brain sHces, the distinctive features that

characterise the microscopic behaviour of native NM DA receptor subtypes can be initially

described and later used as fingerprints in the identification of potential correlations between

the structure and the function of native subtypes of N M D A receptors. Using this kind of

approach, a strong correlation has been found between the presence of subtypes of NM DA

receptors with different single channel properties and the expression of particular NM DA

receptor subunits at specific developmental stages in cerebellar neurons (Farrant et a l, 1994;

Momiyama et a l, 1996). Similar studies have not yet been carried out in the hippocampus and

striatum.

The aims of the experiments conducted in this study were: first, to describe the

functional properties of single NM DA receptor-channels in hippocampal granule cells and

striatal neurons from rats at postnatal day 0 (day of birth) and second, to describe the effects on

their single-channel properties of spermine and ifenprodil, two compounds which have been

shown to have subunit-selective effects on recombinant NM D A receptors. Findings regarding

functional and pharmacological properties will be compared with information from functional

and pharmacological studies in which recombinant NM DA receptors have been used (reviewed

in CoUingridge & Watkins, 1994; Monaghan & Wenthold, 1997); they will then be discussed in

relation to how they correlate with the pattern of expression of NM DA receptor subumts in

these neurons (Monyer e ta l, 1994; Wenzel e ta l, 1997, reviewed by Watanabe, 1997).

1.1 Historical background o f research into glutamate role in the brain

Research into the role of glutamate in the nervous system was initially led by the

description of glutamate oxidisation by homogenates of peripheral nerves and brain slices and

23

by investigations into the enzymatic mechanisms involved in the metabolism of amino acids

in body tissues (reviewed by Weil-Malherbe, 1950).

During the 1930’s, the most influential findings were the description of the ability of

glutamate to increase and sustain the rate of oxygen uptake in brain and retinal slices (Quastel

& Wheatly, 1932; Krebs, 1935a; Weil-Malherbe, 1936) and the presence in brain tissue of

enzymes able to synthesise glutamine, glutamate and ammonia (Krebs, 1935b). The

observation by Krebs (1935b) that glutamine and ammonia did not seem to be the main end

products of the enzymatic process led to the suggestion by Weil-Malherbe (1936) that some of

these enzymes were probably more involved in the synthesis of glutamate than in its

breakdown. More than ten years later, this initial suggestion was partially supported by

evidence showing that the concentration of glutamate is much higher in the brain than in any

other body tissue (Krebs etal., 1949).

The apparent ability of glutamate to increase and sustain oxygen uptake in brain slices,

even in the absence of glucose (Krebs, 1935a) generated interest in its potential role in cellular

respiratory processes in the brain. At that time, the strong correlation between the brain rate

of oxygen consumption and the blood level of glucose was already known and the profound

changes in electroencephalographic activity induced by hypoglycaemia were known to be

relieved by intravenous administration of glucose. Based on this evidence, in vivo experiments

were carried out to test the ability of glutamate to restore brain electro-encephalographic

activity in the absence of glucose. Such experiments showed that glutamate was unable to

maintain or restore to normal levels cortical activity in hypoglycaemic animals (Maddock et

al., 1939). As a result of this lack of in vivo evidence of glutamate effects, the idea of glutamate

involvement in cellular respiratory processes in the brain was soon abandoned.

During the 1940’s, a new hypothesis was put forward. Initial in vitro evidence of the

ability of brain slices to synthesise glutamine from L-glutamate and ammonia through to an

enzymatic process (Krebs, 1935b) led to the suggestion that the role of glutamate in the brain

was probably linked to a metabolic pathway responsible for reducing the concentration of

ammonia in the brain, helping to control brain pH levels. Based on Putnam & Merritt’s (1941)

hypothesis that anticonvulsant drugs exerted their actions by reducing pH levels in the brain,

clinical trials were set up to test the potential anticonvulsive activity of glutamate in epileptic

patients.

Surprisingly, such studies reported evidence suggesting that glutamate was not only

able to reduce the frequency and severity of seizures but it was also able to increase the

patients’ mental and physical alertness as well as improving their mental efficiency (Price et at. ,

1943; Waelsch & Price, 1944). Such reports generated a great deal of interest into the role of

24

glutamate in brain metabolism. A large number of studies were carried out to test the

effectiveness of glutamate in a diverse number of neurologic, psychiatric and metabolic

disorders (reviewed by Weil-Malherbe, 1950) generating a great deal of controversy after

experimental evidence showed that the rate of diffusion of glutamate from the blood to the

brain was very slow when compared with that of glucose (Klein & Olsen, 1947) and that even

intravenous administration of glutamate was not effective in increasing the concentration of

glutamate in the brain (Friedberg & Greenberg, 1947; Waelsch etaL, 1949; reviewed by Weil-

Malherbe, 1950).

It was not until the 1950’s that some of the most influential findings were described.

Glutamic acid descarboxylase (GAD), an enzyme able to catalyse the synthesis of gamma-

aminobutyric acid (GABA) from glutamate, was isolated from rat brain (Roberts & Frankel,

1950; Wingo & Awapara, 1950) and the presence of GABA was first described in brain tissue

(Awapara et al. 1950; Roberts & Frankel, 1950). Nearly twenty years later, GABA was shown

to be the main neurotransmitter at the neuromuscular junction of crustaceans (Otsuka et al.,

1966) and at synapses between Purkinje cells and neurons from the deep cerebellar nuclei of

mammals (Obata & Takeda, 1969).

During the same decade, an even more influential set of findings were reported. The

observation by Hayashi (1952) of the ability of glutamate and aspartate to induce convulsions

when injected into the brain grey matter led him to suggest a direct stimulatory action of

glutamate upon nerve cells (Flayashi, 1954). Five years later, the direct excitatory effect of

glutamate and aspartate on spinal neurons was described by Curtis et at. (1959). At that time,

an equally influential finding was the description by Lucas & Newhouse (1957) of acute

degenerative lesions in the inner retina of normal neonatal mice after parenteral

administration of glutamate. More than ten years later, such effects were confirmed after

glutamate-induced acute neuronal necrosis was described in the brain of neonatal rodents and

primates and the term ‘excitotoxicity’ was introduced to describe the neurotoxic effects of

glutamate and its analogues (Olney, 1969; Olney & Sharpe, 1969; Olney & H o, 1970).

During the 60’s, the results of research into the structure-function relationship of a

large number of neutral and acidic amino-acids led to the idea of an “amino-acid-

receptor complex” present in the extracellular face of the cell membrane of neurons (Curtis et

a l, 1960; Curtis & Watkins, 1960). From that moment, experimentSwere made to determine

what chemical groups within the glutamate and aspartate molecules were involved in the

activation of the ‘amino acid receptor’. A number of glutamate and aspartate analogues were

synthesised and tested helping to elucidate the basis for the excitatory activity of glutamate

and aspartate (Curtis & Watkins, 1960; reviewed by Watkins, 1994; Lodge, 1997). Such work

25

finally produced the synthesis of the N-methyl-D-aspartic acid (NMDA) (Watkins, 1962), a

non-metabolised aspartate analogue which is not a substrate for glutamate transporters and

was later shown to be a remarkably selective excitatory amino acid. Soon after, the existence

of subtypes of excitatory amino acid receptors was suggested after variations in sensitivity to

glutamate and aspartate were observed between thalamic and spinal neurons (McLennan et al.

1968; Duggan, 1974)

During the 70’s, research efforts finally produced evidence of the role of glutamate as a

neurotransmitter and evidence of the existence of subtypes of glutamate receptors. Initially,

high-affinity L-glutamate reuptake systems (Logan & Snyder, 1971; Balcar & Johnston, 1972)

and high affinity binding sites for L-glutamate (Roberts, 1974; Michaelis et al. 1974) were

shown to be present in the brain. By the end of the decade, a fundamental piece of evidence

was the description of the ability of NM DA receptor antagonists to block synaptic

transmission induced by stimulation of afferent pathways in the spinal cord of amphibians and

mammals (Evans et a i, 1979; Davies et al., 1979). From that time until now, a fundamental

role in demonstrating the existence of glutamate receptors and their involvement in synaptic

transmission has been played by the synthesis by Jeff Watkins and co-workers of highly

selective glutamate receptor agonists and antagonists (reviewed by Watkins & Evans, 1981).

Also during the 70’s, the initial suggestion made by Maclennan et al. (1968) of the

existence of subtypes of glutamate receptors was confirmed thanks to the discovery of the

excitatory effects on central neurons of kainic, quisqualic, and domoic acid, three highly

selective and potent glutamate analogues (Shinozaki & Konishi, 1970; Olney et al., 1974;

Biscoe et a l, 1975). The presence of kainate-sensitive and NMDA-sensitive neurons were

described (McCulloch et al., 1974) and the abiHty of Mg^ ions to inhibit selectively NM DA-

induced but not kainate or quisqualate-induced responses was discovered (Evans et al., 1977;

Davies & Watkins, 1977). These findings allowed the classification of glutamate receptors into

two classes: NM DA- and non-NMDA receptors including within the last group kainate- and

quisqualate-activated receptors (Watkins & Evans, 1981).

During the 80’s, research efforts were concentrated on determining the nature and

signalling pathways used by glutamate receptors. Initial ideas about the nature of glutamate

receptors were first put forward by Curtis et al. (1960); they suggested that such receptor sites

would be able to induce an increase in the permeability of the cell membrane to Na" . Direct

evidence of the nature of glutamate receptors appeared for the first time when glutamate-

activated ion channels were described in the neuromuscular junction of invertebrates (Patlak

et a l, 1979; Cull-Candy et a l, 1981) and then with the demonstration, in mammalian central

neurons, that the NMDA-type of glutamate receptor was a glutamate-gated ion channel

26

(Nowak et al. 1984; Cull-Candy & Ogden, 1985) that under physiological conditions was

selectively blocked in a voltage-dependent manner by Mg^ (Nowak et a l, 1984; Mayer et a i,

1984). Such findings finally explained the selective antagonistic effect of Mg^ on NM DA-

induced responses (Evans et a i, 1977; Davies & Watkins, 1977) and the unusual conductance

changes observed during NMDA-induced currents (MacDonald & Wojtowicz, 1980).

Also throughout the 1980’s, an equally important number of findings involving

glutamate receptors were reported. NM DA receptor involvement in the induction of long

term potentiation (LTP) (Collingridge et a i, 1983; Herron et al. 1986). N M D A receptor

participation in synaptic transmission in vivo (Salt, 1986). Glutamate neurotoxicity was shown

to be dependent on Ca (Choi, 1985) and evidence of the high Ca permeability of NM DA

receptors (MacDermott et a i, 1986) appeared. Potentiation of NM DA responses by glycine

(fohnson & Ascher, 1987) was discovered and glycine was later found to be an absolute

requirement for NM DA receptor activation (Kleckner & Dingledine, 1988). Extracellular

concentration of glutamate was shown to rise significantly during hypoxia-ischaemia

(Benveniste et at., 1984, Hagberg et at., 1985) and hypoxic-ischaemic injury was shown to be

blocked in vivo by glutamate receptor antagonists (Simon et al. 1984). In 1987, a tragic

incident of poisoning with the glutamate analogue domoic acid (DA) provided direct evidence

of the involvement of glutamate receptor activation in more than 100 cases of acute toxic

encephalopathy in humans (Perl etaL, 1990; Teitelbaum et at., 1990). Evidence of the presence

in brain of subtypes of NM DA receptors appeared (Monaghan et a l, 1988). By the end of

1980’s, the first glutamate receptor was finally cloned (Hollmann etaL, 1989).

During the 1990’s, major research efforts have been devoted to determining the

structure-function relationship of glutamate receptors. A fundamental role has been played by

the combination of molecular biology and electrophysiological techniques which have allowed

the cloning and functional expression of genes encoding ionotropic (NM DA and non-

NM DA) and metabotropic glutamate receptors (reviewed by Hollmann & Heinemann, 1994).

Thanks to the use of this approach, a large number of glutamate receptors, whose number

seem to exceed those predicted by pharmacological classification, have been cloned, unveiling

the molecular and structural basis for the observed functional and pharmacological diversity of

glutamate receptors in the central nervous system.

1.2 Classification o f glutamate receptors

In the central nervous system, glutamate is thought to be the main excitatory

neurotransmitter. Under physiological conditions, signalling through glutamate receptors

mediates processes of neuronal cell migration (Komuro & Rakic, 1993), synaptogenesis

27

(Constantine-Paton, 1990), synaptic transmission (reviewed by Salt, 1994) and synaptic

plasticity (Bliss & Collingridge, 1993) while their excessive activation is thought to be a major

cause of neuronal cell damage during acute neurologic conditions such as brain trauma,

cerebral ischaemia, hypoglycaemia, hypoxia and epileptic seizures; as well as during chronic

neurodegenerative diseases such as Huntington’s chorea, AIDS encephalopathy and dementia

complex, and neuropathic pain syndromes (reviewed by Choi 1992; Lipton & Rosenberg,

1994).

During these processes, glutamate receptor-mediated signalling occurs through

activation of two different types of receptors: metabotropic glutamate receptors which are

coupled to intracellular effector systems via G-proteins (reviewed by Conn & Pin, 1997) and

ionotropic glutamate receptors which are coupled to ion channels and are responsible for fast

changes in membrane permeability (reviews in Collingridge & Watkins, 1994; Monaghan &

Wenthold, 1997).

1.2.1 M etabotropic glutamate receptors

Metabotropic glutamate receptors (mOluRs) play an important role in the

transduction of glutamate-mediated signalling via coupHng through G-proteins to intracellular

second messenger cascades. They have been classified into groups 1, 2 and 3. Group 1

(mGluRl and mGluR5) receptors are coupled to phospholipase C mediating increases in

intracellular calcium. Group 2 (mGluR2 and mGluR3) and group 3 (mGluR4, mGluR6,

mGluR7, mGluRS) are negatively coupled to adenylate cyclase, producing a decrease in

forskolin-induced cAMP (reviewed by Conn & Pin, 1997).

1.2.2 Ionotropic glutamate receptors

Ionotropic glutamate receptors are cation-selective glutamate-gated ion channels

composed of four or five glycosylated transmembrane proteins or subunits assembled around

a central aqueous pore that span the cell membrane. Each receptor contains three major

domains: an extracellular glutamate-binding domain related to amino acid-binding bacterial

periplasmic proteins, a transmembrane pore-lining domain related to the pore-forming region

or “P-loop” of channels and an intracellular regulatory domain (reviewed by Wo &

Oswald, 1995).

Ionotropic glutamate receptors are essential mediators of fast synaptic transmission

thanks to their ability to generate a response within a millisecond after glutamate

binding. Based on their pharmacology, they have been classified according to their agonist

selectivity into three major sub-classes: AMP A (a-amino-3-hydroxy-5-methyl-4-isoxazole

28

propionate) receptors, Kainate receptors and NM DA (N-methyl-D-aspartate) receptors.

Recently, a fourth and fifth subclasses of glutamate receptor subunits, sequence-related to

ionotropic glutamate receptors have been cloned and termed delta ( Ô ) and chi ( % ) subunits.

Very recently, chi ( % ) subunits have been classified as members of the N M D A receptor sub

class and renamed NR3 subunits (Das et al. 1998). Delta ( 5 ) subunits remain ‘orphan’. Each

receptor sub-class consist of several sub-families of receptor subunits (Table 1.1).

T a b l e l . l Classification o f ionotropic glutamate receptor subunits'^

Pharmacology Subfamily Subunit References

Mouse Rat

AMPA a a l GluRl Hollmann et al (1989), Sakimura et al (1990)a2 GluR2 Keinànen et al (1990), Sakimura et al (1990)a3 GluR3 Keinànen et al (1990), Sommer et al (1992)a4 GluR4 Keinànen et al (1990), Sommer et al (1992)

Kainate P pi GluR5 Bettler et al (1990), Sommer et al (1992)p2 GluR6 Egebjerg et al (1991), Morita et al (1992)P3 GluR7 Bettler et al (1990), Gregor et al (1993)

Kainate y yl KAl Wemeretal (1991)y2 KA2 Herb et al (1990), Sakimura et al (1992)

Unknown 5 51 delta 1 Yamazaki et al (1992a), Lomeli et al (1993)52 delta2 Lomeli et al (1993)

N M DA 8 8l NR2A Monyer et al (1992), Meguro et al (1992)82 NR2B Monyer et al (1992), Kutsuwada et al (1992)83 NR2C Monyer et al (1992), Kutsuwada et a l (1992)84 NR2D Ikeda et al (1992); Ishii et al (1993)

NM DA c ; i N R l Moriyoshi et al (1991), Yamazaki et al (1992b)

NMDAR-L X x i NR3A Ciabarra et al (1995), Sucheretal (1995)(NMDA-like) X2 NR3B Sevarino et al (1996)

"^Modifiedfrom Watanabe, 1997

1.2.2.1 AM P A and Kainate receptors (non-NMDA receptors)

The pharmacological classification of these two ionotropic glutamate receptors

remains ambiguous due to lack of selective agonists able to distinguish between them

(although some partially selective kainate antagonists are available: e.g., GYKI 52466 and

GYKI 53655, reviewed by Huettner, 1997). They are still collectively referred to as non-

29

N M D A receptors. More recently, the terms “AMPA-preferring” and “kainate-preferring”

receptors have been suggested (Partin etaL, 1993). During glutamatergic synaptic transmission,

AMPA-preferring receptors mediate the fast component of synaptic currents while the role of

kainate-preferring receptors in synaptic transmission is still obscure; a slow postsynaptic

current mediated by kainate-preferring receptors has been recently described (Vignes &

Collingridge, 1997; Castillo et a l, 1997). Upon activation by L-glutamate or AMPA these

receptors mediate responses that are characterised by rapid onset, offset and desensitisation

kinetics but when they are activated by kainate or domoate (DA) they produce large non

desensitizing responses (reviewed by Bettler & Mulle, 1995).

In rat brain, four AMPA-preferring receptor subunits with AMPA affinity in the low

nM range have been cloned and termed GluRl, GluR2, GluR3 and GluR4 (Boulter et a i,

1990) or GluR-A, GluR-B, GluR-C and GluR-D (Keinànen et a l, 1990). They are all able to

form homooligomeric receptors as well as heterooligomeric receptors when expressed with

other subunits. Splice variants of each subunit termed Flip or Flop exist as a result of the

presence or absence of a 35 amino acids segment, respectively, close to the T** transmembrane

domain (Sommer etaL, 1990). Splice variants expression is developmentally regulated (Monyer

et aL, 1991). Functionally, they show rapid kinetics and low Ca^ permeability, which is

determined by the GluR-B subunit. Channels composed of Flop subunits show decreased

steady state currents following AMPA or glutamate application (reviewed by Bettler & Mulle,

1995).

In rat brain, two families of kainate-preferring receptor subunits have been cloned. A

family of three sequence-related subunits termed: GluR5, GluR6 and GluR7 and a family of

two also sequence-related subunits termed K Al and KA2. They bind kainate with

intermediate to high affinity. GluR5 and GluR6 are the only kainate-preferring subunits able

to form functional receptors upon in vitro expression while the others can form functional

receptors only when co-expressed with GluR5 or GluR6. Their level of expression in the

brain is lower and more restricted than that of AMPA-preferring subunits. In contrast with

GluRl to 4 subunits, GluR5 and GluR6 strongly desensitise upon activation by kainate

(reviewed by Bettler & Mulle, 1995)

1.2.2.2 NM D A receptors

N M D A receptors are characterised by high Ca permeability, voltage-dependent

block by Mg^ , high single-channel conductances, slow gating kinetics and activation by the

concerted action of glutamate and glycine. During glutamatergic synaptic transmission,

NM DA receptors mediate the slow component of synaptic currents. NM D A receptors result

30

from the assembly of three different subfamiUes of subunits. The N R l family composed of

one subunit with nine different alternatively splice variants, the NR2 family composed of four

sequence-related subunits termed NR2A, NR2B, NR2C and NR2D and the chi ( % ) or NR3

family composed of two subunits termed NR3A and NR3B (Table 1.1)

1.2.2.3 Delta ( S ) subunits (orphan receptors)

Delta (Ô) subunits have been cloned in both rat and mouse brain (Yamazaki et al.

1992, Lomeli et a i, 1993). They consist of two sequence-related subunits termed delta 1 (51)

and delta2 (52) that share 56 % sequence identity (Yamazaki et al., 1992; Lomeli et al., 1993).

Although they show 21 to 25 % amino acid sequence identity with ionotropic glutamate

receptors (Yamazaki et al., 1992), they do not form functional ion channels upon homomeric

expression or heteromeric expression with N R l, NR2A, NR2C, K Al or GluRZ (Lomeli et a i,

1993). The delta2 (Ô2) subunit is highly expressed in cerebellar Purkinje cells while delta 1 (51)

subunit- expression is found throughout the brain during early postnatal development (Lomeli

et a i, 1993). Delta2 (52) subunit-deficient mice show impairment of motor coordination,

Purkinje cell formation and cerebellar long-term depression (Kashiwabuchi et a l, 1995).

1.3 NM D A receptor-mediated synaptic transmission

The contribution of NM DA receptors to central glutamatergic synaptic transmission

was first demonstrated in vitro (Evans et a l, 1979; Davies & Watkins, 1979) and then in vivo

(Salt, 1986). Recently, their vital role in brain functioning and survival has been evidenced by

studies showing that mice lacking N R l or NB2B subunits die soon after birth (Li et a l, 1994;

Forrest et a l, 1994; Kutsuwada et a l, 1996). More recently, pure NM DA receptor-mediated

synaptic transmission has been described during development of the hippocampus (Durand et

a l, 1996), somatosensory cortex (Isaac et a l, 1997), visual system (Wu et a l, 1996) and spinal

cord (Li & Zhuo, 1998) where their activation seem to play a fundamental role in the

formation of AMPA receptor-mediated synaptic transmission (Durand et al., 1996; Wu et al.,

1996) and in controlling neuronal cell migration (Komuro & Rakic, 1993),

NM D A receptors are widely distributed in the brain where they can be found in

nearly every single nerve cell during particular stages of brain development (Moriyoshi et al.,

1991). In central glutamatergic synapses, NM DA receptors are thought to be in a “primed”

state by the presence of glycine in the extracellular space, although its actual concentration is

still unknown (reviewed by Ascher & Johnson, 1994). Upon depolarisation of the

postsynaptic neuron, the voltage-dependent block of the receptor by extracellular Mg^ is

relieved (Nowak et a l, 1984; Mayer et a l, 1984) allowing the binding of synaptically released

31

glutamate to lead to NM DA channel opening and initiation of an excitatory postsynaptic

current (EPSCs) which lasts hundreds of milliseconds (reviewed by Lester et a l , 1994). These

long duration NM D A receptor-mediated EPSCs allow Ca influx and temporal summation

of fast non-NM DA receptor-mediated synaptic currents playing a fundamental role in long

term strengthening of synapses (reviewed by Bliss & Collingridge, 1993) and synchronisation

of neuronal firing (reviewed by Alford & Brodin, 1994).

1.4 Molecular biology o f NM DA receptors

The first cDN A coding for a NM DA receptor subunit isolated from rat brain by

expression cloning techniques was termed NM DARl or N R l. Its expression in Xenopus oocytes

produced functional receptors with many of the properties of NM DA receptors (Moriyoshi et

a i, 1991). From this subunit four new subunits were isolated and grouped into a new subfamily

termed NMDAR2 or NR2 and composed of the subunits: NR2A, NR2B, NR2C and NR2D

(Monyer et a l, 1992; Ishii et a l, 1993). More recently, chi ( % ) or NMDAR-L subunits have been

classified as NM DA receptor subunits and grouped into the NMDAR3 or NR3 subfamily (Das

e ta l, 1998)

1.4.1 N R l gene fam ily

N R l is the most highly expressed gene of all ionotropic glutamate subunits, its

expression can be detected in nearly every neuron in the brain (Moriyoshi et a l, 1991), where

its product, the N R l subunit seems to be indispensable for the assembly, expression and

functioning of NM DA receptors. The N R l gene has been mapped to chromosome 3 in rat

(Kuramoto et a l, 1994) and 9q34 in human (Takano et a l, 1993). Upon expression, N R l

subunits produce functional homomeric assemblies of NM DA receptor-channels with many

of the biophysical and pharmacological properties of native NM DA receptors, providing the

receptor with properties such as: glutamate and glycine sensitivity, antagonist sensitivity, Ca

permeability and voltage-dependent block by Mg^ (reviewed by McBain & Mayer, 1994;

Nakanishi & Masu, 1994). Soon after the rat N R l subunit was cloned (Moriyoshi et a l, 1991),

its mouse (Yamazaki e ta l , 1992), human (Foldes e ta l , 1993; Karp et a l, 1993) and Drosophila

(Ultsch e ta l , 1993) homologues were cloned.

Nine N R l splice variants have been identified (reviewed by Hollman & Heinemann,

1994). They are produced by alternative splicing of exons 5, 21 and 22 (Table 1.2) and all can

be detected in rat brain where they show regional specificity and developmental regulation

(reviewed by Goiter et a l, 1997). All of them, except the truncated form (NRT5), are able to

form functional homomeric NM DA receptors when expressed in Xenopus oocytes. Splicing of

32

T a b l e 1.2 Classification o f N R l splice variants

Nomenclature^ Description

Sugihara et al. Hollmann et al.(1992) (1993)

N R l A N R l- la 1 No deletions at COOH terminal(Cl, SL, NRloii) a Absence of exon 5 or N1 cassette (21 amino acids/NHj terminal)

N R IB N R l- lb 1 No deletions at COOH terminal(LL, NRl 1 1J b Presence of exon 5 or N1 cassette (21 amino acids/NH; terminal)

N R IC N R l-2a 2 Deletion of exon 21 or Cl cassette (37 amino acids/COOH terminal)(Cl-2 , SS, N R I q o i ) a Absence of N1 cassette

N R IF N R l-2b 2 Deletion of exon 2 1 or Cl cassette (37 amino acids/COOH terminal)(NRILS, NRlioi) b Presence of N1 cassette

N R ID N R l-3a 3 Partial deletion of exon 2 2 or C2 cassette (38 amino acids/COOH terminal)( NRIqiq) a Absence of N1 cassette

NRl-3b^ 3 Partial deletion of exon 2 2 or C2 cassette (38 amino acids/COOH terminal)(NRliio) b Presence of N1 cassette

N R IE N R l-4a 4 Deletion of Cl cassette plus partial deletion of C2 cassette(Ic, NRIqoq) a Absence of N1 cassette

N R l G N R l-4b 4 Deletion of Cl cassette plus partial deletion of C2 cassette(NRIioo) b Presence of N1 cassette

N R l-5 5 Generated by use of splice acceptor-donor sites located between exons2 and exon 4. When these sites are used, a stop codon at nucleotide 151of exon 3 terminates transcription of the remaining 3'-sequence producing a polypeptide of only 181 amino acids which is inactive upon expression in Xenopusoocytes^

Names from alternative nomenclatures are given in parentheses (reviewed by Gorter et al.j 1997).Êngineered by Hollmann et al. (1993). ^Sugihara et al. (1992)

33

the N R l subunit seems to play an important role in targeting of NM D A receptors and proton

sensitivity (Ehlers et al. 1995; Traynelis etal.^ 1995).

1.4.2 N R 2 gene fam ily

The NR2 family of NM DA receptor subunits consist of four distinct sequence-related

members termed NR2A, NR2B, NR2C and NR2D (Monyer et a l, 1992; Ishii et a l, 1993).

They show a 40 to 50 % overall amino acid sequence identity. They do not form functional

N M D A receptors when expressed alone but when coexpressed with the N R l subunit. As

N R l, they carry an asparagine residue at the pore forming region that is important in

regulating voltage-dependent Mg^ block. Compared with other glutamate receptors subunits,

they have the longest carboxy-terminal (> 500 amino acids). At their cytoplasmic tail, they

have a conserved SXV sequence (where S is serine, X is any amino acid, and V is valine) that

interacts with PSD-95, a postsynaptic anchoring protein (Kornau et a l, 1995). Their

expression is highly regulated during development and subunit segregation is observed

between brain regions (reviewed by Watanabe, 1997). NR2 subunits confer diversity to the

functional and pharmacological properties of NM DA receptors. They modulate properties

such as strength of Mg^ block (Monyer et a l, 1992), glycine sensitivity (Kutsuwada et a/.,

1992; Stern, et a l, 1992), MK801 sensitivity (Yamakura et a l, 1993), polyamine sensitivity

(Williams, 1997), kinetics of deactivation (Monyer et a l, 1992; Monyer, et a l , 1994) and single

channel properties (Stern e ta l , 1992; Wyllie e ta l , 1996).

1.4.2.1 N R 2A suhunit

The NR2A subunit is composed of 1445 amino acids (Monyer et a l, 1992). Its

predicted molecular weight is around 163 kDa (Hollmann, 1997). The NR2A gene has been

mapped to chromosome 10 in rat (Kuramoto et a l, 1994) and 16pl3 in human (Takano et a l,

1993; Kalsi et a l, 1998). Its brain expression is developmentally regulated, it is highly expressed

in forebrain structures only during postnatal and adult stages (Watanabe et a l, 1992; Akazawa

et a l, 1994; Monyer et a l, 1994). Mice lacking the e l (rat NR2A) subunit show apparently

normal growth, mating behaviour, but an enhanced startle response and reduced hippocampal

LTP and spatial learning (Sakimura e ta l , 1995).

1.4.2.2 NR2B suhunit

It is composed of 1456 amino acids (Monyer et a l, 1992). Its predicted molecular

weight is also around 163 kDa (Hollmann, 1997). The NR2B gene has been mapped to

chromosome 4 in rat (Kuramoto et a l, 1994). NR2B m RNA is widely expressed in the whole

34

embryonic brain and restricted to telencephalic (cortex, olfactory bulb and hippocampus) and

diencephalic (thalamus) structures during postnatal stages and adulthood (Watanabe et a l,

1992; Akazawa et a i, 1994; Monyer et al^ 1994). Mice lacking the z2 (rat NR2B) subunit die

soon after birth showing impairment of suckling response, trigeminal neuronal pattern

formation and hippocampal LTD (Kutsuwada e ta l , 1996).

1.4.2.3 N R 2C suhunit

It is composed of 1218 amino acids (Monyer et a l, 1992). Its predicted molecular

weight is around 133 kDa (Hollmann, 1997). The NR2C gene has been mapped to

chromosome 10 in rat (Kuramoto et a l, 1994) and 17q24-q25 in human (Takano et a l, 1993;

Kalsi et a l, 1998). NR2C m RNA is mainly absent from the embryonic brain and highly

expressed in the adult cerebellum (Watanabe et a l, 1992; Akazawa et a l, 1994; Monyer et a l,

1994). Mice lacking the 83 (NR2C) subunit show apparently normal development and

behaviour (Ebralidze e ta l , 1996; Kadotani e ta l , 1996).

1.4.2.4 N R 2D suhunit

It is composed of 1333 amino acids (Ishii et a l, 1993). Its molecular weight is around

143 kDa (Hollmann, 1997). The NR2D gene has been localised to chromosome 1 in rat

(Kuramoto et a l, 1994) and 19ql3 in human (Kalsi et a l, 1998). There are apparently two

splice variants termed NR2D-1 and NR2D-2 (Ishii et a l, 1993). Expression of NR2D m RNA

is high in the embryonic brain and declines during postnatal development and adulthood

(Watanabe et a l, 1992; Akazawa e ta l , 1994; Monyer e ta l , 1994). Mice lacking the s4 (NR2D)

show apparently normal growth and mating behaviour, but reduced spontaneous behavioural

activity (Ikeda e ta l , 1995).

1.4.3 N R 3 gene fam ily ( xsuhunits )

The x-1 subunit show 27 % and 24 % amino acid sequence identity with N M D A and

non-NM DA receptor subunits, respectively (Ciabarra et a l, 1995; Sucher et a l , 1995). They

consist of two sequence-related subunits termed chil (%-l) and chi2 (%-2 ) (Sevarino et a l,

1996). When expressed alone, %-l subunits do not form functional ion channels in vitro. When

co-expressed with NR2B, NR2D, GluRl, GluR6, KA-1 or Ô2 receptor subunits, the %-l

subunit either fails to generate agonist-activated currents or to affect the current generated by

the coexpressed subunit alone. In contrast, it markedly reduces currents mediated by

homomeric N R l or heteromeric NR1/NR2B and N R 1/N R 2D recombinant NM DA

receptors suggesting that chi (%) subunits interact with NM DA receptors. Because of the

35

presence of a genomically encoded arginine residue in its putative pore region (Ciabarra et a l,

1995; Sucher et a i, 1995) NM DA receptors containing the chil (%-l) subunit show a low

permeability to Ca ions (Das et al., 1998), Chil (%-l) subunit-deficient mice have neurons

with an abnormal dendritic morphology (Takasu et a i, 1997) and increased NMDA-evoked

responses (Rothe e ta l , 1997).

1.5 Expression o f NM DA receptor subunits in hippocampus and striatum o f 0-day-old rats

It is now known that during embryonic and early postnatal development,

glutamatergic synaptic transmission at hippocampal synapses is mediated only by NM DA

receptors (Durand et a i, 1996). Similar evidence has been obtained from thalamocortical (Isaac

et a l, 1997), retino-tectal (Wu et a l, 1997) and spinal (Li & Zhou, 1998) synapses. There is no

evidence yet of ‘pure’ NM DA receptor-mediated synaptic transmission in rat neostriatum.

Subtypes of NM D A receptors composed of different combinations of N M D A receptor

subunits are thought to be present in these brain structures (reviewed by Watanabe, 1997),

1.5.1 N R l suhunit

At PO, m RNA encoding N R la splice variants (lacking N1 cassette) are highly

expressed in dentate gyrus and striatum while very low levels of expression of N R lb splice

variant mRNAs (containing N1 cassette) are observed (Laurie & Seeburg, 1994).

At PO, NR l-1 (containing C l and C l cassettes) and NR 1-2 (lacking C l cassette,

containing C2 cassette) splice variants show also high levels of expression in dentate gyrus and

striatum. NR l-3 splice variants (containing C l cassette, lacking C2 cassette) are expressed at

very low levels in all hippocampal cells and striatum throughout postnatal development.

N R 1-4 splice variants (lacking C l and C2 cassettes) are only weakly expressed until P21, when

expression becomes high in pyramidal and granule cell layers of the hippocampus, at the same

time a very low expression is present in striatum.

1.5.2 N R 2A suhunit

At PO, expression of NR2A m RNA in the C A l region of the hippocampus has been

reported (Monyer etal., 1994; Wenzel etal., 1997). Wenzel etal. (1997) also reported very low

levels of NR2A m RNA expression in the striatum as well as very low levels NR2A protein

immunoreactivity in hippocampus and striatum. In contrast, Portera-Cailliau et al. (1996)

reported an absence of NR2A protein immunoreactivity in striatum before P16.

1.5.3 NR2B suhunit

36

At PO, the hippocampus shows one of the highest levels of expression of NR2B

m RNA and protein in the rat brain while moderate NR2B subunit expression is observed in

the striatum. Expression levels increase progressively in both structures reaching peak levels

between PIG and P21 (Monyer et al., 1994; Riva et a i, 1994; Portera-Cailliau et a l, 1996;

Wenzel e ta l , 1997).

1.5.4 N R 2C suhunit

At PO, NR2C m RNA and protein signals have not been detected in rat hippocampus

or striatum (Pollard e ta l , 1993; Monyer e ta l , 1994; Wenzel e ta l , 1997),

1.5.5 N R 2D suhunit

At PO, NR2D m RNA and protein are present at low levels in hippocampus (Monyer

et a l, 1994; Wenzel et a l, 1996, 1997) while NR2D immunoreactivity is reported to be absent

in PO-striatum (Wenzel e ta l , 1996),

1.5.6 N R 3A or X 'l suhunit

At PI, Ciabarra et a l, (1995) reported abundant %-l transcripts in the C A l field of the

rat hippocampus; in contrast, Sucher et a l, (1995) reported no hybridization in the dentate

gyrus and CA fields of rat hippocampus, %-l transcripts have not been detected in PI striatum

(Ciabarra et , 1995; Sucher ef ^/,, 1995),

In summary, NM DA receptors in PO dentate gyrus could potentially contain the N R l

splice variants: N R l-la , NRl-2a and NRl-4a (all lacking the N1 cassette encoded by exon 5)

plus two different NR2 subunits: NR2B and/or NR2D; while in PO striatum NM DA

receptors can potentially contain N R l-la , NRl-2a and NR2B subunits

1.6 Suhunit composition o f NM DA receptors

Expression of NR2 subunits alone does not produce functional NM DA receptors and

responses produced by expression of homomeric N R l subunits in oocytes are nearly 100

times smaller than those obtained when pairs of N R l and NR2 subunits are co-expressed

(Monyer et a l, 1992), It has therefore been proposed that native NM DA receptor-channels

occur as hetero-oligomeric assemblies of N R l and NR2 subunits. The exact subunit

composition and stoichiometry of native NM DA receptors is not yet known,

Electrophysiological studies using recombinant NM D A receptors were first to show

that co-expression of N R l and NR2 subunits was necessary to produce native-like

37

recombinant NM D A receptors (Monyer et al. 1992; Kutsuwada et al. 1992; Stern et al. y 1992;

Wyllie et a i, 1996). Immunobiochemical evidence of association between N R l and NR2

subunits came from studies in which subunit-selective antibodies were used to precipitate

complexes containing N R l and NR2 subunits. In HEK 293 cells transfected with N R l and

NR2 subunits, Cik et al. (1993) and Chazot et al. (1994) showed immunoprécipitation of

N R 1/N R 2A and N R 1/N R 2C subunit associations. In rat cortex, Sheng et al. (1994) showed

immunoprécipitation of N R 1/N R 2A as well as NR1/NR2B subunit associations. In

cerebellum, Didier et al. (1995) showed immunoprécipitation of e2/Çl (NR2B/NR1) and

s3/Ç l (NR2C/NR1) subunit associations. More recently, Muller et al. (1996) and Lau et al.

(1996) have shown, using antibodies against N R l, inmunoprecipitation of NR 1/N R2B

complexes associated with SAP 102, an anchoring protein present in postsynaptic densities in

rat brain. N R 1/N R 2D subunits associations also seem to be present in rat brain and in HEK

293 cells transiently transfected with N R l and NR2D subunits (Dunah et al.y 1998; A.W.

Dunah, personal communication).

Together with evidence of association between N R l and NR2 subunits, evidence of

association between NR2 subunits has also been described. In rat cortex, Sheng et al. (1994)

reported NR2A/NR2B subunit association. Chazot et al. (1994) showed N R 2C /N R 2A

subunit association in HEK 293 cells transfected with NR2A and NR2C subunits. In

cerebellum, Didier et al. (1995) reported 62/El (NR2B/NR2A) and s2/s3 (NR2B/NR2C)

subunit associations. NR2 subunit associations containing NR2D subunits have not yet been

reported. Given that co-expression of NR2 subunits alone does not produce functional

recombinant NM DA receptors, the presence in rat brain of subunit associations containing

two different NR2 subunits has been interpreted as an indication that native NM DA

receptors can contain three different subunits. Indirect evidence of native N M D A receptors

composed of N R 1/N R 2A /N R 2B and N R 1/N R 2A /N R 2C has been reported (Sheng et al.,

1994; Didier et a l, 1995; Luo et a l, 1997). Evidence of native NM DA receptors composed of

N R 1/N R 2A /N R 2D and N R 1/N R 2B /N R 2D heteromers has also been found (Dunah et al.,

1998).

Recently, Blahos & Wenthold (1996) reported the presence in adult rat forebrain of

NR2A/NR2B subunit associations, but in much lower proportion than N R 1/N R 2A or

NR 1/N R2B subunit associations. They suggested that the fraction of NR2A and NR2B

subunits present in a single receptor complex is probably very small and that most NM DA

receptors in the adult rat forebrain contain only one type of NR2 subunit. In agreement with

this report, Chazot & Stephenson (1997) have identified N R 1/N R 2A /N R 2B NM DA

receptors as a minor subpopulation compared with N R 1/N R 2A and N R 1/N R 2B receptors.

38

In contrast with these reports, Luo et al. (1997) have shown that the majority of native

NM D A receptors in adult rat cerebral cortex are the result of a combination of at least three

different subunits (NR1/NR2A/NR2B) while native NM DA receptors composed of two

different subunits (NR1/NR2B or NR1/NR2A) are present in a much lower proportion. In

summary, native NM DA receptors containing three different subunits seem to be present in

rat brain but evidence regarding the proportion in which they are present is still controversial.

1.6.1 Factors regulating NM DA receptor subunit composition

It has been suggested that the number of potential subunit combinations that can be

found in neurones could be restricted by processes of preferential assembly and differential

expression of NM DA receptor subunits.

1.6.1.1 Preferential assembly between N R l and N R 2 subunits

Sheng et al. (1994) were the first to suggest preferential assembly between N R l splice

variants and NR2 subunits. They showed that NR2A and NR2B subunits were preferentially

associated with N R l^x splice variants (containing exons 5 and 21) rather than with NRliox

splice variants (containing exon 5 but lacking exon 21). At the same time, N R 2B/N R lxix

(containing exon 21) subunit associations were shown to be more abundant than

N R 2B/N R lixx (containing exon 5) subunit associations. N o differences were observed

between associations of N R l splice variants and NR2A subunits (Sheng et al. 1994). In

contrast, a recent study found no evidence of preferential association between N R l splice

variants and NR2A or NR2B subunits (Blahos & Wenthold, 1996). Until more evidence

becomes available, the role of preferential assembly in determining the subunit composition

of NM DA receptors remains unclear.

1.6.1.2 Differential expression of N R l and N R 2 subunits

Recent evidence suggests that differencial expression of NM DA receptor subunits

may depend on subunit-specific or splice variant-specific interactions between N R l and NR2

subunits and cytoskeletal proteins. Currently, two mechanisms of interaction have been

described. One mediated via the alternatively spliced C l cassette encoded by exon 21 that is

present in NR l-1 and N R 1-3 splice variants and another one mediated via a SXV sequence

(where S is serine, X is any amino acid, and V is valine) present at the cytoplasmic tail of

NRl-3 (lacking C2), NRT4 (lacking C l and C2), NR2A, NR2B, NR2C and NR2D subunits

(Kornau et al. 1995). The mechanism involving the C l cassette is still unclear. A recent study

reported an interaction with neuronal intermediate filaments (Ehlers et a i, 1998) while

39

another study reported an interaction with a novel cytoskeleton-associated protein found in

synapses and neuromuscular junctions termed Yotiao (Lin et a l, 1998). The SXV sequence-

dependent mechanism has been shown to be mediated by an interaction with PSD-95, a

postsynaptic protein that co-localises with NM DA receptors in cultured hippocampal neurons

(Kornau etal., 1995). These subunit-specific and splice variant-specific interactions are thought

to be involved in the differential locaHsation of distinct NM DA receptor subtypes, which

could create glutamatergic synapses with distinct physiological and pharmacological

properties even within a single neuron (Rubio & Wenthold, 1997).

In this particular study, the possibility of neurons expressing native N M D A receptors

containing the two highly expressed N R l splice variants NRl-1 and N R l-2 plus the NR2B

and/or the NR2D subunits will be considered. In PO hippocampus, native N M D A receptors

subtypes could potentially be found with subunit compositions such as NR1/NR2B,

N R 1/N R 2D and N R 1/N R 2B /N R 2D plus potential subunit combinations containing

different N R l splice variants. In PO striatum, a simpler situation is found in which subtypes of

NM DA receptors can be the result of subunit combinations such as NR 1/N R2B plus those

produced by differential assembly with different N R l splices variants.

1.7 Suhunit stoichiometry o f NM DA receptors

The exact number of N R l and NR2 subunits present in a single native or

recombinant NM DA receptor is not yet known and it is currently a source of controversy.

Behe et at. (1995) reported that a single recombinant NM DA receptor contains two N R l

subunits and probably three NR2 subunits. Premkumar & Auerbach (1997) have shown

evidence that suggests the presence of at least three (NRl) subunits and two s (NR2)

subunits in a single recombinant NM DA receptor. More recently, Laube et at. (1998) have

reported evidence that suggests a tetrameric structure for recombinant N M D A receptors.

1.8 Properties o f recomhinant NM DA receptors

The functional and pharmacological properties of recombinant NM DA receptors are

determined by their subunit composition (reviews in Monaghan & Wenthold, 1997).

1.8.1 Functional properties

1.8.1.1 Macroscopic currents

Activation, deactivation and desensitisation kinetics are the functional properties of

recombinant NM DA receptors that are more commonly studied at the macroscopic level

(Monyer etal., 1992; Wyllie etal., 1997; Vicini etal., 1998).

40

1.8.1.1.1 N R 1/N R 2A receptors

They show desensitisation when activated by glutamate or NM DA (Monyer et a l,

1994; Vicini et a l, 1998). Deactivation time constants from one or two exponential

components have been reported: 120 ms (Monyer et al., 1994), 33 and 293 ms (Vicini et at.,

1998), 94 ms and 385 ms (Wyllie e ta l , 1997).

1.8.1.1.2 N R 1/N R 2B receptors

NR 1/N R2B receptor-mediated currents show Httle desensitisation when activated by

glutamate or NM DA (Monyer et a l, 1994; Vicini et a l, 1998). Deactivation time constants

from one or two exponential components have also been reported: 400 ms (Monyer et a l,

1994), 71 and 538 ms (Vicini e ta l , 1998).

1.8.1.1.3 N R 1 /N R 2 C receptors

N R 1/N R 2C receptor-mediated currents do not show desensitisation when activated

by glutamate or NM DA and have a slow deactivation time constant of 380 ms (Monyer et a l,

1992; 1994).

1.8.1.1.4 N R 1/N R 2D receptors

N R 1/N R 2D receptor-mediated currents also show no desensitisation when activated

by glutamate or NM DA. N R 1/N R 2D recombinant NM DA receptor-mediated currents show

the slowest deactivation time constants of all N R 1/N R 2 subunit combinations: 4800 ms

(Monyer e ta l , 1994) and 4469 ± 351 ms (Wyllie e ta l , 1997).

1.8.1.2 Single-channel currents

The single-channel properties of recombinant N R 1/N R 2 NM DA receptors that have

been reported so far are single channel conductances, open times and shut times (Stern et a l ,

1992, 1994; Wyllie e ta l, 1996).

The single channel properties of native and recombinant NMDA-receptor channels have

been shown to be strongly affected by the presence of NR2 subunits; while the effects of N R l

splice variants on single channel properties have not been studied (Stern e ta l, 1992, 1993, 1994;

Wyllie et a l, 1996; reviewed by Feldmeyer & Cull-Candy, 1996). Even though expression of

any N R l splice variants, in Xenopus oocytes, produces functional homomeric NM DA

receptors, their single channel properties are not yet known. In contrast, more detailed studies

on the single channel properties of heterodimeric N R 1/N R 2 recombinant N M D A receptors

have been carried out producing a working classification that divides recombinant NM DA

41

receptors in two groups: high conductance and low conductance NM DA channels (reviewed

by Stern & Colquhoun, 1998).

1.8.1.2.1 High and low conductance NM DA channels

The multiplicity of single channel conductance levels observed in NM DA receptors in the

brain is thought to be an expression of differences in NM DA receptor subunit composition

(reviewed by Feldmeyer & Cull-Candy, 1996). Based on their single channel conductance

recombinant heterodimeric NM DA receptors can be grouped into two categories: high

conductance and low conductance NM DA channels. N R 1/N R 2A and NR 1/N R2B

recombinant NM DA receptors produce “high conductance” channels (Stern et al. 1992, 1994)

and N R 1/N R 2C and N R 1/N R 2D recombinants NM DA receptors produce “low

conductance” channels (Stern et al. 1992; WyUie et aly 1996). Recombinant “high conductance”

(NR1/NR2A and NR1/NR2B) NM DA channels have a main conductance level around 50 pS

and a subconductance level around 40 pS while recombinant “low conductance” (NR1/NR2C

and NR1/NR2D) NM DA channels have a main conductance level around 31-35 pS and a

subconductance level around 17 pS (Table 1.3).

1.8.1.2.2 Shut times

Mean time constants and relative areas for the three fastest components of the shut

time distribution have only been reported for N R 1/N R 2A recombinant N M D A receptors

(Stern et a i, 1994). Values from NR1/NR2B and NR 1/N R 2C at -60 mV (Behe & Colquhoun,

personal communication) and from N R 1/N R 2D at -100 mV (Wyllie & Colquhoun, personal

communication) have not yet been published (Table 1.4).

1.8.1.2.3 Open times

The distribution of open times is also dependent on which NR2 subunit is

coexpressed with the N R l subunit. Effects of N R l splice variants on the distribution of open

times have not yet been reported. As evidenced by the number of exponential components in

the open time distribution, “High conductance” (NR1/NR2A and NR1/NR2B) NM DA

channels can exist in at least three different open states (three exponential components) while

“low conductance” (NR1/NR2C and NR 1/NR2D) can exist in at least two open states.

“High conductance” (NR1/NR2A and NR1/NR2B) NM DA channels have longer mean open

times than “low conductance” (NR1/NR2C and NR1/NR2D) NM DA channels (Table 1.5).

1.8.1.2.4 Open times conditional on amplitude

42

At least two different single-channel conductance levels have been identified in all

N R 1/N R 2 subunit combinations, a main level and a sublevel. The mean open time of the

main conductance level is longer in “high conductance” than in “low conductance” channels

while the mean open time of the subconductance level is shorter in “high conductance” than

in “low conductance” NM DA channels (Table 1.6)

T a b le 1.3 Single channel conductance o fN R l/N R 2 recomhinant N M D A receptors

Sub level Main level

conductance area conductance area Sub/Main

(pS) (%) (pS) (%) ratio

High conductance

N R 1/N R 2A ' 38.3 ± 1.3 20.1 ±2.9 50.1 ± 1.4 79.4 ± 2.9 0.76

NR1/NR2B' 38.7 ±1.5 16.9 ±2.9 50.9 ± 0.4 83.1 ± 2 .9 0.76

Low conductance

NR1/NR2C''" 17.9 ± 0.8 25.5 ± 1.2 30.6 ± 0.8 74.5 ± 1.2 0.58

NR1/NR2D" 17.0 ± 2.0 39.0 ± 2.0 35.0 ±2.0 61.0 ± 2 .0 0.49

^Stem e ta l (1992), ^Wyllie etal. (1996).

T a b le 1.4 Time constants and relative areas o f first three shut tim e components for

N R 1/N R 2 recombinant NM DA receptors

^2

(ps) (ms) (ms)

N R 1/N R 2A ' 62 ± 1 0.57 ±0.1 7.0 ± 1.5(28 ± 3 %) (30 ± 4 %) (15 ± 3 %)

NR1/NR2B" 54 ± 1 0.65 ±0.1 8.8 ± 1.3(34 ± 4 %) (23 ± 2 %) (11 ± 2 %)

NR1/NR2C^ 117 ±50 0.66 ±0.1 11.9 ± 1.3(24 ± 2 %) (11 ±4% ) (34 ± 1 %)

NR1/NR2D" 55 ± 6 0.35 ±0.1 6.5 ± 0.7(42 ± 5 %) (19 ± 2 %) (9 ± 1 %)

^Stern et al. (1994), ^Behe & Colquhoun, personal communication (-60 mV), ^Wyllie &Colquhoun, personal communication (-100 mV). Relative areas are :shown in parentheses.

43

T a b l e 1.5 Open times including all amplitude levels in N R 1 /N R 2 recomhinant NM DA

receptors

'Cl 'C2 'C3mean

(ps) (ms) (ms) (ms)

N R 1/N R 2A ’ 67 ± 1 (30 ± 5 %)

1.64 ± 0.4 (40 ± 7 %)

4.27 ± 0.7 (30 ± 4 %)

1.96

NR1/NR2B" 183 ± 300 (23 ± 5 %)

1.83 ±0.3 (41 ± 7 %)

4.99 ±0.1 (36 ± 8 %)

2.59

N R 1/N R 2C ’'" 490 ± 100 (50 ± 8 %)

1.19 ±0.1 (50 ± 8 %)

0.84

NR1/NR2D" 74 + 100 (28 ± 2 %)

1.54 ±0.1 (72 ± 3 %)

0.94

’Stern etal. (1993), ^Wyllie etal. (1996). Relative areas are shown in parentheses.

T a b l e 1.6 Open times conditional on unitary current am plitude in N R 1 /N R 2 recomhinant

NM DA receptors

Subunit composition Conductance levels Mean open times

(pS) (ms)

N R 1/N R 2A ’ 38.3 0.61 ± 0.0550.1 2.70 ± 0.70

N R 1/N R 2B’ 38.7 0.59 ± 0.0750.9 2.80 ±0.30

NR1/NR2C" 17.9 0.91 ± 0.0830.6 1.10 ±0.14

NR1/NR2D" 17.0 1.28 ±0.0635.0 1.01 ± 0.04

’Sterner^/. (1992), ^WyUie etal. (1996).

1.8.2 Pharmacological properties

1.8.2.1 Effects o f spermine

Spermine has inhibitory and stimulatory effects on NM DA receptors that depend on

both N R l subunit splice variants and NR2 subunits (reviewed by Williams, 1997).

44

1.8.2.1.1 Inhibitory effects

Spermine inhibitory effects are thought to be due to two different mechanisms:

reduction of receptor affinity for agonists (Williams, 1994) and voltage-dependent inhibition

(Williams, 1994; Williams etal., 1994).

A. Reduction in receptor affinity for agonists

Reduction in the receptor affinity for agonists is regulated by NR2 subunits. When

N R la subunits are co-expressed with NR2A, NR2C or NR 2 D subunits reduction in the

receptor affinity for agonists is abolished, but not in those receptors containing NR2B

subunits (Williams, 1994). Regulation by N R l subunit splice variants has not been reported.

B. Voltage-dependent inhibition

Voltage-dependent inhibition is also regulated by NR2 subunits. When either N R la

or N R lb subunits are co-expressed with NR2C or NR2D subunits voltage-dependent

inhibition by spermine is abolished (Williams et at.., 1994; Williams, 1995) but not when N R la

or N R lb subunits are co-expressed with NR2A or NR2B subunits (Williams etal.^ 1994).

1 .8 .2 . 1 . 2 Stimulatory Effects

Polyamine-mediated stimulatory effects can be either glycine-dependent or glycine-

independent. In native NM DA receptors, glycine-independent stimulation involves an

increase in the frequency of channel opening (Rock & Macdonald, 1991) and a decrease in

receptor desensitisation (Lerma, 1992) while glycine-dependent stimulation involves an

increase in NM DA receptor affinity for glycine (McGurk et al. 1990; Benveniste & Mayer,

1993).

A. Glycine-independent stimulation

In recombinant NM DA receptors, glycine-independent stimulation by spermine is

regulated by both N R l subunit splice variants (Durand et al. 1992; 1993) and NR2 subunits

(Zhang e ta l , 1994; Williams, 1994; Williams e ta l , 1994; Williams, 1995). In homomeric N R l

receptors, N R la but not N R lb splice variants show glycine independent stimulation

(Traynelis et al , 1995). When the N R la splice variant is co-expressed with NR2A, NR2C and

NR2D but not NR2B subunits, glycine-independent stimulation by spermine is abolished.

When N R lb splice variant is co-expressed with NR2B subunits, glycine-independent

stimulation by spermine is also abolished (Williams e ta l , 1994).

45

B. Glycine-dependent stimulation

Glycine-dependent stimulation by spermine is regulated only by NR2 subunits

(Zhang et a i, 1994; Williams et a l, 1994; Williams, 1995). When either N R la or N R lb

subunits are co-expressed with NR2C (Zhang etal.^ 1994) or NR2D (Williams, 1995) subunits,

glycine-dependent stimulation by spermine is abolished, but not in those receptors containing

N R 2 A or NR2B subunits. (Zhang etal.^ 1994; Williams etal., 1994).

1.8.2.2. Effects ofifenprodil

NR2 subunits have been shown to regulate the inhibitory effects of the non

competitive antagonist ifenprodil on recombinant NM DA receptors. The mechanism

responsible for the subunit-specificity of ifenprodil is not yet known (Gallagher et al., 1996).

Ifenprodil inhibits responses mediated by homomeric N R l receptors with an IC 5 0 of 0.28 pM

(Williams et al., 1993). When N R l and NR2B subunits are co-expressed, IC5 0 values do not

change significantly, 0.27 - 0.34 pM (Williams, 1993; Avenet et al., 1997, Whittemore et al.,

1997). In contrast, when N R l and N R 2 A or NR2C subunits are co-expressed, a 300-fold

increase in IG5 0 is observed, 130 - 145 pM (Williams, 1993; Whittemore et al., 1997). Co

expression of N R l and NR2D subunits abolishes inhibition by ifenprodil (Williams, 1995).

1.9 Experiments conducted in this study

The experiments conducted in this study were aimed firstly, to describe the single

channel properties of NM DA receptor-channels in hippocampal granule cells and striatal

neurons from rats at postnatal day 0 (day of birth) and secondly, to analyse some of the reported

subunit selective effects of spermine and ifenprodil on their single-channel properties.

Techniques of patch-clamp (Hamill et al., 1981) and single channel analysis

(Colquhoun & Sigworth, 1995) were used to record and measure the single-channel properties

of NM DA receptors in outside-out patches from neurons in brain slices. In order to allow

some degree of comparison, single-channel recordings were carried out under ionic conditions

similar to those used by Stern et al. (1992, 1994) and Wyllie et al. (1996) in their studies of the

single-channel properties of recombinant NM DA receptors.

When possible, a detailed analysis of the single channel properties, similar to that

found in Gibb & Colquhoun (1992) was carried out. Additionally, an analysis of aligned

NM DA receptor activations (clusters and super-clusters) similar to that found in Edmonds &

Colquhoun (1992) was also carried out.

46

CHAPTER 2

Materials and Methods

2.1 Solutions

2 . 1 . 1 Slicing solution

A modified Krebs solution (Edwards etal.^ 1989) with the following composition was

used for slicing (in mM): NaCl, 125; KCl, 2.5; CaClz, 1.0; MgCl;, 4.0; NaHzPO^, 1.25;

NaHCO), 24; Glucose, 25; pH 7.4.

2.1.2 External solution for recording

The composition of the external solution used for recording was similar to that of the

slicing solution but with no added Mg^ (Nowak etal.^ 1984). The free Mg^ concentration in

this solution has been estimated to be around 4 pM (Gibb & Colquhoun, 1992). The slicing

and external solution for recording were continuously gassed with a mixture of O 2 (95%) and

CO 2 (5%) (BOG Gases, Manchester, UK). The non-NMDA receptor antagonist D N Q X (5

pM) was added to the external recording solution.

2.1.3 Internal solution for recording

In order to make chloride currents very small at the recording potential used (-60 mV),

patch pipettes were filled with a low chloride (10 mM) internal solution (Gibb & Colquhoun,

1991). The composition of the internal solution was (in mM): NaO H , 140; NaCl, 1 0 ; EGTA,

11; HEPES, 10; Gluconic acid lactone, 140; pH: 7.2. A final pH of 7.2 was obtained only after

38 - 40 mM of NaO H was added, giving a final theoretical Na" concentration of 178 -180 mM.

This solution was stored frozen in 1 ml ahquots.

2 . 2 Drugs and chemicals

NaCl, N aO H , N aH 2 p 0 4 , NaH CO j, KCl, CaCl2 , MgClj and Glucose were

purchased from BDH (Poole, England). HEPES (N-[2-Hydroxyethyl]piperazine-N’-[2-

ethanesulfonic acid]), EGTA (Ethylene Glycol-bis(|3-aminoethyl Ether) N,N,N',N'-Tetraacetic

Acid), D-Gluconic acid lactone (1,2,3,4,5-Pentahydroxycaproic acid 5-lactone), Ifenprodil (2-

(4-Benzylpiperidino)-l-(4-hydroxyphenyl)- 1-propanol), Spermine (N ,N ’-bis[3-Aminopropyl]-

1,4-butanediamine) and Glycine (Aminoacetic acid) were purchased from Sigma (St. Louis,

MO, USA). Glutamate (L-glutamic acid), N M D A (N-Methyl-D-Aspartic acid) and D N Q X

(6,7-Dinitroquinoxaline-2,3-dione) were purchased from Tocris (Bristol, UK).

47

2.3 Drug application

Before drug application, control recordings were made in the absence of agonists or

antagonists. All solutions were superfused into the recording chamber by gravity. The

exchange between solutions was made by manually switching a two way tap. When glutamate

was used as an agonist, the non-NMDA receptor antagonist D N Q X (5 ^M) was added to the

external solution. Each outside-out patch was exposed to a constant low concentration of

glutamate (50 - 100 nM) or NM DA ( 1 - 5 fiM) for periods of time that varied from 2 to 20

minutes in the presence of saturating concentrations of glycine (3 - 10 pM).

2.4 Brain slice preparation

Hippocampal slices were prepared as described elsewhere (Edwards et a i, 1989; Gibb

& Edwards, 1994). Sprague-Dawley rats less than 24 hour-old (postnatal day 0 ) were killed by

decapitation using a pair of surgical scissors (RS6930, Roboz, Germany). The head was

immediately submerged in a 100 ml plastic weighing boat (Fisher Scientific, Loughborough,

UK) containing ice-cold oxygenated slicing solution. The skin was cut along the midline with

a pair of small scissors. The skull was cut along the midline with a pair of fine scissors and

skull bones were removed using fine curved forceps. The brain was exposed, cut along the

midline with a scalpel (N° 11, Swann-Morton, Sheffield, UK) and dissected out using a fine

spatula. Immediately, both brain halves were transferred to a 100 ml plastic weighing boat

previously filled with ice-cold oxygenated slicing solution and continuously bubbled with a

mixture of O 2 (95%) and CO 2 (5%). Both brain halves were allowed to cool down for 3-5

minutes while meninges were carefully removed using fine watchmaker-type forceps (#3,

Ideal-tek, Switzerland). Meninges were removed to reduce brain damage during the slicing

procedure.

For horizontal hippocampal slices, a flat surface was cut across the dorsal side of both

brain halves with a scalpel. This surface was glued, using cyanoacrylate adhesive (RS

Components, Corby, UK), to the tissue block of the specimen bath of a vibroslicer (Vibroslice

752, Campden Instruments LTD, Loughborough, UK) with the lateral side of the brain facing

the cutting blade. For parasagital striatal slices, the medial side of a brain half was glued to the

tissue block of the specimen bath with the dorsal side of the brain facing the cutting blade.

Immediately, the specimen bath was filled with oxygenated ice-cold slicing solution until the

tissue was completely covered. SUces were cut using carbon steel blades (Campden

Instruments LTD, Loughborough, UK) at a thickness of 250 |im. Long hypodermic needles

(Monoject, Ballymoney, UK) were used to dissect out the hippocampal formation and the

48

striatum from the rest of brain slice. Slices were transferred into an incubation chamber

(Edwards & Konnerth, 1992) using a Pasteur pipette (John Poulten Ltd., Barking, UK) cut and

fired polished to a tip opening of 3-5 mm across. The incubation chamber containing standard

slicing solution was kept in a water bath at 32-34 °C and the solution was continuously

bubbled with a mixture of O 2 (95%) and CO 2 (5%). Each slice was transferred in the same way

to a recording chamber fitted to the stage of an upright microscope (Axioscope, Zeiss, Jena,

Germany).

2.5 Cell visualisation and identification

The cell bodies of individual neurons in brain slices were visualised under Normaski

differential interference contrast optics (Yamamoto, 1975; Takahashi, 1978; Edwards et al.

1989) using an upright microscope (Axioskop, Zeiss, Oberkochen, Germany) with an

Achromat 40X water immersion objective with a numerical aperture of 0.75 and a working

distance of 1.6 mm at a total magnification of 600X. Visualisation was carried out on a

monochrome video monitor (VM-902K, Hitachi-Denshi, Tokyo, Japan) connected to GGD

monochrome camera (RS Components, Corby, UK) mounted on top of the microscope

trinocular head. Slice health was visually checked before patching and the presence of a

considerable proportion of neurons with a smooth surface readily evident on the surface of

the slice was used as an indicator of a good healthy slice. Individual healthy cells were

identified by the smooth appearance of their surface. Granule cells in the granule cell layer of

the dentate gyrus were identified by their size and morphology and position in the

hippocampus and striatal cells were identified by their size.

2.6 Patch p ipette fabrication

Patch pipettes were made in a vertical pipette puller (L/M-3P-A, List-Medical,

Darmstadt, Germany or MF-83, Narishige, Tokyo, Japan) from thick-walled borosilicate glass

capillaries containing internal filament (GG150F-7.5, outer diameter 1.5 mm, inner diameter

0.86 mm, Clark Electromedical, Reading, UK). They were coated with an insulating silicon

resin (Sylgard 184®, Dow Corning, Midland, MI, USA) under a dissecting microscope using a

metal or glass hook. Coating started from about 100 pm away from the pipette tip to a

distance of several millimeters up to the pipette shoulder and it was subsequently cured by

moving the coated pipette through a heated coil connected to a variable voltage source. Patch

pipettes were stored, horizontally mounted on a strip of adhesive (Blue-Tack, Bostik,

Leicester, UK) stuck to the bottom of a plastic 90 mm Petri dish (Sterilin, Staffs, UK). The tip

of the pipette was cleaned, smoothed and reduced by fire polishing on a microforge (MF-83,

49

Narishige, Tokyo, Japan). Pipettes were back-filled with the low-chloride internal solution by

using a thin plastic pipette tip connected to a 0,2 pm pore size syringe filter (Minisart-RC4,

Sartorius, Surrey, UK,) attached to a 1 ml sterile syringe (Plastipak, Becton-Dickinson, Spain).

After back-filling, air bubbles remaining in the pipette were removed by tapping the pipette

with the finger. Internal solution level in the pipette was kept to a minimum to prevent

electrical interference produced by solution creeping up the electrode or going into the suction

line.

2.7 Patching procedure

Patch pipettes were positioned on an electrode holder connected to the head-stage of a

patch-clamp amplifier (Axopatch 200A, Axon Instruments, Foster City, CA, USA). Positive

pressure was applied through a piece of tubing (Portex Ltd, Hythe, UK ) connected to the

back of the electrode holder to generate a tiny stream of internal solution at the tip of the

pipette that prevented the accumulation of debris at the tip and the mixing of the internal

solution inside the pipette with the external solution in the recording chamber. The patch

pipette was then lowered into the recording chamber. Once in the solution, the pipette

resistance was measured by passing a 5 mV square pulse through the input using the patch

clamp amplifier. Patch pipettes usually had a final resistance of 2 0 - 30 MQ when filled with

recording internal solution. Positioning of the patch pipette was carried out under the optic

field of the microscope using a micromanipulator (Melles Griot, Cambridge, UK) and under

visual control the patch pipette was lowered further down until visual contact with the slice

was made. At that moment, a piezo-electric device (Melles-Griot, Cambridge, UK) connected

to the micromanipulator was used to approach the cell with the patch pipette. Contact

between the patch pipette and the cell was visually confirmed by the formation of a

characteristic dimple on the cell surface. Immediately, the positive pressure was released and a

high resistance gigaohm seal was formed between the cell membrane and the tip of the

electrode. The membrane under the patch pipette was held at -60 mV and suction was applied

through the tubing connected to the back of the electrode holder to break the membrane and

gain electrical access to the cell interior (Hamill et al. 1981). After the whole cell configuration

was obtained the patch electrode was very slowly withdrawn away from the cell using the

piezo-electric manipulator until an outside out patch was obtained.

Electrical noise was reduced by bringing the patch pipette towards the surface of the

bath leaving only its tip in the solution. The patch pipette was also brought toward the inlet

of the recording chamber to improve contact between the patch and the incoming solutions.

Before recording was attempted, the noise level was checked and an RMS noise level below

50

0.300 pA at a bandwidth of 5 kHz (Butterworth filter) was considered acceptable for

recording. The presence of spontaneous single channel openings was also checked and patches

with spontaneous single channel activity were discarded.

2.8 D ata acquisition and analysis

2.8.1 Detection and recording o f single channel currents

Steady state single channel activity produced by a constant low concentration of

glutamate (50-100 nM) or NM D A (1-5 pM) and a constant saturating concentration of glycine

(3-10 pM) was monitored using a patch-clamp amplifier (Axopatch 200A, Axon Instruments,

Foster City, CA, USA) in the voltage clamp configuration. Signals were filtered at a lOkHz

bandwidth and recorded using a modified Digital Audio Tape recorder (DTR 1202, Bio-Logic,

Claix, France). They were recorded and stored on magnetic tapes (Maxell, DA T 120) for off

line digitisation and analysis. Current-voltage relationships were built by holding the patches

for 2-3 minutes at potentials between -100 and -20 mV. All recordings were done at room

temperature (21-25°C).

2.8.2 Record digitisation

After experiments were carried out, tapes were played back and the signals were

filtered at a cut-off frequency of 2 kHz using a Bessel type low pass filter (-3dB, 8 -pole) to

reduce background noise. The filtered signal was then digitized at 1 0 times the cut-off

frequency of the low-pass filter (20 kHz) using an analogue-to-digital converter (CED

1401plus, Cambridge Electronics Design Ltd, Cambridge, UK) and stored on the hard disc of

a 486D computer (Dell Computer Corporation, Berkshire, UK) using a computer program for

continuous sampling (CONSAM) designed by D. Colquhoun.

2.8.3 Detection and measurement o f events in the digitised record

Each digitised record was then scanned and transitions were detected, measured and

fitted using an interactive computer program (SCAN) that carried out direct fitting of each

event time-course based on the step response of the recording system (Colquhoun &

Sigworth, 1995). Briefly, the digitised recording was displayed and scanned by scrolUng it

across the computer screen under visual inspection. Events were detected after crossing a

threshold placed close to the baseline. Once it was decided that the event could be fitted, the

program made initial guesses for the positions of all the transitions and amplitudes and

performed a least-squares fit on the basis of these guesses; finally, it displayed the fitted curve

superimposed on the digitised event. If the step-response function fitted poorly, the fit was

51

adjusted to obtain the fit that best described the event. Fits were stored as a list of values with

the amplitude and duration of each open period and the duration of each closed period.

Incompletely resolved openings had their amplitude constrained to be the same as that of the

closest opening longer than 2 filter rise-times if such an opening was present in the region of

trace being fitted, if on the contrary, there were no openings with such characteristic in the

section of trace being fitted, they were fitted as openings to the mean amplitude level. After

the record was fitted, a data file containing all the values describing the lifetime and amplitude

of all single channel events present in the record was created and stored as a computer file

which was later used during the analysis of the single-channel data.

2.9 Display and analysis o f single-channel data

Display and analysis of single-channel data was done using EKDIST, a computer

program designed by D. Colquhoun. A fixed resolution of open times and closed times was

imposed that gave a false event rate less than or equal to 10 * events per second (Colquhoun &

Sigworth, 1995). This was usually 50 jis for open and closed times. Before distributions were

built, the data was checked for stabiUty by building stability plots for amplitudes, open times,

shut times and (Weiss & Magleby, 1989).

2.9.1 Stability plots

2.9 .1 . 1 Stability plots for amplitudes

Stability plots for amplitudes were built by plotting the individual single-channel

current amplitude measurements of each opening against the interval number in which the

opening was detected. The amplitude of each single opening longer than 2 or 2.5 filter rise-

times was plotted. Each data point on the plot represented a single observation independently

of its duration.

2.9.1.2 Stability plots for open times, shut times and P pen

Under steady state conditions of temperature, voltage and agonist concentration, the

kinetic behaviour of NM DA receptor-channels can be described by a Markov process in

which the probability of channels existing in a given kinetic state (open or closed) does not

change with time (Gibb & Colquhoun, 1992). Before analysing distributions of open and

closed times, the stability of the channel activity was assessed by building stability plots which

allowed detection of changes in shut times, open times and probability of being open (Popen)

that could undermine the analysis of dwell times and amphtude distributions. Usually,

stability plots for open and shut time intervals were constructed by calculating a moving

52

average of 50 consecutive open or shut time intervals with an overlap of 25 events and plotting

this average against the interval number at the centre of the averaged values. Stability plots for

open probability (Popen) were constructed by calculating a P pea value for each set of 50 open

and shut times as total open time over total length.

Once the stability of the record was confirmed, values were sorted into bins and used

in the construction of frequency distributions histograms.

2.9.2 Distribution o f fitted amplitudes

The amplitude of channel openings can be measured accurately only if the duration is

at least twice the rise-time (T ) of the recording system (Colquhoun & Sigworth, 1995).

Frequency distribution histograms containing individual open-channel amplitudes longer than

2 filter rise-times were constructed and fitted with the sum of two to four Gaussian

components with their standard deviations constrained to be the same. The relative area

occupied by each Gaussian component represents the relative frequency of events to each

particular amplitude level rather than the relative time spent at each level. Each single channel

opening longer than 2 filter rise-times represented one observation independently of its

duration.

2.9.3 Distribution of open times and shut times

Because the duration of closed and open time intervals varied from tens of

microseconds to tens of seconds, frequency distribution histograms were constructed using a

logarithmic transformation of the abscissa (McManus et a l, 1987; Sigworth & Sine, 1987) and

a square root transformation of the ordinate (Sigworth & Sine, 1987). Distributions were

fitted using the maximum likelihood method with probability density functions that were the

sum of one or more exponential components (Colquhoun & Hawkes, 1995).

2.9.4. Distribution of open times conditional on amplitude

In order to minimise the percentage of misclassified openings present in open time

distributions conditional on amplitude, critical amplitude values, between pairs of

adjacent Gaussian components were calculated. Critical amplitude values that gave equal

percentages of misclassified events were used. The presence of openings to more than one

amplitude level makes is difficult to infer the exact amplitude of those events that had a

duration shorter than 2 filter rise-times; they appeared in the recording as incompletely

resolved openings. Such events were excluded by fitting only those bins containing events

longer than 2 filter rise-times.

53

2.9.5 Bursts

Bursts were defined as groups of openings separated by shuttings of duration less than

a critical shut time or which was calculated from the fitted parameters of the distribution

of shut times such that the 2“* and 3’' briefest exponential components of the shut time

distribution were classified as ‘gaps within bursts’. Critical shut time or r rit values were found

by numerical solution by the bisection method using EKDIST. Critical shut time or values

were calculated using three different criteria:

Colquhoun & Sakmann (1985):

A value was calculated so that equal percentages of short and long shuttings were

misclassified. Values for were calculated by solving:

e-“ ' V =

Magleby & Pallota (1983) and Clapham & Neher (1984):

A ïcrit value was calculated so that equal number of short and long shuttings were

misclassified. Values for t rit were calculated by solving:

= slow (1 - e 'cri/' slow )

Jackson etal. (1983):

A value was calculated so that the total number of events that were misclassified

was minimised. Values for were calculated by solving:

crit fast ~ (^slow/ fast) slow

Where 2n^ and a i are the areas of the two exponential components and

between which the value must lie. Values of tcrit obtained using the criterion proposed by

Colquhoun & Sakmann (1985) were usually used. Only when calculation of t rit values using

this criterion failed were alternative values used, calculated using one of the two other

different criteria.

2.9.5.1 Distribution of burst lengths

Values for t^ were calculated between the and 4**" exponential components of the

distribution of shut times. The definition of bursts was imperfect because an unambiguous

classification between shuttings ‘within bursts’ and ‘between bursts’ could not be achieved

54

because the difference between the exponential components of the distribution of shut times

was not big enough.

2.9.5.2 Distribution of total open time per burst

The distribution of the total open time per burst is much simpler than the distribution

of burst lengths because the number of exponential components is expected to be equal to the

number of open states while the effect that the presence of incompletely resolved events may

have on the determination of the number of open states is very much reduced. It is also less

sensitive to missed events than the distribution of open times.

2.9.5.3 Burst P„pg„

The burst or fraction of time during which the channel dwells in any open state

during a burst of openings was calculated for each experiment by dividing the mean total open

time per burst by the mean burst length.

2.9.6 Clusters

Activation of a single NM DA receptor produces clusters of channel openings (Gibb &

Colquhoun, 1991). Clusters of openings were defined as groups of openings separated by shut

times shorter than a critical length or For each experiment, a was calculated from the

fitted parameters of the shut time distribution, assuming the 4 exponential component of the

shut time distribution as being “within-clusters” and the S**' component as being “between-

clusters” . In each case, values were calculated so as to make the percentage of long shut

times that were misclassified as “within-clusters” equal to the percentage of short shut times

that were misclassified as “between-clusters” (Colquhoun & Sigworth, 1995). Cluster length,

total open time per cluster and cluster P ^ were calculated in the same way as for bursts.

2.9.7 Super-clusters

Super-clusters were defined as groups of openings separated by gaps underlying the

first five components of the shut time distribution (Cibb & Colquhoun, 1992). Super-cluster

length, total open time per super-cluster and super-cluster Po n were calculated in the same

way as in bursts and clusters.

2.9.8 Alignment o f activations

Clusters and super-clusters of openings separated by the same value used to build,

distributions of clusters and super-clusters were aligned with the start of the first opening of

55

each cluster or super-cluster occurring simultaneously. Sections of the data record of a fixed

length and containing only one activation (cluster or super-cluster) were visually checked and

stored for subsequent averaging. Those containing simultaneous openings (doubles) were

discarded. Ensemble averages resulting from these alignments were constructed and the time-

course of their decays fitted with a sum of exponentials.

2.9.9 Current-voltage relationship plots

To calculate the slope conductance of the channels, single channel currents were

recorded for 2-3 minutes at holding potentials between -100 and -20 mV. For each holding

potential, an amplitude histogram was built and fitted with Gaussian components to estimate

the mean current amplitude which was plotted against the holding potential. Linear

regressions fitted through the points gave the slope conductance of each unitary current.

2.9.10 Direct transitions between conductance levels

A direct transition was considered as a change in open-channel current level from one

amplitude level to another without intervening closures longer than the shut time resolution.

Under the recording conditions used in this study (-3 dB, 8-pole Bessel type low pass filter), at

least 99,8 % of the amplitude of an opening was resolved when such event had a duration

equal to 2.5 filter rise-times (415 ps, 2 kHz). Critical amplitude values producing an equal

percentage of misclassified events between pairs of adjacent Gaussian components were

calculated. Analysis included openings that were longer than 2 or 2.5 filter rise times, on either

side of a direct transition. Three-dimensional representations of the data were built in which

the volume of each peak indicated the relative proportion of each type of transition. If a

particular type of direct transition, that was not observed during the time-course fitting of the

filtered recording, appeared after imposing the resolution to the idealised record, then each

event was visually inspected to check if they were spurious.

56

CHAPTER 3

S in g le - C h a n n e l P r o p e r t ie s o f NMDA R e c e p t o r s in S t r i a t a l

N e u r o n s f r o m O -D ay-O ld (PO) R a ts

3.1 Su m m a r y

i. The properties of single-channel currents and activations produced by NMDA receptors present

in outside-out patches excised from striatal neurons in brain slices from 0-day old rats were

studied. NMDA receptor single-channel currents were activated by steac^ state application of

low concentrations of glutamate (50 - 100 nM) or NMDA ( 1 - 5 pM) and saturating

concentrations of glycine (3-10 pM).

ii. Glutamate and NMDA activated single-channel currents with similar slope conductance. Two

conductance levels were identified: a main level of 54 pS and a short-lived sublevel of 44 pS.

Direct transitions between these two conductance levels were detected and interpreted as an

indication that both conductance levels were produced by a common receptor population.

Direct transitions between all conductance levels, including the shut level, were symmetric

indicating that the mechanism responsible for producing direct transitions between conductance

levels obeyed microscopic reversibility. This symmetry was apparently not affected by the

membrane potential.

iii. At least 6 exponential components were necessary in order to obtain a satisfactory fit of shut

time distributions. This was interpreted as an indication that in PO striatal neurons NMDA

receptor-channels can exist in at least 6 different shut states. About 50 % of all channel closings

had a mean duration around 20 ps (xi).

iv. In a similar way, the distribution of all individual open times was fitted with 3 exponential

components indicated the existence of at least 3 different open states. Distributions of individual

open times conditional on amplitude indicated that unitary currents to both 54 pS or 44pS

conductance levels can exist in at least 2 different open states.

V. Analysis of distributions of burst length and total open time per burst suggested that bursts of

openings activated by NMDA were longer and had a greater total open time than bursts of

openings activated by glutamate while no differences in mean burst Popen were observed.

57

vi. Analysis of distributions of cluster lengths and total open time per cluster showed no differences

in mean cluster length, mean total open time per cluster. The mean cluster Pojm was larger for

clusters produced by NMDA than for those produced by glutamate.

vii. It was not possible to compare super-clusters produced by glutamate and NM DA because

components 5 and 6 of the glutamate shut time distributions were not well separated. Only

super-cluster produced by NMDA, were analysed. For NMDA, mean super-cluster length and

mean total open time per super-cluster were 116.2 ± 51 and 18.1 ± 2.2 ms, respectively. Mean

super-cluster Popen was 0.30.

viii.No differences in the mean decay time constant of ensemble averages built with clusters of

openings were observed between glutamate and NMDA.

ix. Fitting of the decay time-course of ensemble averages constructed by aligning super-clusters of

openings produced by NMDA gave a mean decay time constant of 132 ± 98 ms.

3.2 In t r o d u c t i o n

Evidence from in situ hybrization and immunohistochemical studies suggests that NR la and

NR2B subunits are the most probable components of NMDA receptors in PO striatum. At PO,

mRNA encoding NRla splice variants are highly expressed in rat striatum with very low levels of

expression of mRNAs encoding NRlb splice variants (Laurie & Seeburg, 1994). Moderate to high

levels of NR2B protein and mRNA encoding the NR2B subunit are also present (Moryer et al,

1994; Wenzel et ï/., 1997) while evidence of expression of NR2A subunits is unclear. Faint signals of

NR2A mRNA with NR2A protein signals hardly exceeding background levels have been recently

reported by Wenzel et (1997); in contrast, Portera-CaiUiau etoL (1996) have reported total absence

of protein encoding NR2A subunits in striatum before P16. So far, no mRNA or protein signals

encoding NR2C or NR2D subunits have been reported in PO-striatum (Monyer etoL, 1994; Wenzel

etal., 1997).

If NMDA receptors in PO-striatal neurons have an NR1/NR2B subunit composition then

they may have single-channel properties similar to those described for NR1/NR2B recombinant

NMDA receptors (Stem et al., 1992). Studies in which the single-channel properties of NMDA

receptors in PO-striatal neurons have been described have not been reported.

58

3.3 R e su l ts

3.3.1 Presence o f functional NM DA receptors in PO-striatal neurons

Ionic currents flowing through individual NMDA receptor-channels were detected after

hath application of glutamate (50 - 100 nM) or NMDA (1 -5 pM) in the presence of glycine (3 - 10

pM) to outside-out patches excised from PO striatal neurons in brain slices. Other characteristics of

NMDA receptor-mediated single channel activity such as occurrence of bursts of openings and

occasional sublevels were also readily observed while recordings were been carried out (Figure 3.1).

3.3.2 Properties o f single-channel currents

3.3.2.1 Stability plots for amplitudes

Stability plots for amplitudes were built to assess the long-term stability of single channel

current amplitudes during each experiment. Figure 3.2A shows a characteristic stability plot in which

two different open channel current levels are readily observed. Their amplitude remained stable

throughout the whole period of recording.

3.3.2.2 Distribution o f single channel current amplitudes

For all patches obtained from PO striatal neurons, frequency distribution histograms

containing the amplitudes of all openings longer than 2 filter rise-times were built. In all cases, they

were best fitted by the sum of 2 Gaussian components with the mean amplitude and relative area of

the Gaussian component being always smaller than those of the 2"* Gaussian component (Figure

3.2B).

T a b l e 3.1 Distribution of single channel current amplitudes

Gaussian 2 Gaussian

amplitude

(pA)

area(0L\

amplitude

(pA)

area(0L\

NM DA 2.61 ±0.12 15 ± 1.3(n=5 patches)

amplitude

ratio

Glutamate 2.61 ±0.08 17 ±1.4 3.42 ±0.10 83 ± 1.4 0.76 ±0.01(n=4 patches)

3.38 ±0.12 85 ± 1.3 0.77 ±0.01

59

VM-4|

LfVNA V/Mv Lv " LW V V)

«"Aiywktfiwfir Ww .,Y ^w~1AAAvr»VWMin|-

-4f»“

frWw .. *4WfW

CO 100 m s

F ig u re 3.1 Functional N M D A receptor-channels in PO striatal neurons. Examples of characteristic ionic currents flowing through single NM DA receptor-channels after hath application of 5 pM NM DA to an outside-out patch excised from a PO striatal neuron in the continuous presence of 3 pM glycine and 5 pM D N Q X . Holding potential was -60 mV. Currents were low-pass filtered at 2 kH z (-3db, 8 pole Bessel filter). Openings are downwards. Traces show 5 seconds of continuous recording. Each trace is 500 ms long and separation between traces is 5 pA.

60

A1

0

Q_

•2

3

•4

•50 1000 2000 3000 4000 5000 6000

Interval Number

B600

<Q_

T-

^ 4000Q.>Ü

0 200DO"2u_

0 1 2 3 4 5Current (pA)

F ig u r e 3.2 Single-channel current amplitude of NM DA receptors in PO-striatal neurons.Stability plot analysis and frequency distribution histogram of single channel current amplitudes from NM DA receptor-mediated openings recorded from a single outside out patch excised from a 0- day-old striatal neuron. Patch was held at -60mV and exposed to a constant concentration of 5 pM NM DA and 3 pM glycine for 10 min. A) Stability plot containing 2934 single channel current amplitudes longer than 2 filter rise-times. B) Frequency distribution histogram built with data shown in A and fitted with the sum of 2 Gaussian components with mean amplitude and relative areas of 2.32 pA (15.6%) and 3.10 pA (84.4 %). Standard deviations were constrained to be the same, 0.185 pA'.

61

N o apparent differences in mean amplitude, mean relative area and Gaussian

amplitude ratio (mean ± S.E.m ) were observed between currents produced by glutamate or NMDA

(Table 3.1).

3.3.2.3 Single channel conductance

The slope conductance of NMDA receptor-mediated single channel currents was estimated

by building current-voltage relationships (I/V plots) with data obtained from 6 to 9 patches for

NMDA and 3 patches for glutamate. Fitting the I/V plots with linear regressions gave slope

conductance values of 44 and 54 pS. No apparent differences in single channel conductance were

observed between openings activated by glutamate (Figure 3.3A) or NMDA (Figure 3.3B), indicating

that in PO striatal neurons both agonists activated NMDA receptors with similar single channel

conductance; similar findings have been described by Howe et oL (1991) in cultured cerebellar

granule cells. NMDA channels with similar conductance levels have also been described for

NR1/NR2A (38 and 50 pS) and NR1/NR2B (39 and 51 pS) but not for NR1/NR2C or NR1/NR2D

recombinant NMDA receptors using similar recording conditions (Stem etoL 1992; 1994; Wylie et

d., 1996).

3.3.2.4 Direct transitions between conductance states

Direct transitions in the data record were identified when the open-channel current changed

from one amplitude range to another without an intervening closure longer than the closed time

resolution. The amplitude range was determined from the Acm values calculated from the fitted

parameters of the amplitude distribution, as described in the Methods. In order to be included in the

analysis both openings on either side of a direct transition needed to be longer than 2.0 filter rise-

times.

Independently of the agonist used, direct transitions between the shut level and 54 pS

currents represented 80 to 82 % of all direct transitions while those between the shut level and 44 pS

currents represented only 8 to 10 %. Direct transitions between 44 and 54 pS currents were also less

frequent but consistently detected in all recordings, they represented 10 to 12 % of all direct

transitions (Table 3.2). The presence of direct transitions between 44 and 54 pS currents (Figure 3.4)

was interpreted as an indication of both conductance levels arising from a common NMDA

receptor-channel population in which 54 pS currents represented the main open channel

conductance state and 44 pS currents represented a subconductance state.

Direct transitions between all conductance states, including the shut state, showed temporal

symmetry with a similar number of direct transitions occurring in either direction

62

/ \Voltage (mV)

-60-120 -100 -80 -40 0-20

44 pS

54 pS

0

-1

-2

-3

-4

-5

-6

-7

1

I3o

B Voltage (mV)

-60-120 -100 -80 -40 0-20

45 pS

53 pS

g3O

-1

-2

-3

-4

-5

-6

-7

F ig u re 3.3 Single channel conductance of N M DA receptors in PO-striatal neurons. Plots showing current-voltage relationships for single channel currents activated by 5 pM N M DA (A) or 50 - 100 nM glutamate (B). Each point represents the mean single-channel current amplitude identified by fitting the amplitude distribution histograms with two Gaussian components at holding potentials between -100 and -20 mV (6 - 9 patches for NM DA and 3 patches for glutamate). Linear regressions, fitted through the points, gave slope conductances values of 43.8 and 54.3 pS for NM DA, and 44.6 and 53.3 pS for glutamate. In both cases, glycine was present (3-10 pM).

63

A

(DT33Q .E<

5

444 pS -> 54 pS

3

2

54 pS ■> 44 pS

1

. /0

0 3 42 51Amplitude ( I ) ( pA )

B

0)E

16

12

8

4

0

F ig u re 3.4 Direct transitions between consecutive open-channel current levels. Plots show the number of direct transitions between consecutive open-channel current levels (shut level not included) in an outside-out patch from a PO-striatal neuron. In A, each dot represent a direct transition, considered as a change in open-channel current level from one amplitude level to another w ithout intervening closures longer than the shut time resolution, 50 ps in this case. Analysis included openings on either side of a direct transition that were longer than 2.5 filter rise-times. B is a three-dimensional representations of data shown in A. The volume of each peak indicates the relative frequency of each type of transition. An amplitude-based separation of unitary currents was carried out by calculating critical amplitudes values (A^„J producing an equal percentage of misclassified events between the two Gaussian components fitted to the amplitude distribution shown in figure 3.2.

64

T able 3.2 Analysis o f direct transitions between a ll conductance levels detected in PO

striatum NM D A receptors and comparison w ith those o f high conductance N R 1 /N R 2 A and

N R 1/N R 2B recombinant NM DA receptors

High conductance High conductancePO striatum recombinant NMDA receptors(this study) (from Stem et oL, 1992)

Type of transition

Glutamate NMDA NR1/NR2A* NR1/NR2B*

(pS) (% ) (% ) (% ) (% )

0Sub 4.4 3.7 5.5 3.9

0 4.4 3.7 5.5 4.1Sub

0

Main 40.2 39.8 38.4 40.3

0 40.3 39.6 38.2 39.8

Main

SubMain 5.4 6.6 6.1 5.9

Sub 5.3 6.6 6.2 6.1Main

Data was obtained from 3 patches exposed to glutamate (50 nM) and NMDA (5 pM) in thecontinuous presence of glycine (3 pM) and DNQX (5 pM). Holding potential was -60 mV. * Dataused for comparison was obtained from Stem et al., 1992

65

(Table 3.2), Presence of temporal symmetry was interpreted as an indication that, under these steady

state conditions, the process responsible for producing direct transitions between conductance levels

obeyed microscopic reversibility (Colquhoun & Sigworth, 1995).

Direct transitions obeying microscopic reversibility have been described between

conductance levels in NR1/NR2A, NR1/NR2B and NR1/NR2C (Stem et Â , 1992; 1994) but not in

NR1/NR2D (Wyllie et 1996) recombinant NMDA receptors. Table 3.2 compares the frequency

of direct transitions between all conductance levels identified in this study and those reported by

Stem etal. (1992) for NR1/NR2A and NR1/NR2B recombinant NMDA receptors. In both studies,

the frequencies of each type of transition were similar and showed temporal symmetry.

Direct transitions at different holding potentials were also analysed, direct transitions

between consecutive conductance levels showed temporal symmetry at all potentials analysed.

Between -20 and -100 mV, a four-fold reduction in the percentage of direct transitions between 44

and 54 pS openings was observed (Table 3.3) while an increment of similar magnitude was observed

in the percentage of direct transitions between the shut level and the subconductance level. A slight

increase in the percentage of direct transitions between the shut and the main open level was also

detected.

3.3.3 Properties o f single-channel activations

The activation properties of NMDA receptors were studied in those patches showing the

lowest Popen after bath application of glutamate (50 nN^ or NMDA (5 pM) in the presence of glycine

(3 pM) and DNQX (5 pM). Data used for analysis of glutamate-induced activations was obtained

from four outside out patches with a mean Popen of 0.006 (0.002 - 0.009) and a mean number of

openings per second of 3.3 ± 1.6 (0.8 - 8.9). For analysis of NMDA-induced activations, data was

obtained from five patches with a mean Popen of 0.013 (0.006 - 0.020) and mean number of openings

per second of 3.2 ± 0.5 (1.6 - 4.8).

3.3.3.1 Stability p lo t for shut tim eSy open times and Popen

Before analysing the distribution of shut and open times, stability plots for shut times, open

times and Popmwere built to verify that under these conditions of steady state receptor activation the

average channel kinetic behaviour did not change over time (Figure 3.5).

3.3.3.2 Distribution of shut times

Distributions of shut times from single channel activity induced by glutamate or NMDA

were generally best fitted with the sum of 6 exponential components with mean time

66

T able 3.3 Direct transitions at different negative membrane potentials

Type of -20 mV -40 mV -60 mV -80 mV -100 mV

0(% ) (% ) (% ) (% ) (% )

44 2.1 3.6 4.0 3.7 4.6

0 1.9 4.0 4.1 3.6 4.644

0

54 37.6 38.9 40.6 42.7 42.9

0 38.5 38.4 39.2 42.9 43.1

54

4454 10.1 7.3 6.1 3.5 2.6

44 9.7 7.8 6.0 3.5 2.354

Data obtained from outside-out patches exposed to NMDA (5 |xM) in the presence of glycine (3 and DNQX (5 pM). 3 - 5 patches analyzed per potential. A resolution of 50 - 200 ps was

used. Data record were filtered at the same bandwidth (2 kHz).

67

A(/)&<DE

05

i

10000

1000

100

10

10 1000 2000 3 0 0 0 4 0 0 0 5 0 0 0 6 0 0 0 7 0 0 0

B

V)

<DE

S.ocs

100

0 1000 2000 6 0 0 04 0 0 0 5 0 0 0 7 0 0 03 0 0 0

c(DQ.0

CL

10.01

0.001

0.00011000 2000 6 0 0 03 0 0 0 4 0 0 0 5 0 0 0 7 0 0 0

Interval number

F ig u r e 3.5 Stability plot analysis of shut times, open times and P„pe„ during activation of NMDA receptors in PO-striatal neurons. Plots show a running average of A) shut times, B) open times and C) during a 572 s recording from a single PO-striatum outside out patch exposed to a constant concentration of 5 |iM NMDA and 3 |xM glycine, and a holding potential of -60 mV. Each bin represents the average of 50 intervals with increments of 25 intervals between averages. Horizontal broken lines represent the average values for the whole recording. The overall mean shut time and open time were 205.7 and 4.16 ms, respectively. Mean overall was 0.01983 and mean opening frequency was 4.76 openings per second. For both openings and shuttings, a resolution of 50 ps was used which gave a false event rate of 1.66 x 10' s h

0)8WOp

(D38"

P

E3

B

<D

1"oO

(U3

8"

<DE

250

160

90

40

10

0.0.1 10000 1000000.01 1 100 100010

Shut time (ms) (log scale)

(D

250

160

40

10

00.01 10 10010.1

Open time (ms) (log scale)

Figure 3.6 D istribution of the duration o f all shut times and individual open tim es. A)Distribution of the duration of 2678 shut time intervals ranging from 0.050 to 61918.95 ms. It was best fitted with the sum of 6 exponential components with time constants and relative areas of 0.0232 ms (55.6 %), 0.323 ms (10.9 %), 1,07 ms (12.8 %), 11.7 ms (5,6 %), 86 ms (4.5 %) and 935,7 ms (10.6 %). The predicted overall mean shut time and number of shut time intervals were: 103.84 ms and 5498, respectively. B) Distribution of the duration of 3747 individual apparent openings. It was fitted with the sum of 3 exponential components with time constants and relative areas of 0.043 ms (11.6 %), 0.717 ms (31 %) and 4.28 ms (57,5 %). The predicted overall mean open time duration and number of openings were: 2.68 ms and 4197, respectively. Openings were separated by shut times longer than 50 ps. A change of 0.318 pA (10 % of the mean full amplitude) in open channel current amplitude was considered significant in determining when one opening finished and a new opening of different amplitude had begun, the two openings being separated by a direct transition between open levels.

69

constants ranging from tens of microseconds to seconds (Figure 3.6A). Table 3.4 shows mean time

constants and relative areas for 6 exponential components detected in patches exposed to glutamate

and NMDA,

The exponential component had time constants and relative areas that ranged from 15 to

28 |LXs and 35 to 76 %, respective^, for glutamate and from 17 to 30 p,s and 34 to 61 %, respectively,

for NMDA. For the exponential component, time constants and relative areas ranged from 0.092

to 0.211 ms and 4 to 17 %, respective^, for glutamate and from 0.208 to 0.378 ms and 6 to 27 %,

respectively, for NMDA. For the component, mean time constants and relative areas ranged

from 0.779 to 0.973 ms and 9 to 19 %, respectively, for glutamate and from 0.730 to 1.78 ms and 10

to 24 %, respectively, for NMDA. For the 4^ component, time constants and relative areas ranged

from 5.3 to 10.8 ms and 4 to 10 %, respectively, for glutamate and from 8.07 to 19.80 ms and 1 to 7

%, respective^, for NMDA.

Time constants and relative areas for the 5* and 6* components showed a greaterO f

variability. For NMDA, a 5* component was consistently detected in 5 out 5 patches and it had time

constants and relative areas ranging from 83 to 577 ms (3 - 9 %). For glutamate 3 out of 4 patches

showed a 5’ component ranging from 47 to 340 ms (1 - 12 %). A 6* component with a longer

duration was detected in all patches exposed to either glutamate or NMDA. It had time constants

and relative areas ranging from 920 to 2407 ms (1 - 22 %) for glutamate and 933 to 1725 ms (8 - 12

%) for NMDA.

3 .3 .3 .3 Distribution of open times to a ll amplitude levels

The lifetimes of individual openings were probably overestimated because the fit to the shut

time distribution predicted that 51 % of shut times had a mean time constant around 20 ps which is

2.5 times shorter than the 50 ps resolution imposed on the idealised record. However, it was still

possible to obtain information about the number and duration of open states because the number of

exponential components fitted to the distribution of apparent open times provides a lower limit to

the number of open states that can be detected (Colquhoun & Sigworth, 1995). Histograms of

apparent open times containing openings to all amplitude levels were built and fitted with the sum of

3 exponential components (Figure 3.6B). Mean time constants and relative areas (mean ± S.E.M) for

each component are shown in Table 3.5.

3 .3 .3 .4 Distribution of open times conditional on am plitude

A critical amplitude value (Aait) calculated from the fit to the amplitude distribution, was

used to differentiate openings to the main level from openings to the sublevel. Frequency

70

( is) (ms)

T a b l e 3 .4 Distribution of shut times

Ti Ta Tc

(ms) (ms) (ms) (ms) (ms)

Mean

(ms)

Glutamate(n= 4 patches )

20 ± 2 51 ± 8 %

0.16 ± 0 .0 2

11 ± 3 %0.87 ± 0.03

14 ± 2 %

8.9 ± 1

7 + 1%+235 ± 77 4 + 3 %

1542 + 270

8 ± 4 %*4980

5 %355 ± 138

(13518)

N M D A(n= 5 patches )

24 ± 2

51 + 4 %

0.31 + 0.03

14 + 3 %

1.03 + 0 .2

14 ± 2 %11.5 ± 1 .9

5 ± 1 %

232 ± 85

5 ± 1 %

1378 ± 122

10 ± 1 %*6258

1%209 ± 51

(12400)

^Detected in 3 out of 4 patches, ^Detected in only 1 patch

71

A250

0)(0 160o(0

I2gS'

40IE3z

0.1 1 10 100

B


160

I2COg- 40V)

.QE3z

0.1 1001 10Open time (ms) (log scale)

F ig u r e 3.7 Distribution of the duration of individual open times conditional on amplitude. A)Distribution of the duration of 2299 individual apparent openings to the main (54 pS) amplitude level (amplitude range = 2.69 to 4.00 pA; duration range = 0.332 to 37.3 ms). It was best fitted with the sum of 2 exponential components with time constants and relative areas of 0.88 ms (18.8 %) and 4.83 ms (81.2 %). The predicted overall mean duration and total number of events were: 4.09 ms and 2592, respectively. B) Distribution of the duration of 549 individual apparent openings to the (44 pS) sublevel (amplitude range = 1.00 to 2.69 pA; duration range = 0.332 to 8.9 ms). It was fitted with the sum of 2 exponential components with time constants and relative areas of 0.49 ms (75.3 %) and 1.48 ms (24.7 %). The predicted overall mean duration and total number of events were: 0.73 ms and 949, respectively. Both plots contained openings separated by shut times longer than 80 ps (0.482 t ; A/Amax = 0.45). A critical amplitude (Ag t) of 2.69 pA that produced an equal percentage (1.86 %) of misclassified events was calculated between the 2 Gaussian components fitted to the distribution of amplitudes shown in figure 3.2. The predicted number of misclassified events from the 1** and 2° Gaussian components were 0.3 and 1.56, respectively. Only openings longer than 0.332 ms (2.0 A/Amax = 98.8 %) were included in the fitting of both distributions.

72

distribution histograms containing individual open times were built (Figure 3.7). Incompletely

resolved openings were not included in the fitting of the distribution because their amplitude is

unknown. Only openings longer than 2 filter rise-times were used.

3.3.3.4.1 Open times to the main level (54 pS)

A maximum of two time constants could be fitted to distributions containing onty openings

to the main 54 pS level (Figure 3.7A). Some distributions were well fitted by one exponential

component, 2 out of 4 for glutamate and 1 out of 5 for NMDA. When fitting an additional

exponential component was necessary, it had a much faster time constant (xi) and a smaller relative

area (Table 3.6). The overall mean time constant was 0.7 ms longer for NMDA- than for glutamate-

evoked openings, even when the resolution was the same in both cases.

3.3.3.4.2 Open times to the sublevel (44 pS)

For glutamate and NMDA, 75% of the distributions containing only openings to the

sublevel were well fitted by two exponential components (Figure 3.7B, Table 3.7).

3.3.3.S Bursts

Bursts are expected to be less affected by the presence of incompletely resolved fast

shuttings than individual open times because they are defined as groups of openings separated by

shuttings of duration less than a critical shut time which was ~20 times longer than the 50 |Lis

resolution imposed on the idealised records.

3.3.3.5.1 Distribution of burst lengths

Frequency distribution histograms containing bursts of openings separated shuttings shorter

than mean tcm values of 1.2 ± 0.05 ms for glutamate and 1.1 ± 0.15 ms for NMDA were built. They

were fitted with the sum of 3 exponential components (Figure 3.8). Although the sum of 6

exponential components should have been necessary to fit a distribution of burst lengths that in

theory included 3 open and 3 shut states; the sum of only 3 exponential components was usually

enough to obtain a satisfactory fit of the distribution of burst lengths (Figure 3.8). The mean burst

length was 2 ms longer for NMDA than for glutamate. There were no apparent differences between

the mean time constants of the exponential components fitted to distributions of burst lengths

activated by glutamate or NMDA. The relative area of the slowest component was larger for NMDA

that for glutamate. The relative area of the intermediate component ranged from 8 to 41 % for

glutamate and from 11 to 20 % for NMDA (Table 3.8).

73

T able 3.5 Distribution of individual open times to a ll am plitude levels

'Cl 'C2 'C3mean

(ps) (ms) (ms) (ms)

Glutamate 5 1 ± 6 0.77 ± 0.1 3.50 ± 0.2 2.11 ±0.3(n= 4 patches) 21 ± 2 % 26 + 4 % 53 + 5 % (8388)

NM DA 50 + 2 0.87 ±0.1 4.10 + 0.1 2.73 ±0.1(n= 5 patches) 10 + 3 % 29 + 4 % 61 + 2 % (9581)

Table 3.6 Distribution o f individual open times to the main level (54 pS)

'Cl 'Ca mean

(ms) (ms) (ms)

Glutamate +0.62 + 0.1 3.17 ±0.3 2.94 ± 0.310 + 8 % 90 ± 6 % (5600)

NM DA ^0.89 ± 0.1 4.10 ±0.1 3.58 ± 0.216 + 5 % 84 ± 4 % (8536)

+2 out of 4 patches, **'4 out of 5 patches

T able 3.7 Distribution of individual open times to the sublevel (44 pS)

Tl 'C2mean

(ms) (ms) (ms)

Glutamate 0.66 ± 0.08 2.34 ± 0.5 0.90 ±0.183 ± 7 % 17 ± 7 % (1524)

NM DA 0.53 ± 0.02 1.96 ± 0.4 0.87 ±0.172 ± 10 % 28 ± 10 % (1602)

74

A

gg(03cr(/)

ËE3

B

<D

IIEgg

E3

90

40

10

00.01 0.1 1 10 100 1000

Burst length (ms) (log scale)

90

40

10

00.01 0.1 1001 10 1000

Open time per burst (ms) (log scale)

F ig u r e 3.8 Distribution of burst lengths and total open time per burst. A) Distribution of the duration of 1321 bursts containing openings to all amplitude levels (duration range = 0.05 to 89.5 ms). It was best fitted with the sum of 3 exponential components with time constants and relative areas of 0.063 ms (30 %), 1.39 ms (15 %) and 13.09 ms (55 %). The predicted overall mean burst duration and total number of bursts were: 7.40 ms and 1594, respectively. B) Distribution of the total open time per burst of the bursts used in A. It was fitted with the sum of 3 exponential components with time constants and relative areas of 0.063 ms (30 %), 1.28 ms (15 %) and 12.44 ms (55 %). The predicted overall mean total open time per burst and number of bursts were: 7.04 ms and 1601, respectively. Openings were separated by shut times longer than 50 fis (0.301 filter rise- times; A/Amax = 0.294). A critical gap length (t^ of 1.37 ms that produced an equal percentage (4.18 %) of misclassified events was calculated between the 3”* and 4** exponential components fitted to the distribution of shut times shown in fig 3.6A. The predicted number of misclassified events from the and 3"" exponential components were 3.2 and 1.01, respectively.

75

3.3.3.5.2 Distribution of total open time per hurst

The distribution of total open time per burst is much simpler than the distribution of burst

lengths because the number of exponential components is expected to be equal to the number of

open states (Colquboun & Hawkes, 1982) while the effect that the presence of incompletely resolved

events may have on the determination of the number of open states is very much reduced. It is also

less sensitive to missed events than the distribution of apparent open times (Table 3.9). Like the

burst length distribution, total open time per burst distributions suggested that bursts activated by

NMDA were significantly longer and bad significantly greater total open time than bursts activated

by glutamate.

3.3.3.5.3 Burst Popen

There was apparently no difference in mean burst Pcpm between glutamate and NMDA

(Table 3.10). Mean burst Popm was higher than the value of 0.86 reported for glutamate-activated

NMDA receptors in dissociated adult hippocampal CAl neurons (Gibb & Colquboun, 1992).

3.3.3.6 Clusters

Clusters included groups of openings separated by shut periods shorter than a critical shut

time (fern) calculated between the 4* and 5* exponential components fitted to the shut time

distribution.

3.3.3.6.1 Distribution of cluster lengths

Frequency distribution histograms containing clusters of openings separated by mean tcri

values of 15.8 ± 1.7 ms for glutamate and 13.6 ± 3.5 ms for NMDA were built and generally fitted

with the sum of at least 4 exponential components (Figure 3.9). For glutamate, in 2 out of 4 patches,

a 4^ exponential component was detected. For NMDA, 3 out of 5 patches showed a 4 ejqjonential

component. It bad a time constant and relative area of 58 ms and 11 %, respectively. For glutamate,

2 out of 4 patches showed a 5* exponential component.

3.3.3.6.2 Distribution of total open time per cluster

Distributions of total open time per cluster were fitted with at least 4 exponential components

(Figure 3.9). For glutamate, 2 out of 4 patches showed a 5* exponential component (Table 3.12).

76

T able 3.8 Distribution o f hurst lengths

' 2 mean

w (ms) (ms) (ms)

Glutamate 55 + 7 2.2 ± 0.7 11.7 + 0.7 4.8 ± 0.7(n= 4 patches) 43 + 5 % 20 + 7 % 37 + 9 % (3835)

NMDA 62 ± 3 1.9 ± 0.2 11.4 + 0.7 6.8 + 0.5(n= 5 patches) 30 + 3 % 12 + 4 % 58 + 3 % (3365)

T able 3.9 Distribution of total open time per hurst

?! ' 3 mean

(ps) (ms) (ms) (ms)

Glutamate 55 + 4 2.3 + 0.5 11.2 + 0.6 4.5 + 0.544 + 4 % 21 + 6 % 35 + 6 % 000

NMDA 61 ± 3 1.9 ± 0.3 11.1 + 0.5 6.5 ± 0.431 + 3% 13 + 4 % 56 + 3 %

Table 3.10 Burst Popen

Mean total open time Mean burst length Mean burst Popenper burst

(ms) (ms)

Glutamate 4.5 ± 0.5 4.8 + 0.7 0.95 ± 0.02

NMDA 6.5 + 0.4 6.8 ± 0.5 0.96 + 0.02

77

A

(0ucr(/)

ËE3

B

I£(03cr(O<DE3

90

40

10

00.01 0.1 1 10 100 1000

Cluster length (ms) (log scale)

90

40

10

00.01 0.1 1 10 100 1000

Open time per cluster (ms) (log scale)

F ig u r e 3.9 D istribution of cluster lengths and total open tim e per cluster. A) Distribution of the duration of 849 clusters (duration range = 0.05 to 226.4 ms). It was best fitted with the sum of 4 exponential components with time constants and relative areas of 0.047 ms (32.6 %), 0.665 ms (12.4 %), 16.2 ms (44.4 %) and 58.1 ms (10.6 %). The predicted overall mean cluster duration and number of events were: 13.41 ms and 1092, respectively. B) Distribution of total open time per cluster of clusters used in A. It was fitted with the sum of 4 exponential components with time constants and relative areas of 0.047 ms (32.7 %), 0.574 ms (13.9 %), 11.26 ms (41.2 %) and 41.9 ms (13.4 %). The predicted overall mean total open time per cluster and number of events were: 10.35 ms and 1089. Individual openings were separated by shut times longer than 50 ps. A critical gap length (t^J of 19.7 ms that produced an equal number (1.1) of misclassified events was calculated between the 4 and 5* exponential components fitted to the distribution of shut times The predicted percentage of misclassified events for the 4** and 5** exponential components were 1.35 % and 6.73 %, respectively.

78

T a b le 3.11 Distribution o f cluster lengths

Tl ' 1 ^4 ?5 mean

W (ms) (ms) (ms) (ms) (ms)

Glutamate 56 ± 6 1.2 ± 0.2 11± 3 +30 ± 2 +92 ± 7 9.1 ± 0.945 ± 5 % 9 ± 3 % 33 ± 10 % 10 ± 5 % 2 ± 1 % (2523)

NM DA 80 ±19 *2.2 ± 0.7 15 ± 4 *45 ± 5 12.2 ± 0.632 ± 2 % 9 ± 4 % 48 ± 4 % 11 ± 5% (2536)

+2 out of 4 patches, '*'3 out of 5 patches

Table 3.12 Distribution of total open time per cluster

Tl ^2 ' 4 mean

(ps) (ms) (ms) (ms) (ms) (ms)

Glutamate 57 ± 3 1.1 ±0.2 10 ±2.1 +25 ± 0.9 +82 ± 9 6.9 ± 0.846 ± 4 % 11 ±4% 32 ± 9 % 9 ± 4 % 2 ± 1 %

NM DA 67 ±10 *1.6 ±0.4 13 ±2.1 *37 ± 3 9.7 ±0.433 ±2% 10 ± 3 % 49 ± 3 % 8 ± 4 %

+2 out of 4 patches; '*'3 out of 5 patches

3.3.3.6.3 Cluster Popen

The mean cluster Popen was larger for clusters of openings produced by NMDA than for

those produced by glutamate (Table 3.13).

Glutamate

NM DA

Table 3.13 Cluster Popen

Mean total open time Mean cluster length per cluster

(ms)

6.9 ± 0.8

9.7 ± 0.4

(ms)

9.1 ±0.9

12.2 ± 0.6

Mean cluster P,open

0.76 ± 0.03

0.83 ± 0.03

79

3.3.3.7 Super-clusters

Super-clusters were defined as groups of openings separated by gaps underlying the first five

components of the shut time distribution (Gibb & Colquboun, 1992). For glutamate, calculation of a

tak between the 5^ and 6^ exponential component of the shut time distribution failed. Onty- in the

case of receptor activations induced by NMDA, calculation of a tcà between the 5* and 6

exponential component of the shut time distribution was possible for all patches.

3.3.3.7.1 Distribution of super-cluster lengths

Distributions of the duration of super-clusters separated by a mean tcà value of 289 + 105

ms for NMDA were built and fitted with 4 exponential components (Figure 3.10). Table 3.14 shows

mean time constants and relative areas for each one of the exponential components.

3.3.3.7.2 Distribution of total open time per super-cluster

The distribution of the total open time per super-cluster was fitted with three exponential

components (Figure 3.10). Table 3.15 shows mean time constants and relative areas for each one of

the exponential components.

3.3.3.7.3 Super-cluster Popen

Table 3.16 shows the mean super-cluster Popm for activations produced by NMDA.

3.3.3.5 Alignment o f activations

3.3.3.8.1 Alignment o f clusters

Clusters of openings separated by mean fait values of 15.8 ± 1.7 ms for glutamate and 13.6 ±

3.5 ms for NMDA were aligned and ensemble averages were constructed (Figure 3.11). The decay of

the ensemble averages were fitted with the sum of up to three exponential components (Table 3.17).

Frequencies with which the 1 (fast), 2"" (intermediate) and 3'" (slow) exponential components could

be fitted were 50 %, 75 % and 100%, respectively.

3.3.3.5.2 Alignment o f super-clusters

For NMDA, super-clusters of openings separated by a mean teat of 139 ± 10 ms were

aligned and ensemble averages were constructed. Their decay time-course was fitted with the sum of

up to three e3q>onential components (Figure 3.12).

80

A

§£gS'

E3

s

64

36

16

4

00.01 0.1 100 1000 100001 10

Super-cluster length (ms) (log scale)

gO'

0)JQI

64

36

16

4

01000 100000.01 0.1 1 10 100

Open time per super-cluster (ms) (log scale)

F ig ur e 3.10 D istribution of super-cluster lengths and total open tim e per super-cluster. A)Distribution of the duration of 583 super-clusters (range = 0.05 to 537.5 ms). It was best fitted with the sum of 4 exponential components with time constants and relative areas of 0,074 ms (25,5 %), 1,26 ms (10 %), 11,66 ms (17,4 %) and 91,1ms (47,2 %), The predicted overall mean super-cluster duration and total number of events were: 45,15 ms and 670, respectively, B) Distribution of total open time per super-cluster of those super-clusters used in A (range: 0,050 to 214,6 ms). It was fitted with the sum of 4 exponential components with time constants and relative areas of 0,073 ms (25,3 %), 1,23 ms (14,2 %), 17,5 ms (42,2 %) and 49,95 (18,4 %), The predicted overall mean total open time per super-cluster and total number of events were: 16,76 ms and 672, respectively. Individual openings were separated by shut times longer than 50 ps, A critical gap length (t J of 132,3 ms that produced an equal number (1,5) of misclassified events was calculated between the 5* and 6** exponential components fitted to the distribution of shut times The predicted percentage of misclassified events for the 5* and 6*** exponential components were 1,67 % and 12,81 %, respectively. The mean number of individual openings and gaps per super-cluster were 6,43 and 3.64, respectively.

81

Glutamate

Table 3.14 Distribution of super-cluster lengths

W (ms) (ms) (ms)

mean

(ms)

NMDA(n= 5 patches)

66+ 12 25 + 2 %

1.2 + 0.4 8 + 2%

14 + 3 25 + 3 %

233 ± 88 42 + 4 %

116.2 + 51 (1614)

Table 3.15 Distribution of total open time per super-cluster

Ti %2 i^Gan

Glutamate

(ps) (ms) (ms) (ms)

NMDA 89 ± 18 5.7 + 2 40 + 9 26 + 1 % 28 + 8 % 46 + 8 %

18.1 ±2.2

T able 3.16 Super-cluster Popen

Mean total open time Mean super-cluster per super-cluster length

Mean super-cluster Popen

Glutamate

NMDA

(ms) (ms)

18.1 ±2.2 116.2 ±51 0.30 ± 0.07

82

Glutamate NMDA

I T L U n j l W1 ---------------------1 --------------------T I U ----------------l l i l — '—

TU ]2pA

i n ------------------------11

TJLJJTinjLJL^TJj---------------------------l ü i u r n ^ j1TÜJ l2pA

0

tIS

I

■1

■2

-3 0 100 200 300

0

1

I ■1

S2-2

-33000 100 200

Time (ms) Time (ms)

F i g u r e 3.11 Alignment of clusters of single NMDA-channel activations. A and B are sections of data record containing clusters of openings from a single outside-out patch exposed to A) glutamate (50 nM) and B) NMDA (5 pM) in the continuous presence of 3 pM glycine and 5 pM DNQX. Each section of data record is 300 ms long and contains a single cluster of openings aligned with the start of the first opening of each cluster occurring simultaneously. Clusters were separated by values of 19.4 ms and19.7 ms for glutamate and NMDA, respectively. B and D are ensemble averages resulting from the alignment of 446 and 798 clusters for glutamate and NMDA, respectively. For B and D, the time-course of the decay of each ensemble average was fitted with the sum of 3 exponential components. For C, the time constants and relative areas for each component were 1.49 ms (28 %), 12.5 ms (61 %) and 88 ms (11 %) and the mean decay time-course was 18 ms. For D, the time constants and relative areas for each component were 1.85 ms (29 %), 16.3 ms (58 %) and 67 ms (13 %) and the mean decay time- course was 19 ms.

83

1f4*%W "WkWiW nr T

y 1jii<l»ml I'HI I i«» i|ii ir t

(H—f«| UÆMnnmnw

]2 p A

B

%cI3c050)

1

0

1

2

35 0 04 0 0 6 0 0200 3 0 00 100

Time (ms)

Figure 3.12 A lignm ent of super-clusters of single channel currents. A) Sections of data record containing characteristic super-clusters of openings from a single outside-out patch exposed to 5 pM NMDA in the continuous presence of 3 pM glycine and 5 pM DNQX. Each section of data record is 600 ms long and contains a single super-cluster of openings aligned with the start of the first opening of each super-cluster occurring simultaneously. Super-clusters were separated by a t„it value of 132.3 ms. B) Ensemble average resulting from the alignment of 532 super-clusters. The time-course of the decay was fitted with the sum of 3 exponential components with time constants and relative areas of 1.24 ms (1.1 %), 10.7 ms (21.6 %) and 93 ms (77.3 %). Mean decay time-course was 74.4 ms.

84

T able 3.17 Decay time-course o f aligned clusters

Glutamate

NM DA

'Cl Î2 'C3 mean

(ms) (ms) (ms) (ms)

1.3 ±0.1 8.7 + 1.6 47 + 12 33.2 ± 6.51.7 ± 0.9 % 28.3 ± 9 % 70 + 8 % (1799)

2.4 + 0.4 13.2 ± 2.2 54 + 9 36.0 ± 2.22.9 ± 0.1 % 38 + 7 % 59 + 7 % (1254)

Glutamate

NM DA

Table 3.18 Decay time-course o f aligned super-clusters

(ms)

2.6 ± 0.9 2 + 1%

(ms)

16.3 ± 4 33.5 ± 6 %

(ms)

181 + 93 64 + 6 %

mean

(ms)

131.8 + 67

85

3.4 D is c u s s io n

In this discussion, I will try to compare the single channel properties of NMDA receptors in

0-day old striatal neurons with those reported from studies using recombinant NM DA receptors

which have been shown to have properties remarkably similar to those reported for native NMDA

receptors (Stem etoL 1992; 1993; 1994; W)Æe etal. 1996). Except for one stuc^ (Stem etal. 1994),

most of the reported information on the single-channel properties of recombinant NM DA receptors

has been obtained by expression of pairs of NMDA receptors subunits in Xenopus oocytes. Very

recently, the use of Xenopus oocytes in the characterization of NMDA receptors has been questioned

by evidence showing that XerKpîs oocytes endogenously ejq)ress a glutamate receptor subunit

(XenUl) which can functionally interact with N R l subunits (Soloviev & Bamard, 1997). Assembly

of functional X enU l/N R l hybrid receptors in Xenopus occytes is thought to be the reason why

homomeiic expression of N R l subunits produces functional NMDA receptors in Xenopus oocytes

but not in mammalian cells such as HEK 293 cells (discussed by Soloviev & Bamard, 1997). So far,

results from co-immunoprecipitation studies showing evidence of X enU l/N R l subunit associations

in Xenopus oocytes have not yet been reported. It is not yet known what effects association of

XenUl and NMDA receptor subunits may have had on the reported single channels properties of

recombinant NMDA receptors.

3.4.1 Single channel conductance

In 0-day old striatal neurons, activation of NMDA receptors produced high conductance

single-channel currents with two conductance levels, a main level of 54 pS and subconductance level

of 44 pS. This finding is consistent with evidence from in situ hybridization and

immunohistochemical studies which suggest that NMDA receptors in PO striatum may contain

NR2B subunits, and possibly NR2A subunits. Indeed, NMDA receptors producing high

conductance single-channel currents have only been described after heteromehc expression of N R l

and NR2A or NR2B subunits (Stem etd.^ 1992; 1994; Table 1.3). In addition, the inability to detect

in PO-striatal neurons low conductance single-channel currents also agrees with the biochemical

evidence that there is an absence of NR2C and NR2D subunits in PO striatum (Monyer et d. 1994;

Wenzel et d^ 1997). NR2C and NR2D subunits produce only low conductance single-channel

currents upon heteromeiic expression with N R l subunits (Stem et cL 1992; W )ê et d^ 1996; see

also Table 1.3).

Whether NMDA receptors from PO striatum contained NRla or NR lb splice variants

could not be determined based on the analysis of the single channel conductance. The presence

(NRlb) or absence (NRla) of the 21 amino acids cassette encoded by exon 5 in N R l subunits have

apparently no effect on the single channel conductance of NMDA receptors. Araneda et d (1997)

has recently reported identical single channel conductances (52 pS in 1 mM extemal Ca^+) for

86

recombinant NMDA receptors produced after co-expression of NR2B/NRla or NR2B/NRlb

subunits in Xenopus oocytes.

3.4.2 Shut times

For recombinant rat NRl/mouse e l (mouse homologue of NR2A) NMDA receptors

expressed in HEK 293 cells, mean time constants and relative areas for the three fastest components

of shut time distributions in the presence of glutamate have been reported (Stem etoL 1994). Similar

mean time constants and relative areas have also been obtained for the three fastest components of

shut time distributions from NR1/NR2B recombinant NMDA receptors in the presence of

glutamate (Behe & Colquhoun, personal communication) (See Table 3.19). When data from the

distribution of shut times from recombinants and data presented in this study were compared, the

main difference was apparently the presence in PO-striatum NMDA receptor activations of an

additional shut state as indicated by the number of fitted exponential components (Table 3.19).

T able 3.19 Comparison between shut times from PO striatum and recombinant N M D A

receptors

(ps) (ms) (ms) (ms)

PO Striatumi 20 ± 2 0.16 ± 0.02 0.87 ± 0.03 8.9 ± 1(51 ± 8 %)* (11 + 3%) (14 + 2%) (7±1% )

NR1/NR2A2 39 ± 4 0.54 + 0.04 9.9 ±1.3(37 + 4%) (21 ±2% ) (15 + 1 %)

NR1/NR2B3 54 + 1 0.65 + 0.1 8.8 ±1.3(34 ±4% ) (23 ±2% ) (11 ±2% )

' This study; ^Stem et oL (1994); Bdoe & Colquboun (personal canmunkatim)

Even when the mean time constants of the 1^( %i) and 4‘ (1 4 ) exponential components of

the shut time distribution from PO-striatum NMDA receptors were similar to those of the 1* ( Xi )

and 3* ( 1 3 ) exponential components of shut time distributions from NR1/NR2A and NR1/NR2B

recombinant NMDA receptors (Table 3.19), in recombinant receptors only 1 exponential

component ( 1 2 ) was fitted between these components while in PO-striatum NMDA receptors 2

components had to be fitted in order to obtain a satisfactory fit. The presence of an additional shut

state in PO-striatum NMDA receptors can be interpreted either as a real difference in receptor

activation kinetics or as a difference in criteria when fitting shut time distributions. This wiU have to

be investigated in more detail. It should be noted, however, that the shut time distribution from

87

NR1/NR2A recombinant NMDA receptors shown in Figure 2A in Stem et al (1994) could have

probably also been fitted, between X\ (32.6 ps) and T3 (7.35 ms), with two rather than one

exponential components.

3.4.3 M ain level openings

In PO-striatal neurons, glutamate-activated main level openings (54 pS) had a mean time

constant similar to that reported for glutamate-activated main level openings from NR1/NR2B and

NR1/NR2A recombinant NMDA receptors (Stem etal, 1993).

An additional open state with a mean time constant ( ti ) in the microsecond range was

reported by Stem et al. (1993) for NR1/NR2A and NR1/NR2B. In contrast, in this stuc^ such fast

openings were not detected because events with a duration less than 2 filter rise-times (332 ps) were

excluded from the fitting of the distribution. Such fast events appeared as incompletely resolved

openings and their amplitude can not be determined.

Even though glutamate-activated main level openings (54 pS) also showed the two other

open states reported in recombinant NMDA receptors their mean time constants were faster and

their relative areas different.

Table 3.20 Comparison between main level openings from PO striatum and recombinant

NM DA receptors

mean'Cl 'C2 'C3

(ps) (ms) (ms)

PO Striatum^ 0.62 ± 0.1 (10 ± 8 %)

3.17 ± 0.3(90 ± 6 %)

NR.1/NR2AP 67 ± 1 (30 ± 5 %)

1.64 ± 0.4 (40 ± 7 %)

4.27 ± 0.7 (30 ±4% )

NR1/NR2B2.3 183 ± 300 (23 ± 5 %)

1.83 ± 0.3 (41 ± 7 %)

4.99 ± 0.1(36 ± 8 %)

(ms)

2.94 ± 0.30

2.70 ± 0.703

2.80 ± 0.303

TMs stu(fy;^frcmStemetaL (1993)y rneans are from Stern et oL (1992)

3.4.4 Sublevel openings

Glutamate-activated sublevel openings (44 pS) had a mean open time similar to that

reported for glutamate-activated sublevel openings (39 pS) from NR1/NR2A and NR1/NR2B

recombinant NMDA receptors (Stem 1992; 1994).

Table 3.21 Comparison between sublevel openings from PO striatum and recombinant

NM DA receptors

Tl ' 2 m ea n

(ms) (ms) (ms)

PO-Striatum^ 0.66 ± 0.08 (83 ± 7 %)

2.34 ± 0.5 (17 ± 7 %)

0.90 ±0.10

NR1/NR2A2^ 0.18 ± 0.02 (87 ±1% )

1.31 ±0.2 (13 ± 1 %)

0.61 ± 0.053

NR1/NR2B3 n.r. n.r. 0.59 ± 0.07

This study, ^fian Stem et oL (1994, HEK 293 cdls), values from Stem et oL (1992)nr. = no reported

3.4.5 Bursts, clusters and super-dusters

Unfortunately, lack of information about the properties of burst, clusters and super-clusters

from NR1/NR2A and NR1/NR2B recombinant NMDA receptors did not allow a more detailed

comparison.

The functional evidence presented in this stuc^ seems to suggest that at the single channel

level native NMDA receptors from PO-striatal neurons share many of the properties of a NMDA

receptor population composed of N R l, NR2B and probably NR2A subunits.

Because evidence about the presence of NR2A subunits in PO-striatum is still unclear and

the high degree of similarity in single channel properties between NR1/NR2A and NR1/NR2B

recombinant NMDA receptors made them apparently indistinguishable at the single channel level, it

is not possible to estimate the relative proportion of the NMDA receptors that may have contained

NR2A or NR2B subunits. Use of sub-unit selective drugs will be necessary in order to have a more

clear idea about the potential subunit composition of PO-striatum NMDA receptors.

89

CHAPTER 4

E f f e c t s o f S p erm in e o n t h e S in g le - C h a n n e l P r o p e r t i e s o f

NMDA R e c e p t o r s f r o m P O -S tr ia ta l N e u r o n s

4.1 Su m m a r y

i. The effects of spermine (100 pM) on the single-channel properties of PO-striatum NM D A

receptors were studied in outside-out patches.

ii. Spermine (100 pM) produced a 20 % reduction of the mean single channel current

amplitude of PO-striatum NM DA receptors

iii. Spermine (100 pM) produced a reduction in mean shut time without reducing the time

constants of any of the six exponential components. It produced a 38 % increase in the

relative area of the fastest exponential component (xi= 26 ps) and a reduction in the

relative areas of all other exponential components.

iv. Even while spermine (100 pM) had profound effects on each one of the three identified

open states, this had apparently only minor effects (only a 7 % reduction) in mean open

period.

V. In contrast, spermine (100 pM) produced a 95 % increase in mean burst length and an 84

% increase in mean total open time per burst without increasing the time constants of any

of the exponential components fitted to burst distributions or the mean Apen per burst. It

was calculated that such an increase in mean total open time per burst produced a 48 %

increase in mean charge transfer per burst.

vi. In addition, spermine (100 pM) produced a 145 % increase in mean cluster length and a

180 % increase in mean total open time per cluster. The estimated mean charge transfer

per cluster increased by 93 %.

vii. Spermine (100 pM) produced a 50 % increase in mean decay time-course of ensemble

averages built using clusters of openings from single NM DA receptor-channel activations.

90

viii.In summary, these data suggest that at the single channel level native N M D A receptors

from PO striatal neurons respond to spermine with an increase in their overall activation

properties which suggests that these receptors share properties found in recombinant

N M D A receptors containing N R la and NR2B subunits.


Spermine produces a glycine-independent potentiation of macroscopic currents

mediated by NM DA receptors which is both N R l splice variant-selective and NR2 subunit-

selective (reviewed by Williams, 1997). Upon homomeric expression in Xenopus oocytes,

N R la but not N R lb splice variants show glycine-independent potentiation by spemine

(Durand et al., 1992; 1993). When N R la splice variants are co-expressed with NR2A, NR2C

or NR2D subunits glycine-independent potentiation by spermine is abolished. In contrast,

when N R la splice variants are co-expressed with NR2B subunits glycine-independent

potentiation by spermine can be rescued. When NR2B subunits are then co-expressed with

N R lb splice variants no glycine-independent stimulation by spermine is observed (Zhang et

a i, 1994; Williams, 1994; Williams et a l, 1994; WiUiams, 1995). Glycine-independent

potentiation of N R la/N R 2B recombinant NM DA receptors has not yet been studied at the

single-channel level. It has been suggested, however, that at the single channel level glycine-

independent potentiation by spermine probably involves an increase in the channel opening

frequency (Williams, 1994). If PO-striatum NM DA receptors potentially contain N R la splice

variants and NR2B subunits, as suggested by biochemical evidence (Monyer et at., 1994; Laurie

& Seeburg, 1994) and by the functional evidence described in the previous chapter, then

spermine in the presence of a saturating glycine concentration should be able to produce an

increment in single channel activity.

4.3 R e s u l t s

4.3.1 spermine affects NMDA receptor-channels in PO-striatal neurons

The effect of spermine (100 pM) on single NM DA receptor activations induced by

glutamate (100 nM) and glycine (3 pM) was studied in detail in a single outside-out patch

excised from a PO striatal neuron. Similar results were observed in two other patches. Figure

4.1 shows characteristic NM DA receptor activations before and during bath application of

spermine. On the data record, it is possible to observe that in the presence of spermine, single

channel currents appeared smaller and bursts longer (Figure 4.IB).

91

I 4*

B

.#*W#'ww#W *4“»»

ft

1

100 ms 100 ms

"Mr

F igu re 4.1 Effect of spermine on N M D A receptor single-channel currents from PO striatal neurons. Examples of characteristic single NMDA-channel activations obtained from a single outside-out patch in the presence of A) 100 nM glutamate and B) after application of 100 pM spermine, both in the presence of 3 pM glycine and 5 pM D N Q X . Holding potential was -60 mV. Currents were low-pass filtered at 2 kHz (-3db, 8 pole Bessel filter). Openings are downwards. Each panel contains ten contiguous 500 ms sweeps of single channel recording with a separation of 5 pA between sweeps.

92

4.3.2 Effects on steady state single-channel behaviour

A stability plots analysis of single channel current amplitudes, shut time intervals,

open time intervals and Popg„ was carried out before analysing in more detail frequency

distributions for each one of these parameters. Figure 4.2 shows a reduction in current

amplitude, mean shut time interval and mean open time intervals and an increase in mean

Popen- A large increase in the number of intervals was also observed.

4.3.3 Effects on single channel current amplitudes

The effect of spermine on the amplitude of single-channel currents was studied by

building frequency distribution histograms containing all fitted amplitudes that were longer

than 2 filter rise-times.

In the presence of spermine, a 20 % reduction in mean single-channel current

amplitude and a 175 % increase in the number of fitted amplitudes (in an 125 seconds long

analysis period) were observed (Figure 4.3) while no apparent changes in their relative areas

and amplitude ratio were detected (Table 4.1).

T a b le 4.1 Distribution of single channel current amplitudes

Glutamate

Spermine

T* Gaussian Gaussian st !

amplitude area amphtude area amplitude

(pA) (%) (pA) (%) ratio

2.62 ± 0.22 14.3 3.40 ± 0.22 85.7 0.77

2.04 ± 0.23 13.5 2.75 ± 0.23 86.5 0.74

Total number of fitted amplitudes were 987 for glutamate and 2718 for glutamate + spermine

4.3.4 Effects on shut times

Because of the reduction in mean shut time detected in stability plot analysis, the

effect of spermine on the distribution of shut times was studied in more detail. Distributions

of shut times imder control conditions and in the presence of spermine were generally fitted

with the sum of 6 exponential components (Figure 4.4). Table 4.4 shows the mean time

constants and relative areas for all six components. Spermine produced no significant changes

in their time constants but variations in the relative areas of nearly all components were

detected. The relative area of the 1** exponential component increased 38 % while the relative

areas of the 2“*, 4***, 5‘** and 6*** components were reduced.

93

A 1

Î 0

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B^ 10000 £ 1000 g 100

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0.00016000 80002000 4000

Interval number

F ig u re 4.2 Stability plot analysis of the effect of spermine on amplitudes, shut times, open times and Popen.- Stability plots for A) amplitudes, B) shut times, C) open times and D) before and after application of 100 fiM spermine to a single outside-out patch in the continuous presence of 100 nM glutamate and 3 pM glycine. Holding potential was -60 mV. For B,C and D, each bin represents the average of 50 intervals with increments of 25 intervals between averages. For B, C and D, values represent the overall mean shut time, open time and P ^ , respectively. The mean opening frequencies were 5.95 openings per second under control conditions and 16.08 openings per second during bath application of 100 pM spermine. A resolution of 50 ps was used for openings and shuttings. Data records with similar duration (125 s) were analysed in each case.

94

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S' 100L I - 2.62 pA

Current amplitude (pA)

5002.75 pA

400

300

200

2.04 pA100cr

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Current amplitude (pA)

Figure 4.3 Effect o f spermine on the amplitude o f NMDA receptor single-channel currents from PO striatal neurons. Panels A and C show sections of data record containing NMDA receptor single-channel currents obtained from a single outside-out patch under control conditions (A) and after addition of 100 pM spermine (B). Holding potential was -60 mV. Currents were low-pass filtered at 2 kHz (-3db, 8 pole Bessel filter). Openings are downwards. Histograms in B and D are frequency distributions of current amplitudes longer than 2 filter risetimes (2rj fitted with the sum of 2 Gaussian components. Mean amplitude values for each component are shown on top of each curve and their relative areas are shown in table 4.1. Data records with similar duration (125 s) were analysed in each case.

95

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1000.01 0.1

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0.01 0.1 1 10 Open period (ms) (log scale)

100

F i g u r e 4.4 Distribution of shut times and open periods under control conditions (A, B) and after 100 pM spermine (C, D). Histograms in A and C are frequency distributions of shut times fitted with the sum of 6 exponential components. Time constants and relative areas for each exponential component were in A) 0.0262 ms (50.4 %), 0.257 ms (16.8 %), 1.16 ms (8.1 %), 7.83 ms (4.2 %), 114.3 ms (6.4 %) and 572.8 ms (14.1 %), and B) 0.031 ms (69.7 %), 0.232 ms (10.8 %), 0.886 ms (8.8 %), 5.55 ms (3.4 %), 123 ms (3.8 %) and 575.4 ms (3.5 %). Predicted overall mean shut time and number of shut intervals were in A) 88.57 ms and 1390, respectively and in B)25.08 ms and 4759, respectively. Histograms in B and D are distributions of open periods fitted with the sum of 3 exponential components. Time constants and relative areas for each exponential component were in B) 0.044 ms (38.8 %), 3.52 ms (51.2 %) and 7.76 ms (10.0 %) and in D) 0.061 ms (15.7 %), 1.64 ms (27.2 %) and 3.43 ms (57.1 %). Predicted overall mean open period and number of open intervals were in A) 2.59 ms and 1021, respectively and in B) 2.41 ms and 2212, respectively.

96

The relative area of the component showed no apparent changes

T a b le 4.2 Distribution of shut times

' 3 ^4 ' 5 ' 6


Glutamate 26.2 0.257 1.16 7.83 114.3 572.850% 17% 8% 4% 6% 14%

Spermine 31.1 0.232 0.89 5.55 122.9 575.470% 11% 9% 3% 4% 4%

4.3.5 Effects on open periods

Distributions of the duration of contiguous open times (open periods) produced by

openings to all amplitude levels were built and fitted with the sum of 3 exponential

components (Figure 4.4). Table 4.3 shows the time constants and relative areas of each

exponential component.

T a b le 4.3 Distribution of open periods

mean

(ps) (ms) (ms) (ms)

Glutamate 44.3 3.52 7.76 2.5938.8 % 51.2 % 10.0 %

Spermine 61.2 1.64 3.43 2.4115.7% 27.2% 57.1%

Total number of open periods analysed were 745 for glutamate and 1982 for glutamate + spermine

In the presence of spermine, a 166 % increment in the number of fitted open periods

was observed. The predicted time constant for the 1” exponential component did not show

significant changes but a 60 % reduction in its relative area was observed. In contrast, the

and 3" exponential components showed a 55 % reduction in their time constant together with

a 250 % and 471 % increment in their relative areas, respectively. Overall, these changes

produced only a 7 % reduction in mean open period.

97

64

S 36

II

IZ10000.1 1000.01

Burst length (ms) (log scale)

S 36

I 16

Iz0.1 10000.01 I 10

Burst length (ms) (log scale)100

D64

i 36

Ii*

10001 10 1 Open time per burst (ms) (log scale)

1000.01 0.1

64

362i

IsI

0.01 10 100 10000.1 1Open time per burst (ms) (log scale)

Figure 4.5 Distribution of burst lengths and total open time per burst under control conditions (A, B) and in the presence of 100 pM spermine (C, D).Histograms in A and C are frequency distributions of burst lengths fitted with the sum of 4 exponential components. Time constants and relative areas for each exponential component are shown in table 4.5 were in A) 0.033 ms (60.7 %), 0.428 ms (7.3 %), 8.13 ms (23.5 %) and 26.6 ms (8.5 %), and in B) 0.047 ms (37 %), 0.489 ms (12.6 %), 8.31ms (34.4 %) and 33 ms (16.1 %). Predicted overall mean burst length and number of bursts were in A) 4.29 ms and 648, respectively and in B) 8.25 ms and 668, respectively. Histograms in B and D are distributions of total open time per burst fitted with the sum of 4 exponential components. Time constants and relative areas for each exponential component were in B) 0.034 ms (59.5 %), 0.4 ms (7.5 %), 7.6 ms (24.4 %) and 26 ms (8.5 %), and in D) 0.046 ms (36.9 %), 0.44 ms (14.5 %), 8.5 (35 %) and 33 ms (13.6 %). Predicted overall mean total open time per burst period and number of open intervals were in A) 2.59 ms and 1021, respectively and in B) 2.41 ms and 2212, respectively.

98

4.3.6 Effects on bursts

In contrast with the small effect of spermine on the mean lifetime of open periods, a

nearly 100 % increase in mean burst length and mean total open time per burst was observed

with no apparent changes in mean burst P pea

In the distribution of burst lengths, the relative area but not the time constants of all

four exponential components changed. The relative area of the 1” component was reduced by

~ 40 % while those of the 2“*, 3'’' and 4* exponential components increased by 73 %, 46 % and

89 %, respectively (Table 4.4).

Table 4.4 Distribution o f burst lengths

'Cl 'C2 'C3 'C4mean

(ps) (ms) (ms) (ms) (ms)

Glutamate 33 0.428 8.1 27 4.261 % 7% 24% 9%

Spermine 47 0.489 8.3 33 8.337% 13% 34% 16%

Total number of bursts analysed were 648 for glutamate and 668 for glutamate + spermine

In the distribution of total open time per burst, a similar situation was observed with

no changes in the time constants of all exponential components but changes in their relative

areas (Table 4.5). The relative area of the component was reduced by ~ 40 % while those of

the 2"* , and 4 exponential components increased by 48 %, 30 % and 38 %, respectively.

The mean burst Popen, calculated by dividing the mean total open time per burst by the mean

burst length, was apparently unchanged with values of 0.97 and 0.92 for glutamate and

glutamate + spermine, respectively.

Table 4.5 Distribution of total open time per burst

'Cl 'C2 3 'C4mean


Glutamate 34 0.40 7.6 26 4.160% 8% 24% 9%

Spermine 46 0.44 8.5 33 7.637% 15% 35% 14%

99

In NM DA receptor-mediated macroscopic currents, a ~ 50 % increase in mean total

open time per burst produced by spermine may not be evident because spermine also

produced a ~ 20 % reduction in mean single channel current amplitude (Table 4.1), so the

mean charge transferred during each burst of single channel activity may not have changed.

The mean charge transfer per burst, calculated by multiplying the mean current amplitude by

the mean total open time per burst, increased by 33 % in the presence of spermine (Table 4.6).

Glutamate

Spermine

T ab le 4.6 Mean charge transfer per hurst

Mean open time per burst

(ms)

4.1

7.6

Mean current amplitude

(pA)

3.29

2.65

Mean charge transfer per burst

(fC)

13.52

20.03

to,4.3.7 Effects on clusters

In the presence of spermine, clusters showed changes similar^those observed in bursts.

The mean cluster length and mean total open time per cluster increased by 144 % and 139 %,

respectively while the mean per cluster showed no apparent change.

In the distribution of cluster lengths, the time constants of the 1 and 2“‘* exponential

components showed no apparent changes while those of 4 and 5' components increased by

-110 % and —70 %, respectively (Table 4.7). A 3'”' exponential component detected in the

presence of spermine could not be detected under control conditions. The relative area of the

component showed a 45 % reduction while no apparent changes in the relative areas of the

T^, 4'* and 5 , exponential components were observed.

T a b l e 4.7 Distribution of cluster lengths

Glutamate

Spermine

W

31.8 60.1 %

44.9 33.2 %

(ms)

0.343 9.4 %

0.380 10.5 %

(ms)

3.892 1 %

(ms)

10.4 26.1 %

21.8 27.6 %

(ms)

60.6 4.4 %

102.8 6.6 %

mean

(ms)

5.6

13.7

Total num ber of clusters analysed were 276 for glutamate and 331 for spermine

100

In the distribution of total open time per cluster, a similar situation was observed. The

time constants of the 1®* and 2"“ exponential components showed no apparent changes while

those of 4* and components increased by 89 % and 67 %, respectively (Table 4.8). A 3'”'

exponential component detected in the presence of spermine could not be detected under

control conditions. The relative area of the component showed a 49 % reduction while that

of the 2“* and 5* component showed a 68 % and 53 % increase, respectively. N o apparent

change in the relative area of the 4 component was observed.

Table 4.8 Distribution o f total open time per cluster

T. T. T, T. Tr mean


Glutamate 35.3 0.423 8.92 52.3 4.959% 9.4% 26.9% 4.7%

Spermine 54.4 0.329 3.08 16.9 87.2 11.730 % 15.8 % 18.6 % 28.5 % 7.2 %

As for bursts, the mean cluster was apparently unchanged with values of 0.90 and

0.85 for glutamate and spermine, respectively. The mean charge transfer per cluster increased

by 48 % in the presence of spermine (Table 4.9).

T ab le a.9 Mean charge transfer per cluster

Mean open time per Mean current Mean charge transfercluster amplitude per cluster

(ms) (pA) (fC)

Glutamate 4.9 3.29 16.1

Spermine 11.7 2.65 31.0

4.3.8 Effects on the decay time-course o f aligned clusters

Clusters of openings are known to occur within a single N M D A receptor activation

(Gibb & Colquhoun, 1991). An increase in the mean cluster length, such as the one produced

by spermine could have a significant effect on the activation kinetics of the receptor which is

known to underlie the decay of NM DA receptor-mediated synaptic currents (Lester et a l.

101

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ControlS p erm ine

Figure 4.6 Alignment of clusters of single NMDA-channel activations under control conditions (A, B) and after 100 |iM spermine (C, D). A and C are sections of data record containing characteristic clusters of openings from a single outside-out patch under A) control conditions (100 nM glutamate) and C) after application of 100 pM spermine, both in the presence of 3 pM glycine and 5 pM DNQX. Each section of data record is 300 ms long and contains a single cluster aligned with the start of the first opening of each cluster occurring simultaneously. Clusters were separated by values of 11.90 ms and 11.65 ms for glutamate and spermine, respectively. B and D are ensemble averages resulting from the alignment of 169 and 319 clusters for glutamate and spermine, respectively. In B and D, the time-course of the decay of each ensemble average was fitted with the sum of 3 exponential components. The time constants and relative areas of each component were 0.72 ms (0.9 %), 10.6 ms (53.4 %) and 123.1 ms (45.6 %) under control conditions and 2.83 ms (5.4 %), 23.7 ms (38.8 %) and 149.3 ms (55.8 %) in the presence of spermine. The mean decay time-courses were 62 ms and 93 ms for control and spermine, respectively. In E, the first 100 ms of the ensemble averages shown in B and D are normalised to peak to show the effect of spermine on the decay.

102

1990). The potential effects that a spermine-induced increase in mean cluster length may have

on the decay of NM DA receptor mediated currents were studied by aligning clusters of single

NM D A receptor activations and by measuring their decay (Figure 4,6),

T ab le 4,10 Decay time-course o f aligned clusters

Tl %2 ' 3 mean

(ms) (ms) (ms) (ms)

Glutamate 0.719 10,6 123,1 61.90,9 % 53,4 % 45,6 %

Spermine 2,83 23,7 149,3 92,75,4 % 38,8 % 55,8 %

4.4 D is c u s s io n

Spermine (100 pM) had a stimulatory effect on the activation properties of PO-

striatum N M D A receptors. Stimulatory effects of spermine at saturating concentrations of

glycine has only been observed in recombinant NM DA receptors containing N R la and

NR2B subunits. Although, it is not possible to compare directly the data presented in this

study which was obtained from single-channel currents with the reported data from

macroscopic currents, the stimulatory effect of spermine on PO-striatum N M D A receptors

suggests that these receptors share a property only found in N R la/N R 2B recombinant

NM DA receptors.

Although the overall effect of spermine was stimulatory, spermine (100 pM) also had

inhibitory effects. It produced a 20 % reduction in mean single channel current amplitude

with no change in the sublevel/main level amplitude ratio suggesting that spermine produced

a similar degree of reduction in the amplitude of both main level and sublevel openings. In

addition, no changes in the relative areas of the main level and sublevel were observed,

suggesting that the effect of spermine was not produced by either a selective increase in the

number of sublevel openings or a selective reduction in the number of main level openings.

Similar effects have been described in single NM DA receptor-channels from cultured spinal

cord neurons (Rock & Macdonald, 1992) and cultured hippocampal neurons (Araneda et a l ,

1993),

Spermine produced a reduction in mean shut time without reducing the time

constants of any of the six exponential components in the shut time distribution. Spermine

produced a 38 % increase in the relative area of the fastest exponential component (xi= 26 ps).

103

An increase in the proportion of brief closures has been described for NM DA receptors in

cultured hippocampal neurons (Araneda et aL, 1993). Even when spermine (100 pM) had

profound effects on each one of the three identified open periods, such effects produced only a

7 % reduction in mean open period.

In contrast, spermine produced a 95 % increase in mean burst length and an 84 %

increase in mean total open time per burst without increasing either the time constants of any

of the exponential components fitted to the burst distributions or the mean Pope„ per burst. It

was calculated that such an increase in mean total open time per burst would produce a 48 %

increase in mean charge transfer per burst.

In addition, a 145 % increase in mean cluster length and a 180 % increase in mean

total open time per cluster were observed. The calculated mean charge transfer per cluster

increased by 93 %. Ensemble averages built using clusters from single N M D A receptor

activations showed a 50 % increase in mean decay time-course as a result of spermine

application.

In summary, these data suggest that at the single channel level native NM DA

receptors from PO striatal neurons respond to spermine with an increase in their overall

activation properties which suggests that these receptors do share properties found in

recombinant N M D A receptors containing N R la and NR2B subunits.

104

CHAPTER 5

S in g le - C h a n n e l P r o p e r t ie s o f NMDA R e c e p t o r s in H ip p o c a m p a l

G r a n u le C e l l s f r o m O -D ay-O ld (PO) R a ts

5.1 Su m m a r y

i. The properties of single-channel currents produced by activation of NMDA receptors were

studied in outside-out patches excised from hippocampal granule cells in slices from 0-day-old

rats,

ii. Activation of NMDA receptors by glutamate (50 - 100 nM or NMDA ( 1 - 5 |iM) produced

either only high conductance or a mixture of both high and low conductance single-channel

activity. The number of single-channel amplitude levels was independent of the agonist used. In

patches containing only large amplitude unitary currents, two conductance levels with

magnitudes of 42 and 51 pS were identified. In patches containing large and small amplitude

unitary currents, four conductance levels were identified with magnitudes of 17, 33, 42 and 49

ps.

iii. Direct transitions between the shut and all but 17 pS currents were symmetric suggesting that

direct transitions to and from the shut level obeyed microscopic reversibility. Nearly all direct

transitions between open-channel current levels were also symmetric, except those involving the

17 pS level. Asymmetry was particularly evident for direct transitions connecting 17 pS and 42

pS single-channel currents.

iv. Distributions of all open times were usually fitted with 3 exponential components, indicating the

existence of at least 3 different open states. Independently of the agonist used, glutamate or

NMDA, 17 pS currents showed a single open state while 33, 42 and 50 pS currents showed at

least two different open states. N o differences in the duration of open times were observed

between single channel currents produced by glutamate or NMDA.

V . Independently of the agonist used, glutamate or NMDA, distributions of burst length and total

open time per burst were fitted with 3 exponential components. In patches with only high

conductance channels, no differences in mean burst length, total open time per burst and mean

burst Popen were observed between bursts of openings produced by glutamate and NMDA.

vi. For clusters, the number of exponential components that could be fitted to distributions of

cluster length and total open time per burst was dependent on the agonist used. In the presence

105

of glutamate, they could be fitted with up to five different exponential components while for

NMDA three exponential components were alw^s fitted. Apparently, no difference in mean

cluster length and total open time per burst was observed between clusters of openings

produced by glutamate or NMDA. In contrast, the mean cluster Popen was larger in clusters

produced by NMDA (0.85 ± 0.02) than in those produced by glutamate (0.71 ± 0.04).

vii. In patches containing only high conductance single-channel activity, information about super

cluster properties could be obtained only from a smaller number of patches, 2 out of 4 for

glutamate and 2 out of 3 patches for NMDA. Large variations in mean tcm were observed from

patch to patch. Apparently, mean super-cluster length and total open time per super-cluster were

larger in super-clusters produced by glutamate than in those produced by NMDA. In contrast,

mean super-cluster Popen for NMDA was ~3 times larger than for glutamate.

viii.In summary, NMDA receptors mediating two patterns of single-channel activity were found in

patches from PO-hippocampal granule cells. A pattern of high conductance single-channel

activity which was characterised by 42 and 51 pS currents and a pattern consisting of a mixture

of both high and low conductance single-channel activity which showed, in addition to high

conductance 42 and 49 pS currents, low conductance 17 and 42 pS currents. Absence of direct

transitions connecting the smallest (17 pS) and largest (49-51 pS) conductance unitary currents

as well as absence of direct transitions connecting 17, 42 and 50 pS currents suggested that high

and low conductance single channel activity may have been produced as a result of the activation

of two different NMDA receptor-channel populations.

ix. Finally, it is suggested that high conductance single-channel activity was probably the result of

the activation of NMDA receptors containing NR2B subunits which are highly expressed in PO-

hippocampus while low conductance single-channel activity was probably produced by

activation of NMDA receptors containing NR2D subunits which are also expressed in PO-

hippocampus.

106

5.2 I n t r o d u c t i o n

At PO, one of the main differences between the pattern of expression of NMDA receptor

subunits in striatum and dentate gyrus is the presence in PO-dentate gyrus of mRNA and protein

encoding the NR2D subunit (Monyer et al., 1994; Wenzel e toL, 1996, 1997). At PO, striatum and

dentate gyrus express the NRla splice variants: NR l-la, NRl-2a and NRl-4a plus the NR2B

subunit (Laurie & Seeburg, 1994; Monyer et at., 1994), but PO-dentate gyrus additionally expresses

mRNA and protein encoding the NR2D subunit (Monyer etoL, 1994; Wenzel e ta l, 1996, 1997 . To

investigate whether the presence of NR2D subunits in PO-dentate gyrus may have any effects on

single-channel activity mediated by NMDA receptors, receptor-channel activations produced by

glutamate or NMDA, were studied in patches excised from dentate gyrus granule cells in

hippocampal slices form 0-day-old (PO) rat brain.

5.3 R e s u l t s

5.3.1 Presence o f functional NM DA receptors in PO-hippocampal granule cells

In outside-out patches from PO-hippocampal granule cells, NMDA receptor-channel

activations were observed after bath application of glutamate (50-100 nM) or NMDA (1-5 pM) and

glycine (3-10 pM) in the presence of DNQX (5 ph^ (Figure 5.1). In contrast with PO-stiiatal

neurons, in PO-hippocampal granule cells differences in the number of amplitude levels to which

NMDA receptor-channels opened were observed between patches. The main difference was the

presence of patches with either only high amplitude single-channel currents (Figure 5.1A) or a

mixture of high and low amplitude single-channel currents (Figure 5.IB).

5.3.2 Properties o f single-channel currents

As in PO-striatum NMDA receptors, the amplitude and conductance of single-channel

currents produced by activation of NMDA receptors in PO-hippocampal granule cells were studied.

5.3.2.1 Stability o f single-channel current amplitudes

In patches containing onty" high amplitude single-channel currents, stability plot ana^sis of

single-channel current amplitudes showed the presence of two different current amplitude levels

(Figure 5.2A), a similar behaviour was observed in PO-striatum NMDA receptor-channels (Figure

4.2A).

107

A

! [

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100 ms

5tr*“IT

mh

I'I I'lll IM <

-p'-iv'TTTI

I**#'

m u \„ i r ,

T l ' ~ t■4*** urn.

[100 ms

Fig ure 5.1 Types of single-channel activity m ediated by N M D A receptors in PO hippocampal granule cells. Examples of single-channel currents from NM DA receptors in outside-out patches excised from PO hippocampal granule cells. Patches containing only high amplitude single-channel currents (A) and a mixture of high and low amplitude single-channel currents (B) were observed. Openings are downwards. Currents were low-pass filtered at 2 kH z (-3db, 8 pole Bessel filter). Each panel contains ten contiguous 500 ms sweeps of single channel recording. The solution bathing the extracellular face of the patches contained glutamate (0.05 pM) and glycine (3 pM) as agonists plus the non-NM DA receptor antagonist D N Q X (5 pM). Holding potential was -60 mV.

108

41

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-2 ■3 -4 -50 ■1Current (pA)

4000 8000 12000 16000 20000

Interval number

D800

600

400

200

■30 •1 -2 -5-4

Current (pA)

F ig ure 5.2 Single channel-current am plitudes of N M D A receptors in PO-hippocampal granule cells. Stability plots and amplitude distributions of NM DA receptor single-channel currents recorded in outside-out patches containing only high amplitude currents (A,C) or a mixture of high and low amplitude currents (B,D). A and B are stability plots built with 3798 and 7656 single-channel current amplitudes, respectively. Amplitudes of events shorter than 0.415 ms (2.5 filter rise-times) were excluded from the analysis. C and D are amplitude distribution histograms built with the same amplitudes plotted in A and B, respectively. C and D were fitted with 2 and 4 Gaussian components, respectively. The mean current amplitude, standard deviation and relative area of each Gaussian component were: 2.48 ± 0.2 pA (13 %) and 3.32 ± 0.1 pA (87 %) (C) and 1.10 ± 0.1 pA (15.2 %), 2.24 ± 0.3 pA (3.7 %), 2.72 ± 0.1 pA (38 %) and 3.39 ± 0.2 pA (43 %) (D). Glutamate (0.05 pM) and glycine (3 pM) were used as agonists. Holding potential was -60 mV.

109

In patches containing a mixture of high and low amplitude single-channel currents, stability

plots revealed the presence of an additional lower amplitude single-channel current level (Figure

5.2B). Unexpectedly, stability plot analysis of amplitudes also revealed that in patches containing a

mixture of high and low amplitude single-channel currents, amplitude levels were not evenly

distributed throughout the whole recording. Current amplitudes appeared to be grouped into periods

of either high or low amplitude. Single-channel current amplitudes switched from periods of high

amplitude to periods of low amplitude and vice-versa. The amplitude of the single-channel currents

within each period remained apparently constant. An analysis of the kinetic behaviour of channel

activations within these periods was later carried out (Section 5.3.3.1).

5.3.2.2 Distribution of single-channel current amplitudes

The mean amplitude of the single-channel currents was estimated by building amplitude

distribution histograms and then fitting these with sums of multiple Gaussian components. For each

patch, an amplitude distribution containing single-channel current amplitudes longer than 2.5 filter

rise-times were built and fitted with the sum of 2 and 4 Gaussian components (Figures 5.2C-D). N o

apparent differences in the mean amplitude of each Gaussian component were observed between

patches; in contrast, variations in their relative areas were evident.

Amplitude distributions built with data obtained from patches containing only high

amplitude single-channel currents (n = 3 - 4) were best fitted by the sum of 2 Gaussian components.

The relative area of the smaller Gaussian component (16 ± 2 %) was smaller than that of the larger

amplitude component (Figure 5.2C, Table 5.1). A similar observation was made for NMDA

receptors in patches from PO-striatal neurons (Figure 4.2B, Table 4.1).

Amplitude distributions obtained from patches containing a mixture of high and low

amplitude single-channel currents (n = 3) were usually best fitted by the sum of 4 Gaussian

components. The mean amplitude of the 1 Gaussian component was smaller than that of the 2"«*

component but its relative area was larger. The Gaussian component had the smallest relative

area of all Gaussian components, and this may be the reason why in some distributions this

component was not detected. The mean amplitudes of the 3'' and 4 Gaussian components were

also constant but their relative areas varied from patch to patch. In some patches, the relative area of

the 3'‘‘* Gaussian component was sometimes larger than that of the 4 component. N o apparent

differences in mean current amplitude and mean relative area (mean ± S.E.M) were observed when

single-channel currents were recorded in the presence of glutamate or NMDA (Table 5.1).

110

T able 5.1 Distribution of single channel current amplitudes

Gaussian Components

%st 3rd

(pA) (pA) (pA) (pA)

GlutamateOnly High 2.50 ± 0.07 3.26 ± 0.08

(n= 4 patches) 16 ± 2 % 84 ± 2 %

High & Low 1.08 ±0.02 2.01 ±0.07 2.67 ±0.03 3.37 ±0.04(n= 3 patches) 9 ±3% 3 ± 1 % 3 7 ± 3 % 51 ± 6 %

NM DAOnly High 2.50 ± 0.09 3.24 ± 0.09

(n= 3 patches) 16 ± 2 % 84 ± 2 %

High & Low 1.09 ±0.01 2.02 ±0.00 2.68 ±0.02 3.33 ±0.04(n= 3 patches) 11 ± 6% 3 ± 1 % 36 ± 9 % 50 ± 14%

5.3.2.3 Conductance o f single<hannel currents

The slope conductance of the each one of the identified single-channel current levels was

estimated by building current voltage relationships (I/V plots) in which mean single-channel current

amplitudes (pA) were plotted against the holding potential (mV) (Figure 5.3). In patches containing

only high amplitude single-channel currents, two conductance levels with magnitudes of 42 and 51

pS were identified (Figure 5.3A,C). In patches containing a mixture of high and low amplitude

single-channel currents four conductance levels were identified with slope conductances of 17, 33,

42 and 49 pS (Figure 5.3B,D). In patches containing either only high conductance or a mixture of

high and low conductance single-channel currents, no difference in single-channel conductance was

observed when currents were activated by glutamate or NMDA suggesting that the differences in the

number of conductance levels between patches were apparently agonist-independent.

5.3.2.4 Direct transitions between conductance levels

Direct transitions between the shut and all identified open-channel current levels were

analysed as well as direct transitions between open levels. In patches with either only high

conductance or high and low conductance single-channel activity, direct transitions between the shut

and open levels were the most common representing ~90 % of all direct transitions (Table 5.2).

I l l

BVoltage (mV)

-120 -100 -60 -40 -20 0-80

Voltage (mV)

0-100 -80 -60 -40 -20

17 pS

33 pS

42 pS

49 pS

-3

- -4

- -5

L -6

gO

D

...w

W42 pS 51 pS

» Shut

- 42 pS

F ig u r e 5.3 Conductance of NMDA receptor single-channel currents in PO-hippocampal granule cells. Current-voltage relationships from patches containing only- high amplitude single-channel currents (A) and a mixture of high and low amplitude single-channel currents (B). Each point shows the average ± S.E.M (n = 1 - 7) of mean current amplitude values obtained by fitting multiple Gaussian components to amplitude distributions. Patches were held at potentials between -100 and -20 mV. Linear regressions fitted through the points gave slope conductances values of 42 and 51 pS (A) and 17, 33, 42 and 49 pS (B).

112

T able 5.2 Frequencies o f direct transitions between shut level and open levels

H igh & Low Only H igh

Sequence(pS)

Glutamate (patches = 3)

(%)

NMDA (patches = 3)

(%)Sequence

(ps)

Glutamate (patches = 4)

(%)

NMDA (patches = 3)

(%)

0-^17 3.9 ± 1.1 (999)

4.2 ± 1.5 (392)

17-+0 4.7 ± 1.3 (1202)

5.2 ± 2.1 (494)

0-^33 0.9 ± 0.2 (169)

1.3 ± 0.3 (79)

3 3 - + 0 1.1 ± 0.2 (196)

1.2 ± 0.4 (81)

0 -+42 17.2 ± 1.8 (3953)

16.8 ± 3.8 (1507)

0->42 5.8 ± 2.0 (209)

4.1 ± 0.9 (223)

42 -+ 0 16.7 ± 1.8 (3838)

16.5 ± 3.4 (1456)

42-+0 5.9 ± 2.1 (212)

4.2 ± 0.9 (227)

0-+49 23.1 ± 2.5 (4478)

23.0 ± 5.0 (1476)

0-^51 39.7 ± 2.2 (2859)

40.5 ± 1.5 (2573)

49 -+ 0 23.3 ± 2.4 (4516)

23.3 ± 5.2 (1475)

51-+0 40.0 ± 2.3 (2878)

41.1 ± 1.4 (2615)

Sub-totalsn

90.9 % (19352)

91.4 % (6960)

Sub-totalsn

91.4 % (6158)

89.9 % (5638)

113

In patches with either only high conductance or high and low conductance single-channel

currents, the frequency of occurrence of direct transitions between pairs of consecutive events

involving the shut level was: 0^49,51 > 0*-»42 > 0^ 17 > CK->33 pS. In patches with high and low

conductance single-channel currents, direct transitions between the shut and all but the 17 pS

conductance level were symmetric with a similar proportion of direct transitions going in either the

closing or opening direction (Table 5.2). This suggested that direct transitions to and from the shut

level obeyed microscopic reversibility.

Direct transitions between open-channel current levels were also analysed (Figure 5.4). The

order of occurrence of direct transitions between pairs of consecutive events involving only open-

channel current levels was: 42<-> 49,51 > 17^42 > 33^49 > 33^ 42 events (Table 5.3). As with

direct transitions to and from the shut level, nearly all direct transitions between open-channel

current levels, with the exception of those involving the 17 pS level, were symmetric. Asymmetry

was particularly evident for direct transitions connecting 17 and 42 pS single-channel currents. The

percentage of direct transitions going from 42 to 17 pS was larger than the percentage of transitions

going from 17 to 42 pS (Figures 5.4B, D). This suggested that direct transitions involving 17 and 42

pS single-channel currents did not obey microscopic reversibility.

5 .3 .3 Properties o f single-channel activations

As in patches from PO striatal neurons, the activation properties of NMDA receptors in PO-

hippocampal granule cells were studied in those patches that showed the lowest Popen after bath

application of glutamate (50 nM) or NMDA (5 in the presence of g^cine (3 - 10 pM) and

DNQX (5 |uM). Data used for analysis of glutamate-induced activations in patches showing only

high conductance single-channel activity were obtained from four outside-out patches with an

overall mean Popen of 0.012 ± 0.004 (0,002 - 0.019) and a mean number of openings per second of

2.9 ± 0.8 (0.6 - 4.6). For patches showing high and low conductance single-channel activity, data was

obtained from three patches with an overall mean Popen of 0.014 ± 0.004 (0.009 - 0.024) and a mean

number of openings per second of 4.9 ± 1.8 (2.6 - 9.3).

For analysis of NMDA-induced activations, data was obtained from three outside-out

patches with an overall mean Popen of 0.040 ±0.018 (0.005 - 0.060) and a mean number of openings

per second of 8.24 ± 3.5 (1.2 - 12.7) in patches with only high conductance single-channel activity.

For patches with high and low conductance single-channel activity, data was

114

■5

42 -*■ 51 pS-4

1■3

c -2

I -151 - * 42 pS

00 ■ 1 2 ■3 -4 -5

Current ( i ) ( pA )

D

1

I

42 -» 49 pS

L 17 ^ 42 p S ,

49 ■ * 42 pS

42 -*■ 17 pS

LI I I I 1 I I I 1 1 I I I I I I I j 1 1 1 1 I i-2 -3 -4

Current ( i ) { pA )

F igu re 5.4 Direct transitions between consecutive open-channel current levels. Plots show the number of direct transitions between consecutive open-channel current levels (shut level not included) in outside-out patches containing only high conductance unitary currents (A,C) or high and low conductance unitary currents (6,D). Each dot represent a direct transition, considered as a change in open-channel current level from one amplitude level to another w ithout intervening closures longer than the shut time resolution, 50 ps in these examples. Analysis included openings on either side of a direct transition that were longer than 2.5 filter rise-times. B and D are three-dimensional representations of data shown in A and C, respectively. The volume of each peak indicates the relative frequency of each type of transition. An amplitude-based separation of unitary currents was carried out by calculating critical amplitude values (Ag^J producing an equal percentage of misclassified events between the four Gaussian components fitted to the amplitude distribution shown in figure 5.2D. A,. , values were 1.71, 2.35 and 3.05 pA.

115

T able 5.3 Frequencies o f direct transitions between open-channel current levels

High & Low Only High

Glutamate NMDA Glutamate NMDASequence (patches = 3) (patches = 3) Sequences (patches = 4) (patches = 3)

(pS) (%) (%) (pS) (%) (%)

17-)" 33

33->17

17 -> 42 0.6 ± 0.3 0.9 ± 0.4(164) (91)

42 ^ 17 1.1 ± 0.4 1.6 ± 0.9(292) (163)

17->49

49->17

33->42

42->33

33->49

49->33

42->49 2.6 ±0.5 2.1 ± 0.7 42->51(489) (155)

49->42 2.5 ±0.4 2.2 ± 0.6 51->42

0.3 ± 0.1 0.5 ± 0.3(42) (43)

0.5 ± 0.2 0.5 ± 0.3(67) (39)

0.7 ± 0.4 0.4 ± 0.1(106) (41)

0.8 ± 0.5 0.5 ± 0.2(114) (43)

2.6 ± 0.5 2.1 ± 0.7(489) (155)

2.5 ± 0.4 2.2 ± 0.6(462) (162)

Sub-totals 9.1 % 8.6 % Sub-totalsn (1736) (737) n

4.3 ± 0.5 5.1 ± 1.3(335) (329)

4.3 ± 0.5 4.9 ± 1.3(334) (320)

8.6 % 10.0 %(669) (649)

116

obtained from three patches with an overall mean Popen of 0.041 ± 0.026 (0.007 - 0.1) and a mean

number of openings per second of 13.7 ± 8.7 (2.3 - 35.1).

5.3.3.1 S ta b ility p lo t analysis o f shut timeSy open times a n d Popen

Stability plots from patches with only high conductance single-channel activity were

different from those obtained from patches with high and low conductance single-channel activity.

Figure 5.5 shows stability plots for amplitudes, shut times, open times and Popen from a patch

containing only high conductance single-channel activity (Figures 5.5A, C, E, G) and from a patch

with high and low conductance single-channel activity (Figures 5.5B, D, F, FÏ). In both cases, large

reductions in mean shut time accompanied by high Popen periods were observed (Figures 5.5C, D).

The mean duration of the open intervals was much more stable in patches containing only high

conductance single-channel activity than in patches containing high and low conductance single

channel activity in which variations in mean open time (Figure 5.5F) seemed to coincide with the

changes in current amplitude detected by the stability plot for amplitudes (Figure 5.5B).

In patches with high and low conductance single-channel activity, the apparent correlation

between the changes in amplitude and mean open time (Figure 5.5) was examined by dividing the

original data into sections based on the changes in current amplitude (Figure 5.6). These sections

were then pooled and analysed by building new stability plots (Figure 5.7), amplitude distributions,

shut time distributions (Figures 5.8A1, A2) and open time distributions (Figures 5.10A1, A2) with

data containing either mainly high conductance or low conductance single-channel events. Pools of

sections containing mainly high amplitude single-channel currents or mainly low amplitude single

channel currents were ana^sed separately before comparing them with the activity observed in the

whole data record.

The results of stability plot analysis for shut times, open times and Popen are shown in Figure

5.7. Surprisingly, in both sets of pooled data sections, the mean duration of the open intervals was

stable (Figure 5.6). The mean open time was larger for sections containing mainly high amplitude

single-channel activity than for sections containing mainly low amplitude single-channel activity

(Figures 5.7C1, C2). Stability plots for shut time intervals showed large reductions in mean shut time

whenever a high Popen period occurred and that high Popen periods were associated with the activity of

high amplitude single-channel activity (Figures 5.7Bi, Di), while during low amplitude single-channel

activity, the mean shut time and Popen remained stable (Figures 5.7Bz, Di). The overall mean shut

time was larger during high amplitude single-channel activity than during low amplitude single

channel activity

117

Only high conductance High & Low conductance

1 p- 0 -1 -2 -3

-4

-5

^ 10000 è. 1000

mean = 387.03 ms

100

E

m ean = 4.90 ms

m ean = 0.0125

0.10.01

0.001OOOOlL 2000 4000 6000 8000

Interval numtter

_ lOOOOg- “ lOOO ijiI 100 L

mean = 95.15 m s

F

H

^ 100 Em ean = 2.32 m s

0.0001

m ean = 0.0238

8000 12000

interval number

F ig u re 5.5 Stability plot analysis of shut times, open times and Popen* Stability plots for amplitudes (A,B) are the same as those shown in figure 5.2A-B; they are shown here for comparison. In C to H , plots show running averages of shut time intervals (C,D), open intervals (E,F) and (G,H). Each bin represents the average of 50 intervals with increments of 25 intervals between averages. Horizontal dashed lines represent overall mean values for the whole data record. Overall mean shut time, open time and P p n values are shown for each plot. Resolution for openings and shuttings was 50 ps. Patches were exposed to a constant concentration of glutamate (0.05 pM) and glycine (3 pM). Holding potential was -60 mV.

118

<Q_

Q)"D

Q_

E<

4000 8000 12000

Interval number16000 2 0 0 0 0

F ig u re 5.6 Sectioning of stability plot for amplitudes in patches containing high and low conductance single-channel activity. Sections of the data containing mainly high amplitude currents (point line boxes, n = 9), low amplitude currents (continuous line boxes, n = 7) and a mixture of low and high amplitude currents (no box, n = 5) are shown. Sections were used for further analysis. Data was obtained from a single outside-out patch in the continuous presence of 50 nM glutamate and 3 pM glycine. Holding potential was -60 mV. Plot contains 8143 single-channel current amplitudes longer than 2 filter rise times.

119

‘High Amplitude’ ‘Low Amplitude’

0

1

D.

m ean = 173.5

C

m ean = 3 54 m s

m ean = 0 .0200

0.012 0.001

0.0001 1000 2000 4000 50003000

Interval num ber

10000m ean = 35.2 ms

100

0.1c.

m ean = 1.48 ms

1I 0.1 001

ii 0.001

00001

„ m ean = 0.0404 p,

1000 2000 3000 4000 5000 6000 7000

Interval number

F ig u re 5.7 Stability plot analysis of amplitudes, shut times, open times and for sections of data containing mainly high amplitude openings (A„ B„ Cj, Di) and mainly low amplitude openings (A^, B^, Q , Dj). Stability plots for amplitudes (Aj, Aj), shut times (Bj, B ), open times (Cj, Cj), and (Dj, Ô 2). O n Bj, B2 , Cl, C 2 , Di and D 2 , bins represent the average of 50 intervals with increments of 25 intervals between averages. Dashed lines shown on B;, B2 , Cj, Q , D; and D 2 represent the overall mean shut time, open time and respectively. Dashed lines shown on A; and A; represent the shut level. Pooled sections of mainly high amplitude openings contained a total of 5141 intervals and sections of mainly low amplitude openings contained a total of 6911 intervals.

120

(Figures 5.7Bi, B2 ). In contrast, the overall mean Popen was larger during low amplitude single

channel activity than during high amplitude single-channel activity (Figures 5.7Di, D 2 ).

Under steac^ state recording conditions, large reductions in mean shut time accompanied by

high Popen periods have been shown to be a characteristic feature of the kinetic behaviour of native

and recombinant NMDA channels (Gibb & Colquhoun, 1991; Stem etoL 1992). They could also be

an indication of instability in the kinetic behaviour of this particular NMDA receptor-channel

population. In order to test this possibility, all shut time intervals used to build stability plots for

pooled sections containing mainly high amplitude single-channel activity and on which large

reductions in mean shut time and high Popen periods were observed were also used to build

distributions of shut times (Figures 5.8A1, A2). Such distributions were similar to shut time

distributions built with shut intervals obtained from patches containing only high amplitude single

channel activity (Figure 5.8B) suggesting that the large reductions in mean shut time and high Popen

periods observed on sections of the data containing mainly high amplitude single-channel activity

were quite likely produced by a receptor-channel behaviour similar to that producing channel

opening in patches containing only high amplitude single-channel activity and not due to instability

in kinetic behaviour.

Because sectioning of the data did not tota% exclude intervals containing low conductance

single channel currents, shut time distributions were built with only shut time intervals that were

bordered on each side by openings to the highest, 50 pS, level. These distributions were built for

both patches with high and low conductance single-channel activity, and for patches with only high

conductance single-channel activity. Figure 5.9 shows the results of this analysis. Notably, the time

constants and relative areas of the first three components were identical between patches with high

and low conductance single-channel activity and patches with only high conductance single-channel

activity (Figure 5.8A1,B). These three components have been shown to occur within individual

NMDA receptor-channel activations (Gibb & Colquhoun, 1991; 1992). This result suggests that the

time constant and relative frequency of shut states occurring within periods of activation of 50 pS

openings were apparently independent of them occurring in patches with high and low conductance

single-channel activity or in patches with only high conductance single-channel activity.

5.3.3.2 Distribution o f shut times

Distributions of shut times from patches with only high conductance single-channel activity

(Figure 5.8A; Table 5.4) were different from those of patches with high and low

121

Patch with high and iow amplitude currents360 I—

CO 250

Ï 40

0.01 1 10 100 1000 Shut time (ms) (log scale)

10000 100000

A2 Sections with mainly low amplitude currents

T (m«) area (%) 360 — T (ms) area (%)0.0316 14.5 'oT 0.152 15.10.123 12.2 S 250 — vTK 0.995 13.20.568 13.2 (/} I / U 12.0 44.7

1.52 11.1 "S 2 160 \] 76.7 24.6

14.0 25.7 2 369.6 2.5101.7 16.2 (0845.1 7.2 § . 90

10 —

0.01mil mil lljlL mil mill

1 10 100 Shut time (ms) (log scale)

1000 10000 100000

Sections with mainly high amplitude currentsT (ms) area (%)0.0389 38.8

0.720 32.510.5 11.1

100.1 6.1946.4 11.4

ID 40

I I II

Patch with only high amplitude currents

0.01 1 10 100 1000 Shut time (ms) (log scale)

10000 100000

T (m«> crea (%)0.02650.410

1.2813.4

430.72688.

w 360

......1 10 100 1000 Shut time (ms) (log scale)

10000 100000

Figure 5.8 Distribution of the duration of shut times. Histograms are shut time distributions from a patch containing a mixture of high and low amplitude openings (A), pooled sections of data from A with mainly high amplitude openings (Al) and mainly low amplitude openings (A2), and from a different patch containing only high amplitude openings (B). Histograms were fitted with seven (A) and five (Al, A2, B) exponential components. Time constants (t) in ms and relative areas (%) for each fitted exponential component are shown on each histogram. The overall mean shut time and predicted number of shut intervals were: (A) 80.8 ms, 9728; (Al)115.9 ms, 3528; (A2) 33.6 ms, 3460; and (B) 210.4 ms, 5618.

122

conductance single-channel activity (Figure 5.8B; Table 5.4). Because of their different relative areas,

exponential components were much easier to identify in shut time distributions from patches with

only high conductance single-channel activity than in those from patches with high and low

conductance single-channel activity in which exponential components had more similar relative

areas.

5.3.3.2.1 Patches w ith only high conductance single-channel activity

In patches with only high conductance single-channel activity that were exposed to

glutamate, 2 out of 4 shut time distributions were satisfactorily fitted by the sum of 5 exponential

components; while for the rest of the distributions, fitting a 6 exponential component (around 1.0

ms) improved the fitting. For patches that were exposed to NMDA (5 pM), shut time distributions

may be less informative because of their higher mean Popen (0.040 ± 0.018). High mean Popen did not

allow a good separation between single channel activations. These patches usually contained a larger

than desirable number of superimposed single-channel activations.

5.3.3.2.2 Patches w ith high and low conductance single-channel activ ity

In contrast, in patches with high and low conductance single-channel activity (n=2) that

were exposed to the same NMDA concentration (5 pM) a ~ 4 times lower mean Popen (0.009) was

observed. A satisfactory fit of these distributions was obtained by the sum of 6 exponential

components. For those that were exposed to glutamate, 3 out of 4 shut time distributions were best

fitted by the sum of 7 exponential components. The remaining patch was fitted with 6 exponential

components (fitting an extra 7* component did not improve the fit).

In all patches, the magnitude of the time constants ranged from tens of microseconds to

seconds (Figure 5.8; Table 5.1). For the 1 exponential component, no apparent differences in mean

time constant were observed between patches. The slowest and more variable mean time constant

(59 ± 19 ms) was observed in patches with high and low conductance single-channel activity ejqjosed

to NMDA. Mean relative areas were also similar between patches except for patches with only high

conductance single-channel activity exposed to NMDA which showed the largest mean relative area

(63 ± 7 %). For the 2" exponential component no apparent differences in mean time constants and

relative areas were observed between patches. The 3" exponential component showed two-fold

slower mean time constants in patches with high and low conductance single-channel activity than in

patches with only high conductance single-channel activity but mean relative areas were similar

between patches. The 4^ exponential component had slower mean time constants (18 + 1 ms) in

patches with high and low conductance single-channel activity exposed to NMDA than in patches

123

Glutamate

Only High (n= 4 patches )

High & Low (n= 3 patches)

N M D A


High & Low (n= 2 patches )

(fis)

34 ± 3 40 + 7 %

34 ± 2 25 + 7 %

23 ± 4 63 + 7 %

58 + 19 21 + 8 %

T a b le 5.4 Distribution of shut times for glutamate and NM DA

%2 ' 3 ^4 "5

(ms)

0.3 ± 0.04 14 + 2 %

0.4 ± 0.07 16 ± 2 %

0.3 ± 0.08 9 + 2%

0.4 + 0.07 20 + 6 %

(ms)

1.0 ±0.3 10 ± 1 %

1.8 ± 0.2 1 1 ± 1 %

0.8 ± 0.05 9 ± 6 %

2.0 ± 0.3 8 ± 1 %

(ms)

11 ± 1 8 ± 1 %

12 ± 1 15 ± 5 %

7 ± 1 4 ± 1 %

18 ± 1 11 ± 1 %

(ms)

678 ±331 7 ± 2 %

65± 17 11 ± 3 %

394 ± 297 6 ± 0.4 %

458 ± 73 34 ± 3 %

(ms)

2049 ± 719 21 ± 11 %

281 ± 50 10 ± 1 %

910 ±622 8 ± 2 %

1625 ± 34 6 ± 1 %

(ms)

1316 ±300 12 ± 2 %

Mean

(ms)

445 ± 243

190 ± 56

91 ±61

258 ± 24

124

with only high conductance single-channel activity ( 7 + 1 ms). Mean relative areas were larger and

more variable (16 ± 5 %) in patches with high and low conductance sing)e-channel activity exposed

to glutamate and smaller ( 4 + 1 %) in patches with onty" high conductance single-channel activity

exposed to NMDA.

The 5^ exponential component showed large variations in mean time constant and relative

area between patches. For glutamate, mean time constants were slower (867 + 331 ms) in patches

with only high conductance single-channel activity and faster (65 + 17 ms) in patches with high and

low conductance single-channel activity, no apparent differences in mean relative areas were

observed. In contrast, for NMDA-induced single-channel activity mean time constant for the 5

component were similar between patches but mean relative areas showed large variations. Larger

variations in the mean time constant of the 6* exponential component were observed between

patches. For glutamate, the slowest mean time constant (2049 ±719 ms) was obtained from patches

with only high conductance single-channel activity and the fastest (281 ± 50 ms) from patches with

high and low conductance single-channel activity.

5.3.3.2.3 Distribution of shut times bordered on each side by 50 pS currents

Shut time distributions containing only shut time intervals that were bordered on each side

by openings to the highest conductance levels: 49 - 51 pS (50 pS level) were built for both patches

with only high conductance single-channel activity, and for patches with high and low conductance

single-channel activity. Before distribution of shut times bordered on each side by openings to the 50

pS level were built, an amplitude-based separation of unitary currents to the 50 pS level was carried

out by calculating critical amplitudes values (Amt) between adjacent Gaussian components fitted to

the amplitude distribution. Aait values producing an equal percentage of misclassified events were

used. In patches with only high conductance single-channel activity, single-channel currents with

amplitudes ranging between 2.96 + 0.02 pA and 4.05 ± 0.06 pA and between 2.88 ±0.11 pA and

4.07 ± 0.07 pA were used for glutamate and NMDA, respectively. In patches with high and low

conductance single-channel activity, single-channel currents with amplitudes ranging between 3.05 ±

0.05 pA and 4.13 ± 0.07 pA and between 3.03 ± 0.04 pA and 4.20 ± 0.00 pA were used for glutamate

and NMDA, respectively. Distribution of shut times bordered on each side by openings to the 50 pS

level were fitted with sum of 4 or 5 exponential components (Figure 5.9, Table 5.5).

125

Patch with high and low amplitude currents360 I—

(ms) area (%)0.0267CD 2 5 0

92 4 0

I I 111000 10000 100000

Shut time (ms) (log scale)

B3 6 0 I—

Patch with only high amplitude currents

T (ms) area (%)0.0217

0.82910.9

1272.2600.

1 10 100 1000 Shut time (ms) (log scale)

62.327.6

6 . 0

0 . 8

3.3

CD 4 0

I I III I III I III10000 100000

Figure 5.9 Distribution of the duration of shut times bordered on each side by 50 pS currents in patches with high and low conductance (A) and only high conductance (b) single-channel currents. Histograms are distribution of the duration of all shut times bordered on each side by 50 pS openings, amplitudes ranges were 3.05 to 4.10 pA (A) and 2.99 to 3.95 pA (B). Histograms were fitted with five exponential components. Time constants (t) in ms and relative areas (%) for each fitted exponential component are shown. The overall mean shut time and predicted number of shut intervals were: 35.9 ms, 3359 (A) and 93.7 ms, 5437 (B).

126

T a b le 5.5 Distribution of shut times bordered on each side by 50 pS currents

Glutamate



N M D A



(ps)

27 ± 3 51 ± 10 %

25 ± 8 55 + 8 %

27 ± 2 51 ± 10 %

2 7 + 1 47 ± 4 %

(ms)

0.69 ± 0.08 28 + 4 %

0.75 ± 0.04 28 + 6 %

0.74 ± 0.08 32 ± 6 %

0.76 + 0.04 37 + 3 %

(ms)

8.0+1.47 + 2%

6.7 ± 2.08 + 2%

4.9 + 0.9 7 ± 3 %

5.8 + 1.4 9 + 4%

(ms)

120 + 78 3 + 1%

27*1 %

102 ± 65 4 ± 1 %

(ms)

1577 ± 442 13 ± 6 %

1280 ± 269 6 + 3%

593 ± 369 9 ± 2 %

1428 + 656 5 + 2%

Mean

(ms)

242 + 147 9112

70 + 23 7558

50 ±28 4484

41 + 19 1032

^Detected in only 1 out of 3 patches

127

Except for the larger mean shut time obtained from patches with only high conductance

single-channel activity that were exposed to glutamate, the overall mean shut time was similar

between all patches. N o apparent differences in the mean time constants and relative areas of 2

and 3''' exponential components were between patches with only high conductance and those with

high and low conductance single-channel activity or between patches exposed to glutamate or

NMDA. A 4^ exponential component with a mean time constant around 100 ms was clearly evident

only in patches with high and low conductance single-channel activity but not in patches with only

high conductance single-channel activity. A S**’ exponential component was detected in all patches, it

hada similar time constant between nearly all patches, except in patches with high and low

conductance single-channel activity exposed to NMDA (Table 5.6).

5.3.3.3 Distribution of individual open times

Distributions of the duration of individual openings to all amplitude levels were built and

fitted by the sum of three exponential components (Figure 5.10). The overall mean duration of

individual openings was ~1 ms (28 - 41 %) longer in patches with onty high conductance single

channel openings than in patches with high and low conductance single-channel openings (Table

5.6).

T able 5.6 Distribution of individual open times to all am plitude levels

?! 3 mean

(ps) (ms) (ms) (ms)

Glutamate

Only High (n = 4 patches)

45 ± 4 17 + 6 %

0.73 ± 0.2 29 + 5 %

4.1 ± 0.6 54 + 5 %

2.50 ± 0.6 (9293)

High & Low (n = 3 patches)

45 ± 2 10 + 5 %

1.08 + 0.1 59 + 5 %

4.1 + 0.331 + 1 %

1.95 + 0.2 (19332)

N M DA

Only High (n = 3 patches)

65 + 3 7 + 5%

0.73 ±0.1 31 + 3%

4.4 + 0.462 ± 1 %

2.94 + 0.3 (4213)


48 + 12 24 + 9 %

1.08 + 0.1 44 + 9 %

4.1 + 0.432 + 9 %

1.73 + 0.2 (8616)

128

Patch with high and low amplitude currents A1 Sections with mainly low amplitude currents2000

T (ms) area C%) 10.7

1.03 57.43.31 32.0

0.0666O1280

2 720

320

0.01 0.1 10 100Open time (ms) (log scale)

640T (m <rea (%)

12.60.931 61.02.06

<Ds« 360

2

0.0658

26.4

160

E


A2 Sections with mainly high amplitude currents 8 Patch with only high amplitude currents640

T (mO area <%) 9.9

1.22 39.63.81 50.6

0.0651

(O 360

3 160


1000T (ms)

0.0396 1.34 4.07

1 (%) 4.9

28.0 67.0

g 640

360

160

0.01 0.1 100Open time (ms) (log scale)

Figure 5.10 Distributions of the duration of individual apparent openings. Histograms are individual open time distributions from a patch with both high and low amplitude openings (A), from pooled sections of A containing mainly high amplitude openings (Al) and mainly low amplitude openings (A2), and from a patch containing only high amplitude openings (B). Histograms were all fitted with three exponential components. Time constants (♦) in ms and relative areas ( %) for each fitted exponential component are shown on each histogram. The overall mean duration of individual openings and predicted true number of openings were: (A) 1.66 ms, 11411; (Al) 2.41 ms, 3751; (A2) 1.12 ms, 4393 and (B) 3.10 ms, 4941.

129

The most noticeable difference between patches was in the relative areas of the 2"* and 3'' *

exponential components. In patches with on^ high conductance single-channel activity, the relative

area of the 3 ' exponential was always larger than the relative area of the exponential component;

in contrast, in patches with high and low conductance single-channel activity the relative area of the

2"(l exponential component was larger than that of the exponential component.

For the exponential component, there were no apparent differences in mean time

constant and relative area between glutamate and NMDA or between patches. For the 2 '

exponential component, mean time constants were -0.4 ms ( -30,% ) slower and mean relative areas

were larger in patches with high and low conductance single-channel openings than in patches with

only high conductance openings. For the 3* exponential component, differences in relative areas but

not in mean time constants were observed between patches. Relative areas were larger in patches

with high and low conductance single-channel openings than in patches with only high conductance

openings (Table 5.6).

5.3.3.3.1 Distribution of open times conditional on current am plitude

An amplitude-based separation of unitary currents was carried out by calculating critical

amplitude values (Acdt) producing an equal percentage of misclassified events between adjacent

Gaussian components fitted to the amplitude distribution. Open time distributions containing

openings to each one of the identified conductance levels were built and fitted with exponential

components (Figure 5.11).

a) 17 pS currents

Openings to the 17 pS level were detected in all patches with high and low conductance

single-channel activity. Open time distributions were fitted with the sum of 1 exponential

component (Figure 5.11A). There were no apparent differences in mean open time between

openings activated by glutamate or NMDA (Table 5.7).

h) 33 pS currents

Openings to the 33 pS single-channel currents were not detected in all patches. In those

patches in which 33 pS openings were detected, distributions were fitted with the sum of 1 and

2 exponential components for glutamate or NMDA. (Figure 5.1 IB). There were no apparent

differences in mean open time between glutamate and NMDA. The 1* (fastest) exponential

component had the largest relative area (Table 5.8),

130

17 pS 33 pS250

T (ms) d e a (%) 1.71 100.0

I2 90

I40

I10

0.1 100


160 |—T (ms) area (%) 0.3538

“ 90

100.0

2

ÈE

0.1 100Open time (ms) (log scale)

42 pS 49 pS640

T (ms) area (%) 0.893

1.7586.513.5w 360

ZJ 160

40

1000.1Open time (ms) (log scale)

360

T (ms) area (%) 1.46 25.33.55 74.7

160

40

01 10

Open time (ms) (log scale)1000.1

F ig u r e 5.11 D istribution o f the duration o f individual openings conditional on amplitude in a patch w ith high and low conductance single-channel activity.An amplitude-based separation of unitary currents was carried out by calculating critical amplitudes values (A^j producing an equal percentage of misclassified events between the four Gaussian components fitted to the amplitude distribution shown in figure 5.2D. A values were 1.71, 2.35 and 3.05 pA. Amplitude ranges were: 0.4 - 1.71 pA (A), 1.71 - 2.35 pA (B), 2.35 - 3.05 pA (C) and 3.05 - 4.20 pA (D). Histograms are distributions of 1241 (A), 247 (B), 3438 (C) and 3363 individual open times fitted with 1 (A,B) and 2 (C,D) exponential components. Time constant and relative areas for each fitted exponential component are shown. The overall mean duration and predicted number of openings on each distribution were: 1.71 ms, 1479 (A); 0.35 ms, 578 (B); 1.01 ms, 4698 (C) and 3.02 ms, 3768 (D). Resolution for openings and shuttings was 50 ps. Only bins containing events longer than 300 ps are shown. Glutamate (50 nM) and glycine (3 pM) were used as agonists.

131

Table 5.7 Distribution o f open times for 17p S currents

Glutamate


NM DA


(ms)

1.51 ±0.19 (1928)

1.60 ±0.25 (811)

Table 5.8 Distribution of open times for 33 pS currents

mean

Glutamate


NM DA


(ms)

0.40 ± 0.03 81 ± 10 %

0.46 ± 0.23 88 ± 6 %

(ms)

1.46 ± 0.01 19 ± 10 %

2.14 ±0.8 12 ± 5 %

(ms)

0.60 ±0.13 (1148)

0.62 ± 0.20 (262)

132

c) 42 pS currents

For the 42 pS single-channel currents, 2 out of 4 open time distributions for glutamate and

3 out of 3 distributions for NMDA were fitted with the sum of 2 exponential components in patches

with only high conductance single-channel activity. In contrast, in patches with high and low

conductance single-channel activity all distributions could be fitted with 2 exponential components

(Figure 5.11C). There were no apparent differences in mean open time between patches with only

high conductance single-channel activity and patches with high and low conductance single-channel

activity. For the 1 component, there were no apparent differences in mean time constant or relative

area between all patches. The 1 (fastest) exponential component had the largest relative area. In

patches with only high conductance single-channel activity exposed to glutamate, the 2"‘ e3q)onential

component was not detected in 2 out of 4 distributions (Table 5.9).

d) 49 - 51 pS currents (50 pS)

For the largest conductance single-channel currents (50 pS currents), a satisfactory fit of

open time distributions was usually but not always obtained by the sum of 2 exponential

components (Figure 5.1 ID). There were no apparent differences in mean open time between

patches with only high conductance and patches with high and low conductance single-channel

activity (Table 5.10). For the 1 exponential component, time constants were ~0.5 ms (40 - 48 %)

slower in patches with high and low conductance single-channel activity than in patches with only

high conductance single-channel activity. For the 2" exponential component, no differences in mean

time constants and relative areas were observed between patches (Table 5.10).

5.3.3.4 Bursts

By analogy with previous reports in which bursts of openings mediated by high conductance

(40/50 pS) single-channel activity were also studied at low agonist concentrations (Gibb &

Colquhoun, 1991, 1992), in patches with only high conductance single-channel activity, bursts were

defined as groups of openings separated by shuttings or gaps shorter than mean tak values (1.41 ±

0.10 ms and 1.13 ± 0.11 ms for glutamate and NMDA, respectively) calculated between the 3*"* (1.0 ±

0.3 ms and 0.8 + 0.05 ms for glutamate and NMDA, respectively) and 4 (11 ± 1 ms and 7 ± 1 ms

for glutamate and NMDA, respectively) exponential components of the shut time distribution (Table

5.4).

133

Table 5.9 Distribution of open times for 42 pS currents

'Cl 'C2 mean

(ms) (ms) (ms)

Glutamate

Only High 0.77 ± 0.09 *4.82 ± 1.6 1.00 ±0.15(n= 4 patches ) 93 + 4 % 7 ± 4 % (1701)

High & Low 0.85 ± 0.05 2.39 ± 0.3 0.99 ± 0.02(n= 3 patches) 90 + 2 % 10 ± 2 % (7526)

NM DA

Only High 0.59 ±0.11 3.68 ± 0.4 1.00 ± 0.07(n= 3 patches) 86 + 4 % 14 ± 4 % (954)

High & Low 0.86 ±0.15 4.39 ± 1.4 1.18 ± 0.03(n= 3 patches ) 80 ± 16 % 20 ± 16 % (2826)

’‘’Onty’ detected in 2 out of 4 patches

T a b le 5.10 Distribution of open times for 50 pS currents

'Cl mean

(ms) (ms) (ms)

Glutamate

Only High 0.60 ± 0.2 4.15 ±0.5 3.44 ± 0.6(n= 4 patches ) 22 ± 7 % 78 ± 7 % (6465)

High & Low 1.42 ± 0.2 4.34 ± 0.2 3.32 ±0.1(n= 3 patches) 35 ± 7 % 65 ± 7 % (7114)

NM DA

Only High 0.55* 4.14 ±0.3 3.83 ± 0.2(n= 3 patches) 7% 93 ± 7 % (3505)

High & Low 0.94 ± 0.5 4.29 ± 0.6 3.61 ± 0.4(n= 3 patches ) 20 ± 15 % 80 ± 12 % (2494)

’'’Detected in only 1 out of 3 patches. Total number of events are shown in parentheses

134

5.3.3.4.1 Distribution of hurst lengths

Burst length distributions containing burst of openings separated by mean tcHt values of 1.41

± 0,10 ms for glutamate and 1.13 ± 0.11 ms for NMDA were built from patches with only high

conductance single-channel activity. They were fitted with the sum of 3 exponential components

(Figure 5.12). N o differences in mean burst length were observed between burst of openings

induced by glutamate or NMDA (Table 5.11). In addition, no apparent differences in the mean time

constants and relative areas of the three exponential components fitted to burst length distributions

were observed between glutamate and NMDA (Table 5.11).

5.3.3.4.2 Distribution of total open time per hurst

As for the burst length distribution, total open time per burst distributions were fitted by the

sum of 3 exponential components (Figure 5.12). There were no apparent differences in mean total

open time per burst between bursts produced by NMDA or glutamate. There were also no apparent

differences in the mean time constant and relative areas of the three exponential components fitted

to the total open time per burst distribution between burst of openings produced by glutamate or

NMDA (Table 5.12).

5.3.3.4.3 Burst Popen

There were no apparent differences in mean burst Popen between burst of openings

produced by glutamate or NMDA (Table 5.13). The mean burst Popm was similar to that of NMDA

receptor activations from PO-striatal neurons and higher than the value of 0.86 reported for

glutamate-activated NMDA receptors in dissociated adult CAl neurons (Gibb & Colquhoun, 1992).

S.3.3.5 Bursts o f 50 pS currents

To find out whether bursts of high conductance single-channel activity were different

between patches containing only high conductance single-channel activations and patches containing

a mixture of high and low conductance single-channel activations, distributions of burst length and

total open time per burst containing bursts that consisted of any series of openings with

conductances within the 50 pS level were built (Figure 5.13). Shut intervals and openings outside the

50 pS level were considered as ‘within bursts’ only if they had a total duration less than a critical shut

time or tcm. Openings with conductances outside the 50 pS level that were considered as ‘within

bursts’ were treated as shut intervals. Critical shut time or tent values were calculated between the 2"‘

and exponential components fitted to distributions of the duration of shut times bordered on

each side by openings with conductances within the 50 pS level. Mean ïcrit values were 2.02 ± 0.3 ms

for patches with only high conductance and 1.94 ± 0.2 ms for patches with high and low

conductance single-channel activity.

135

Glutamate NMDA

250 r T (ms) area (%) : 0.0906- 2.78- 12.7

30.524.6 44.9

S 160

^ 40

10000.01 0.1 100Burst length (ms) (log scale)

T (ms) area (%) - 0.0771

4.72 r 13.6

35.325.639.1

I

I

0.01 10000.1Burst length (ms) (log scale)

250 r T (ms) area (%): 0.0927 30.5- 2.61 24.0: 12.1 45.5

160

1902

§

IIz

10000.01Open time per burst (ms) (log scale)

E 10

T (ms) area (%)- 0.0763 35.4

4.62 26.2r 13.3 38.5 t r - R

r

l! 1 I 1 I Mill 1 I I I Mill 1 I I I

Vsuil 1 t 1 mil

0.1 1 10 100 Open time per burst (ms) (log scale)

Figure 5.12 Distribution of burst lengths and total open time per burst in the presence of glutamate and NMDA. A and B are distributions of the duration of 1442 (A) and 803 (B) bursts of single-channel openings produced by 50 nM glutamate and 5 pM NMDA, respectively. Distributions were fitted with the sum of 3 exponential components. Predicted mean burst duration and number of events were: 6.41 ms, 1668 (A) and 6.55 ms, 1049 (B). C and D are distributions of total open time per burst from the bursts used in A and B, respectively. They were fitted with the sum of 3 exponential components. Predicted mean total open time per burst and number of events were: 6.14 ms, 1664 (C) and 6.34 ms, 1051 (D). Critical gap lengths (tcrit*= 123 ms for A, C; 1.26 ms for B and D) that produced an equal percentage of misclassified events were calculated between the 3’’* and 4*‘' exponential components fitted to shut time distributions. Time constant and relative areas for each fitted exponential component are shown. Individual openings were separated by shut times longer than 50 ps (A,C) and 80 ps (B,D).

136

T able 5.11 Distribution o f burst lengths

Ti %2 mean

(ps) (ms) (ms) (ms)

Glutamate


95 ± 12 3.02 ± 0.7 33 ± 4 % 23 + 2 %

11.7 + 2.4 44 ± 4 %

6.3 ± 1.8 (3291)

NMDA

Only High (n= 3 patches)

92 ± 9 4.54 ± 0.3 32 + 5 % 20 + 10 %

12.6 ± 1.7 47 + 4 %

6.7 + 0.8 (2239)

T able 5.12 Distribution of total open time per burst

Tl mean

(ps) (ms) (ms) (ms)

Glutamate

Only High 85 ± 8 2.83 ± 0.7 34 + 5 % 24 + 2 %

11.5 ±2.4 42 ± 6 %

6.0 ± 1.9

NMDA

Only High 82 + 4 4.22 + 0.332 + 5 % 29 + 2 %

13.1 + 0.7 39 ± 4 %

6.4 ± 0.8


Mean total open time Mean burst length per burst

Mean burst Popen

(ms) (ms)

Glutamate 6.0 ± 1.9 6.2 + 2.0 0.98 ± 0.01

NMDA 6.4 + 0.8 6.7 ± 0.8 0.95 ± 0.01

137

Bursts of 50 pS currents

High conductance High & Low conductance

160T (ms) area <%)

1.55 8.89

23.077.090

40

10

0,0.1 10001 10

Burst length (ms) (log scale)100

160

T (ms) a rea (%) 1.70 29.913.0 70.190

40

10

0, 10Burst length (ms) (log scale)

10000.1 1 100

160T (m»>

1.29 S.04

I C%) 21.9 78.1

90

40

10

00.1 1 10 100 Open time per burst (ms) (log scale)

10 1000burst

160

T (ms) a rea (%) 1.63 12.0

29.270.8s

IS

I


Figure 5.13 Distribution of burst lengths and total open time per burst for bursts of 50 pS currents. A and B are burst length distributions containing bursts of 50 pS openings from patches with only high conductance and high and low conductance single-channel activity, respectively. They were fitted with the sum of 2 exponential components. Predicted mean burst length and number of bursts were: 6.20 ms, 885 (A) and 9.65 ms, 544 (B). C and D are distributions of total open time per burst from the bursts used in A and B, respectively. They were fitted with the sum of 2 exponential components. Predicted mean total open time per burst and number of events were: 6.56 ms, 884 (C) and 8.96 ms, 545 (D). Critical gap lengths or t , values were: 2.20 ms for A and C; 1.85 ms for B and D). Time constant and relative areas for each fitted exponential component are shown. Individual openings were separated by shut times longer than 50 ps. Glutamate 50 nM and glycine 3 mM were used as agonists.

138

Bursts with duration less than 2 filter rise-times (0.332 ms) were not included in the fitting of the

distributions because their amplitude was unknown.

In patches with only high conductance or high and low conductance single-channel activity,

burst length distributions and distributions of total open time per burst were fitted with the sum of 2

exponential components (Figure 5.13). N o apparent differences in mean burst length and mean total

open time per burst were observed between patches with only high conductance and patches with

high and low conductance single-channel activity (Tables 5.13, 5.14). In addition, no apparent

differences in the mean time constant and relative areas of the two fitted exponential components

were observed between distributions of burst lengths or total open time per burst (Tables 5.14, 5.15).

No apparent differences in mean burst Popen were observed between bursts of 50 pS openings in

patches with only high conductance and patches with high and low conductance single channel

activity (Table 5.16).

5.3.3.6 Clusters

5.3.3.6.1 Distribution of cluster lengths

Cluster length distributions containing clusters of openings that were separated by mean tcri

values of 30 ± 5.4 ms for glutamate and 13 ± 0.5 ms for NMDA were built from patches containing

only high conductance single-channel activity. They were usually fitted with 3 and 4 exponential

components for NMDA and glutamate, respectively (Figure 5.14).

There were no apparent differences in mean cluster length between clusters produced by

glutamate or NMDA. The mean cluster length showed larger variations in clusters of openings

produced by glutamate than in those produced by NM DA For ûtamate but not for NMDA, patch

to patch variations in the number of exponential components were observed. For glutamate, cluster

length distributions sometimes were fitted with up to five different exponential components while

for NMDA cluster length distributions were always fitted with three exponential components. For

the 1 exponential component, no apparent differences in mean time constant and relative areas

were observed between glutamate and NMDA. A 2"< exponential component with a mean time

constant of 2.4 ± 0.3 ms was detected in clusters produced by glutamate but not in clusters produced

by NMDA (Figure 5,14, Table 5.17). A 3" exponential component, which was detected in aU patches

had similar mean time constants and relative areas for glutamate and NMDA. A 4 exponential

component was detected in all patches exposed to NMDA but only in 2 out of 4 patches exposed to

glutamate; it had similar time constant and relative areas for glutamate and NMDA. A 5

exponential

139

Table 5.14 Distribution o f hurst lengths for hursts o f 50 p S currents

mean

Glutamate(ms) (ms) (ms)


2.2 ± 0.433 + 5 %

11.0 ±2.7 67 + 5 %

8.4 ± 2.4


1.6 ± 0.6 32 ± 7 %

11.111.0 68 + 7 %

8.2 + 1.0

T a b l e 5.15 Distribution of total open time per hurst in hursts o f 50 pS currents

l2 mean

Glutamate(ms) (ms) (ms)

Only High 2.0 ± 0.4 31 + 4%

10.0 ± 2.5 69 + 4 %

7.8+ 2.1

High & Low 1.5 ± 0.5 33 + 7 %

10.7 ± 0.9 67 + 7 %

7.9 ± 0.9

T a b le 5.16 Burst Popen in hursts o f 50 pS currents

Mean total open time per burst

Mean burst length Mean burst

Glutamate(ms) (ms)

Only High 7.8 + 2.1 8.4 ± 2.4 0.93 + 0.01

High & Low 7.9 ± 0.9 8.2 ± 1.0 0.96 + 0.02

140

Glutamate NMDA

250

O.OS82S 160 1.6013.644.0

16.943.113.5I

902I40

I 10

0,0.01 1 10

Cluster length (ms) (log scale)10000100

160

T (m*) area (%) 0.120 24.7

10.5 37.9

8“ 90 69.6

15.71£

I 10

0,0.01 1 10 100

Cluster length (ms) (log scale)1000 10000

250T (m*) (¥60 (%) 0.102 27.0

1.66 12.3 35.1

19.646.47.12

g 90

I 10

00.1 1 10

Open time per cluster (ms) (log scale)1000.01 1000 10000

160

T (mi) (¥00 (%) 0.127 8.22 30.6

90

1I 40

I 10

0Open time per cluster (ms) (log scale)

0.01 0.1 10000

Figure 5.14 Distribution of cluster lengths and total open time per cluster in the presence of glutamate and NMDA. A and B are distributions of the duration of 911 (A) and 560 (B) clusters of single channel openings produced by 50 nM glutamate and 5 pM NMDA, respectively. Distributions were fitted with the sum of 4 (A) and 3 (B) exponential components. The predicted overall mean cluster duration and number of events were: A) 12.1 ms and 1036; B) 12.2 ms and 614. C and D are distributions of total open time per cluster from the clusters used in A and B, respectively. They were fitted with the sum of 4 (C) and 3 (D) exponential components. The predicted overall mean total open time per cluster and number of events were: A) 8.6 ms and 1025 and B) 10.2 ms and 613. Critical gap lengths 25.2 ms for A and C; 12.5 ms for B and D) that produced an equal number of misclassified events were calculated between the 4^ and 5 exponential components fitted to the distribution of shut times. The predicted number of misclassified events for the 4^ and 5 exponential components was 0.8 in all cases (A, B, C, D). This value represented predicted percentages of misclassified events of 1% (4 ) and 5% (5 ) for A and C and 1% (4 ) and 7 % (5***) for B and D. Time constant and relative areas for each fitted exponential component are shown. Individual openings were separated by shut times longer than 50 ps.

141

component with a mean time constant of 136 ms was detected only in 1 out of 4 patches exposed to

glutamate, a similar component was not detected in patches exposed to NMDA.

5.3.3.6.2 Distribution o f total time per cluster

A situation similar to that described for cluster length distributions was observed in

distributions of total open time per burst. There were no apparent differences in mean total open

time per cluster but in the number of exponential components that could be fitted to open time per

cluster (Table

5.18).

The mean total open time per cluster showed larger variations for glutamate than for

NMDA. For the 1 e3q)onential component, no apparent differences in mean time constant and

relative areas were observed between glutamate and NMDA. A 2"* exponential component with a

mean time constant of 2.1 ± 0.2 ms was detected in the presence of glutamate but in the presence of

NMDA (Figure 5.14, Table 5.18). A 3* exponential component, which was detected in all patches

had similar mean time constants and relative areas for glutamate and NMDA. A 4 exponential

component was detected in all patches exposed to NMDA but only in 2 out of 4 patches exposed to

glutamate; it had similar time constant and relative areas for glutamate and NMDA. A 5

exponential component with a time constant of 117 ms was detected in only 1 out of 4 patches

exposed to glutamate and it was not detected in patches exposed to NMDA (Table 5.18).

5.3.3.6.3 Cluster Popen

Apparent differences in mean cluster Popen were observed between glutamate and NMDA

with mean cluster Popen being larger in the presence of NMDA than in the presence of glutamate

(Table 5.19). This difference was probab^ due to the larger mean tent value obtained for glutamate.

5.3.3.6.4 Clusters o f 50 pS currents

Glutamate-induced clusters containing openings with conductances within the 50 pS level

were also analysed. Critical shut time or toit values were calculated between the 3'"' and 4

exponential components fitted to distributions of the duration of shut times bordered on each side

by openings to the 50 pS level (Figure 5.15, Table 5.20). Mean tcm values were 28.8 ± 5.7 ms for

patches with only high conductance and 18.9 ± 5.4 ms for patches with high and low conductance

single-channel activity. As in the analysis of bursts, clusters with a duration less

142

T able 5.17 Distribution of cluster lengths

Tl T2 T3 T4 T5mean

(ms) (ms) (ms) (ms) (ms) (ms)

Glutamate


0.11 ±0.01 31 + 4%

2.4 + 0.3 19 + 7 %

12 ± 2 32 ± 4 %

+42 ± 1 15 ± 13 %

*1363%

14.8 ± 6.2 (2523)

N M DA


0.12 ± 0.03 33 + 8 %

8 ± 1 45 ± 5 %

36 ± 4 22 ± 8 %

10.4 ± 1.7 (2536)

+detected in 2 out of 4 patches; “‘‘detected in 1 out of 4 patches

T able 5.18 Distribution of total open tim e per cluster

Tl T2 T3 T4 T5mean

(ms) (ms) (ms) (ms) (ms) (ms)

Glutamate

Only High 0.12 + 0.14 32 + 4 %

2.1 ±0.2 19 ± 8 %

9.5 ± 1.234 ± 4 %

+33 ± 2 13 ± 13 %

*1172%

10.3 ± 4.3

NM DA

Only High 0.12 ± 0.02 32 + 9 %

6.1 ± 1.143 ± 6 %

27 ± 3.0 25 ± 10 %

8.8 ± 1.5

+detected in 2 out of 4 patches; ’‘‘detected in 1 out of 4 patches

T able 5.19 Cluster Popen

Mean total open time per cluster

Mean cluster length Mean cluster Popen

(ms) (ms)

Glutamate 10.3 ± 4.3 14.8 ± 6.2 0.71 ± 0.04

NM DA 8.8 ± 1.5 10.4 ± 1.7 0.85 ± 0.02

143

Clusters of 50 pS currents

High conductance High & Low conductance

90 T (ms) area (%)I.62 20.9II.6 63.9 44.8

I26.1I 40

II 10

0,0.1 10001 10

Cluster length (ms) (log scale)1 100

T (ms) area (%) 2.16 36.49.63 49.636.2 14.9

1 10 100

Cluster length (ms) (log scale)

T (ms) area (%)1.269.19

1 10 100 Open time per cluster (ms) (log scale)

21.862.3

28.9 16.9

160 T (ms) area (%) 2.02 7.60 26.9

33.263.113.790

40

10

0100 10001 10

Open time per cluster (ms) (log scale)0.1

Figure 5.15 Distribution of cluster lengths and total open time per cluster for clusters of 50 pS currents. A and B are cluster length distributions containing clusters of 50 pS openings from patches with only high conductance and high and low conductance single-channel activity, respectively. They were fitted with the sum of 3 exponential components. Predicted mean cluster length and number of clusters were: 17.78 ms, 569 (A) and 10.95 ms, 823 (B). C and D are distributions of total open time per cluster from the clusters used in A and B, respectively. They were also fitted with the sum of 3 exponential components. Predicted mean total open time per cluster and number of events were: 10.59 ms, 549 (C) and 8.21 ms, 828 (D). Critical gap lengths or values were: 28.92 ms for A and C; 13.44 ms for B and D. Time constants and relative areas for each fitted exponential component are shown. Individual openings were separated by shut times longer than 50 ps. Glutamate 50 nM and glycine 3 pM were used as agonists.

144

than 2 filter rise-times (0.332 ms) were not included in the fitting of the distributions because their

amplitude was unknown

In both, patches with only high conductance and patches with high and low conductance

single-channel activity, cluster length and total open time per cluster distributions were fitted with

the sum of 2 or 3 exponential components (Figure 5.15). N o apparent differences in mean burst

length and mean total open time per burst were observed between patches with only high

conductance and patches with high and low conductance single-channel activity (Table 5.20). N o

apparent differences in the mean time constant and relative areas of the three exponential

components were observed between distributions of burst lengths or total open time per burst

(Tables 5.20, 5.21).

No apparent differences in mean cluster Popen were observed between clusters of 50 pS

openings in patches with only high conductance and patches with high and low conductance single

channel activity (Table 5.22).


The characteristics of super-clusters were studied in 2 out of 4 patches for glutamate and 2

out of 3 patches for NMDA. Critical gap lengths (toit) that produced an equal number of

misclassified events were calculated between the 5* and 6 ejqjonential components fitted to the

distribution of shut times (See Methods). Super-clusters were separated by mean critical gap lengths

(tcrit) of 1704 ± 660 ms for glutamate and 589 ± 303 ms for NMDA. Large variations in critical gap

lengths (tcrit) were observed between patches.

5.3.3.7.1 Distribution of super-cluster lengths

Frequency distribution histograms containing the duration of super-clusters were built and

fitted with the sum of 3 or 4 exponential components (Figure 5.16). The overall mean super-cluster

length was ~5 times larger for glutamate than for NMDA. Mean time constants and relative areas for

the 1 and 2" exponential components were similar between super-clusters produced by glutamate

or NMDA. Mean time constants for the 3* exponential component were larger for glutamate than

for NMDA, but their relative areas were similar. (Table 5.23)

5.3.3.7.2 Distribution of total open time per super-cluster

Distributions of the total open time per super-cluster were also fitted with the sum of three

or 4 four exponential components (Figure 5.16). The overall total open time per super-cluster length

was ~3 times larger for glutamate than for NMDA. For the 1 exponential

145

T able 5.20 Distribution of cluster lengths for clusters o f 50 pS currents

Tl T2 T3 T4Mean

Glutamate(ps) (ms) (ms) (ms) (ms)

Only High 2.1 ±0.2 11.2 + 2.5 +41 ± 2.3 ’"140 20.6 ± 7.9(n= 4 patches ) 29 ± 10 % 45 ± 7 % 21 ± 12 % 4%

High & Low 1.0 ±0.5 11.2 ±1.4 52 ± 10.4 16.4 ± 2.9(n= 3 patches ) 22 ± 5 % 60 ± 4 % 18 ± 2 %

^Detected in 2 out of 4 patches, ’‘‘Detected in 1 out of 4 patches

T a b le 5.21 Distribution of total open time per cluster in clusters o f 50 pS currents

Tl l2 T3 T4

Mean

Glutamate(ps) (ms) (ms) (ms) (ms)

Only High 1.8 ±0.2 8.1 ±1.1 +28 ± 0.7 ’"119 13.4 ±5.128 ± 9 % 50 ± 7 % 19 ± 12 % 3%

High & Low 0.9 ± 0.4 8.9 ± 0.627 ± 2 % 59 ± 3 %

39 ± 5.8 14 ± 1 %

11.1 ±1.3

^Detected in 2 out of 4 patches, ’‘‘Detected in 1 out of 4 patches

T a b le 5.22 Cluster Popen in clusters o f 50 pS currents

Mean total open time per cluster

Mean cluster length Mean cluster Popen

(ms) (ms)

Glutamate

Only High 13.4 ±5.1 20.6 ± 7.9 0.67 ± 0.04

High & Low 11.1 ±1.3 16.4 ± 2.9 0.70 ± 0.05

146

Glutamate NMDA

T (mi)I 9.47 16.9II


T (ms) area (%) 26.4

4.69 23.3898.8

0.0776S

50.31£I

I100000


64

T (m*) oraa (%) 14.6 24.0S(O 36

0.0677

ïI 16

V

00.01 0.1 1 10 100 1000

Open time per super-cluster (ms) (log scale)10000

36T (ms) area (%)

34.2 3.82 22.126.5

0.0930S

43.7i£

4

I00.01 0.1 1 10

Open time per super-duster (ms) (log scale)10O 1000

F ig u r e 5.16 Distribution of super-cluster lengths and total open time per super-cluster in the presence of glutamate and NMDA. A and B are distributions of the duration of 151 (A) and 102 (B) super-clusters of single channel openings produced by 50 nM glutamate and 5 pM NMDA, respectively. Distributions were fitted with the sum of 4 (A) and 3 (B) exponential components. The predicted overall mean super-cluster duration and number of events were: A) 2027.8 ms and 161; B) 452.8 ms and 117. C and D are distributions of total open time per cluster from the clusters used in A and B, respectively. They were fitted with the sum of 4 (C) and 3 (D) exponential components. The predicted overall mean total open time per cluster and number of events were: A) 80.4 ms and 165 and B) 12.5 ms and 119. Critical gap lengths 2638 ms for A and C; 1017.0 ms for B and D) that produced an equal number of misclassified events were calculated between the 5 and 6 exponentialcomponents fitted to the distribution of shut times.

147

component, there were no differences in mean time constant between glutamate and NMDA but

relative areas were larger for glutamate than for NMDA. For the 2" exponential component, mean

time constants and relative areas were similar between glutamate and NMDA. For the 3 rd

exponential component, the mean time constant and relative areas were larger in the presence of

glutamate than for NMDA. (Table 5.24)

5.3.3.7.3 Super-cluster Popen

The mean super-cluster Popen was ~3 times larger for NMDA than for glutamate. (Table

5.25)

5.4 D i s c u s s i o n

5.4.1 NM D A receptors mediate two patterns o f single-channel ac tiv ity in PO hippocam pal

granule cells

In patches from PO-hippocampal granule cells, two patterns of single-channel activity were

observed. One pattern of activity was similar to that observed in patches from PO-striatum (Figure

3.2), with onty" high conductance (42 and 51 pS) single-channel activity (Figure 5.2A) while the

second pattern of single-channel activity showed, in addition to high conductance (42 and 49 pS)

single-channel activity, low conductance (17 and 42 pS) single-channel activity (Figure 5.2B).

5.4.1.1 High conductance pattern o f single-channel activ ity

In the high conductance pattern of single-channel activity, unitary currents opened to a

main conductance level of 51 pS and a short-lived subconductance level of 42 pS. In recombinant

NMDA receptors, a similar high conductance pattern of single-channel activity has onty' been

observed when N R l subunits are co-expressed with NR2A or NR2B subunits Stem et oL 1992;

Table 1.3). This finding suggests that NMDA receptors in PO hippocampal granule cells probably

contain NR2B and/or NR2A subunits and it agrees with evidence from in situ hybrization and

immunohistochemical studies which describe the presence in PO hippocampus of mRNA and

protein signals encoding NR2B subunits. In contrast, evidence of NR2A subunit expression in PO-

hippocampus is still unclear. At PO, expression of NR2A mRNA has been reported in the CAl

region of the hippocampus (Monyer et ai, 1994; Wenzel et oL, 1997) while NR2A protein

immunoreactivity has been shown to be either absent (Okabe et al., 1998) or present at very low

levels (Wenzel et oL 1997) in hippocampus before postnatal day 7 (P7). Taken together, evidence

seems to suggest that NR2B subunits are probably the major NR2 subunit contributing to the high

conductance pattern of single- channel activity observed in PO hippocampal granule cells. However,

it is important to point out that NMDA receptors containing NR2A and NR2B subumts produce

148

Table 5.23 Distribution of super-cluster lengths

Tl T2 T] T4mean


Glutamate(n= 2 patches)

60 ±13 10 ± 1 %

5.4 ± 3 11 ±3%

45 ±21 7 ± 1

2150 ± 735 71 ± 6 %

1452 ±407 (571)

NM DA(n= 2 patches)

93 ± 8 21 ± 3 %

5.8 ± 0.6 20 ± 2 %

525 ± 187 59 ± 4 %

278 ± 124 (350)

Table 5.24 Distribution of total open time per super-cluster

Tl T2 T) T4mean


Glutamate 118 ±43 11 ± 2 %

14 ±2.1 22 ± 1 %

66 ±17 60 ± 8 %

*2776 %

57.2 ± 14

NM DA 135 ± 30 25 ± 6 %

7.9 ± 2.9 30 ± 6 %

33 ± 5 45 ± 1 %

17.6 ± 2,6

T a b le 5.25 Super-cluster Popen

Mean open time per super-cluster

Mean super-cluster Mean super-cluster length

Glutamate

(ms)

57 ±14

(ms)

1454 ± 406 0.04 ± 0.0004

NM DA 18 ± 3 278 ± 123 0.12 ±0.07

149

produce single-channel currents with similar conductance (Table 1.3), so a functional contribution by

NR2A or NR2B subunits cannot be detected by looking only at the single-cbannel conductance.

5.4.1.2 High and low conductance pattern o f single-channel activity

bi patches with a high and low conductance pattern of single-cbannel activity, high

conductance single-cbannel activity was also characterised by unitary currents with a main

conductance level of 49 pS and a sublevel of 42 pS while low-conductance single-cbannel activity

was mediated by unitary currents with a main conductance level of 42 pS and a sublevel of 17 pS.

The presence of low conductance single-cbannel activity agrees with expression in PO-bippocampus

of mRNA and protein signals encoding NR2D subunits (Monyer etaL 1994; Wenzel et oL 1997)

which are known to produce, upon co-expression with N R l subunits, low conductance unitary

currents of 17 and 35 pS (W)ilie et d ., 1996). Recombinant NMDA receptors containing NR2C

subunits also produce low conductance single-cbannel currents (17 and 31 pS; Stem et al., 1992;

Wyllie et al., 1996) but expression of mRNA or protein encoding the NR2C subunit have not been

detected in PO rat hippocampus (Pollard etoL, 1993; Monyer etoL, 1994; Wenzel e td , 1997).

It was somehow unexpected to find that patches with a high and low conductance pattern

of single-cbannel activity currents bad not only either high (NR2A- or NR2B-like) or low (NR2C- or

NR2D-like) single-cbannel conductance but single-cbannel activations appeared to be segregated

into periods of either high and low conductance. During periods of high conductance single-cbannel

activity, single-cbannel current amplitudes and kinetics were strikingly similar to those observed in

patches with only high conductance single-cbannel activity suggesting that the functional behaviour

of the NMDA receptors producing the high conductance (NR2A- or NR2B-like) pattern of single-

cbannel activity were apparently independent of their being present in patches with high and low

conductance or patches with only high conductance single-cbannel activity. The reason for this

apparent segregation was not known and it will need further investigation. Single-cbannel data from

recombinant NMDA receptors produced by co-expression of three rather than two NMDA receptor

subunits may prove helpful in understanding this single-cbannel behaviour.

5.4.2 Presence o f direct transitions between conductance levels

Even where currents have either high (NR2A- or NR2B-like) or low (NR2C- or NR2D-like)

conductances and single-cbannel activations contain either high or low conductance currents; it was

still possible that the high and low conductance pattern of single-cbannel activity may have been

produced by a single NMDA receptor species. A good indication of currents being produced by a

single receptor species is the presence of direct transitions between open-cbannel current levels. The

analysis of direct transitions showed that in patches with high and low conductance single-cbannel

activity, only 8.6 - 9.1 % of all direct transitions that were detected appeared as connecting one open

150

level to another open level. This indicated a larger tendency of the channels to make sojourns to and

from shut states rather than to and from open states. Per patch, mean percentages of direct

transitions connecting two consecutive open-channel current levels were: 5.1 % (42 ^ 49 pS), 1.7 %

(17 42 pS), 1.5 % (33 49 pS) and 0.8 % (33 ^ 42 pS).

Even where direct transitions connecting events such 17 pS and 49 pS currents are not

observed, the absence of this type of direct transition does not necessarily exclude the possibility of

17 and 49 pS currents being connected by a shut state or by an intermediate open state. CuU-Cand^

&Usowicz, (1987) suggested that 18 and 48 pS could be indirectly connected through 38 pS currents

which showed clear direct transitions to both 18 and 48 pS currents. In that sense, the 38 pS currents

reported by CuU-Canc^ and Usowicz (1987) were very similar to the 42 pS currents observed in this

study which also showed clear direct transitions to both the 17 and 49 pS level. The possibility of 42

pS currents linking 17 and 49 pS currents was assessed by analysing direct transitions involving three

rather than two conductance levels and on which currents to the 42 pS level occurred between two

different conductance levels. Table 5.26 shows an analysis of more than 5000 of these transitions

involving three conductance levels with currents to the 42 pS level being in between. Table 5.26 shows

that in no case did 42 pS events appeared as connecting 17 and 49 pS events. This finding suggested

that either 42 pS currents were common to two different receptor species, one producing 17 and 42 pS

currents and another one producing 42 and 49 pS currents or that 17, 42 and 49 pS currents were

actually produced by the same receptor species but they were actually connected via a shut state rather

than by an intermediate open state. The possibility of 17 and 49 pS being connected by a shut state is

difficult to test and it should not be discarded because analysis of direct transitions showed that these

channels had a very high probability of making sojourns to and from shut states rather than to open

states.

5.4.3 Properties o f single-channel activations

5.4.3.1 Shut times

When compared with data from recombinant NR1/NR2A and NR1/NR2B NMDA

receptors, shut time distributions from glutamate-mediated NMDA receptor activations in patches

containing only high conductance single-channel activity showed an additional shut state (Table

5.27). A similar observation was made for PO-striatum NMDA receptors.

151

Table 5.26 Direct transitions involving three consecutive conductance levels

Type of Sequence

17 - 42 - 49

49 - 42 - 17

17 - 42 -17

0 - 4 2 - 4 9

n

0.00

0.00

96 1.80

17

49

42

42

42

49

17

77 1.44 17 1742

49

4 9 - 4 2 - 0 100 1.87

4942

49 - 42 - 49

1 7 - 4 2 - 0

217 4.06 4249

353 6.6117

42

49

0 - 4 2 - 1 7 579 10.84

4217

0 - 4 2 - 0 3918 73.37 0 0

42

152

S.4.3.2 Open times

a) 17 pS currents

In PO-dentate gyms NMDA receptors, glutamate-activated 17 pS currents had a mean open

time similar (Table 5.28) to that reported for 17-18 pS currents produced by NR1/NR2C and

NR1/NR2D recombinant NMDA receptors (Wyllie et al,, 1996). Because mRNA and protein

signals encoding NR2D but not NR2C subunits have been reported in PO-bippocampus (Pollard et

aL 1993; Monyer et 1994; Wenzel etaL, 1997), 17 pS currents in PO-bippocampus were probably

produced by activation of NMDA receptors containing NR2D subunits.

h) 33 pS currents

In PO-dentate gyrus, 33 pS currents bad a mean open time of 0.60 ± 0.13 ms. The presence

of 0.3 to 0.8 % of direct transitions between 33, 42 and 49 pS currents suggested that 33 pS currents

were probably associated with high conductance single-cbannel activity rather than with low

conductance single-cbannel activity. Direct transitions between 33 and 17 pS currents were very low,

between 0.00 to 0.03 %. Single-cbannel currents with a similar chord conductance (28 pS) and a low

frequency of occurrence (3 %) has been reported for NR1/NR2A recombinant NM DA receptors

which are known to produce high conductance single-cannel currents; unfortunately, information

about its mean open time was not reported (Stem et al., 1992). For NR1/NR2C recombinant

NMDA receptors, glutamate-activated single-cbannel currents with conductances of 31 - 36 pS but

with a much higher relative frequency of occurrence (relative area of 74.5 %) and a mean open time

of 0.91 ± 0.08 ms have been reported (Stem et d.^ 1992, WjHie etd.^ 1996). Whether these 33 pS

currents were the result of the activation of NMDA receptors containing NR2A subunits cannot be

discarded, evidence of expression of NR2A in PO-bippocampus is still controversial (Moiyer et d^

1994; Wenzel etd.^ 1997; Okabe etd.^ 1998). In contrast, detectable expression of mRNA or protein

encoding NR2C subunits have not been found in PO-bippocampus (Pollard et À , 1993; Monyer etoL

1994; Wenzel 1997).

c) 42 pS currents

In PO-dentate gyrus, glutamate-activated 42 pS currents bad mean open times similar to

those of 44 pS currents from PO-striatum NMDA receptors but larger than those reported

153

T able 5.27 Comparison o f shut times between PO-hippocampus, PO-striatum and

recombinant N R 1/N R 2A and N R 1/N R 2B NM DA receptors

'Cl l 2 'C3 'C4

W (ms) (ms) (ms)

PO Dentate gyms^ 34 ± 3 0.3 ± 0.04 1.0 ± 0.3 11 ± 1(Only high) (40 ± 7 %)* (14 + 2 %) (10 ± 1 %) (8 ± 1 %)

PO Striatum^ 20 ± 2 0.16 ±0.02 0.87 ± 0.03 8.9 ± 1(51 ± 8 %) (11 ± 3 %) (14 ±2%) (7 ± 1 %)

NR1/NR2A2 39 ± 4 0.54 ± 0.04 9.9 ± 1.3(37 + 4%) (21 ± 2 %) (15 ± 1 %)

NR1/NR2B3 54 ± 1 0.65 ± 0.1 8.8 ± 1.3(34 + 4%) (23 ± 2 %) (11 ±2%)

^This stucfy;^StemetaL (1994); Behe & Colquhœtn (persond ca?mu?ikationJ " Relative areas are grêm in parenûxses

T able 5.28 Individual open times for 17pS currents in PO-dentate gyrus and N R 1 /N R 2 D

recombinant NM DA receptors

(ms)

PO-Dentate gyrus 1.51 ± 0.19

NR1/NR2C2 0.91 ± 0.06

NR1/NR2D2 1.28 ±0.06

^This stu(^, ^WyWie et al. (1996).

154

for glutamate-activated 38 and 39 pS currents from NR1/NR2A and NR1/NR2B recombinant

NMDA receptors, respectively (Table 5.29). Unfortunately, no mean time constants and relative

areas have been reported for NR1/NR2B receptors. It would be interesting to investigate whether

this difference is consistent between native and recombinant NMDA receptors.

T a b l e 5.29 Comparison of open times for 40 pS currents from PO-dentate gyruSy PO-striatum

and recombinant NM DA receptors

^2 mean

(ms) (ms) (ms)

PO-Dentate gyrus^

Only High 0.77 ±0.09 (93 ± 4 %)

+4.82 ± 1.6 (7±4% )

1.00 ±0.15

High & Low 0.85 ± 0.05 (90 ± 2 %)

2.39 ± 0.3 (10 ± 2 %)

0.99 ± 0.02

PO-Striatum^ 0.66 ± 0.08 (83 ± 7 %)

2.34 ± 0.5 (17 ± 7 %)

0.90 ±0.10

NR1/NR2A2.3 0.18 ±0.02 (87 ± 1 %)

1.31 ±0.2 (13 ± 1 %)

0.61 ± 0.05

NR1/NR2B2.3 n.r. n.r. 0.59 ± 0.07

This study; ' jrcm Stem et d . (1994, HEK 293 cells), ^msan vdues fian Stem et oL (1992; Oocytes), n. r. = no reported - 2 out of 4patches.

d) 50 (49 - 51)pS aments

In patches containing either only high conductance or high and low conductance single

channel activity, glutamate-activated main level currents (49-51 pS) had a mean open time slightly

larger than that reported for glutamate-activated main level openings from NR1/NR2B and

NR1/NR2A recombinant NMDA receptors (Stem et aL, 1993) (Table 5.30). The fact that in the

reported data from recombinant NMDA receptors the calculation of the mean open time included a

fast exponential component with a magnitude in the microsecond range made aT ore direct comparison

d-iç^lcDit . In this stu(fy, these fast components were not included because they represented

incompletely resolved openings withai unknown amplitude.

155

T ab le 5.30 Comparison of open times for 50 pS currents from PO-dentate gyruSy PO-striatum

and recombinant NM DA receptors

'Cl 'C2 'C3

mean

(ps) (ms) (ms) (ms)

PO Dentate gyrus Only Hi J i 0.60 ± 0.2

(22 ± 7 %)4.15 + 0.5 (78 ± 7 %)

3.44 ± 0.6

High & Low 1.42 ± 0.2(35 + 7%)

4.34 ± 0.2 (65 + 7 %)

3.32 + 0.1

PO Striatum! 0.62 + 0.1 (10 ± 8 %)

3.17 ±0.3 (90 ± 6 %)

2.90 ± 0.30

NR1/NR2A2.3 67 + 1 (30 ± 5 %)

1.64 + 0.4 (40 + 7 %)

4.27 + 0.7 (30 ±4%)

2.70 ± 0.70

NR1/NR2B2.3 183 ± 300 (23 ± 5 %)

1.83 ± 0.3 (41 ± 7 %)

4.99 + 0.1 (36 + 8 %)

2.80 ± 0.30

This study; ^From Stem et oL (1993), ^Means are from Stem et oL (1992)

5.4.3.3 Bursts

Because there is no reported data on the structure of bursts of openings from

recombinant NMDA receptors, bursts of openings from PO-dentate gyrus NMDA receptors were

compared with data obtained from PO-striatum NMDA receptors. Only bursts from patches with a

high conductance pattern of single-cbannel activity were compared with bursts of high conductance

single activity observed in PO-striatum.

No apparent differences in mean burst length, mean total open time per burst and meanof

burst Popen were found between bursts of openings produced by activation NMDA receptors by

glutamate or NMDA in striatum and dentate gyrus (Tables 5.31, 5.32). There were also no apparent

differences in the mean time constant and relative area of the three exponential components fitted to

distributions of burst length and total open time per burst. Values of mean burst Popen were also

similar tareer than the value of 0.86 reported for glutamate-activated NMDA receptors in

dissociated adult hippocampal CAl neurons (Gibb & Colquboun, 1992).

156

T able 5.31 Comparison of burst lengths between dentate gyrus and striatum

mean

(|Lis) (ms) (ms) (ms)

Glutamate

PO-Dentate gyrus 95 ± 12 3.0 ±0.7 12 ±2.4 6.3 ± 1.833 ± 4 % 23 ± 2 % 44 ± 4 %

PO-Striatum 55 ± 7 2.2 ± 0.7 12 ± 0.7 4.8 ± 0.743 ± 5 % 20 ± 7 % 36 ± 9 %

NM DA

PO-Dentate gyms 92 ± 9 4.5 ± 0.3 13 ± 1.7 6.7 ± 0.83 2 ± 5 % 20 ± 10 % 4 7 ± 4 %

PO-Striatum 62 ± 3 1.9 ±0.2 11 ±0.7 6.8 ± 0.530 ± 3 % 12 ± 4 % 57 ± 3 %

T able 5.32 Comparison of total open time per burst between dentate gyrus and striatum

mean

(fis) (ms) (ms) (ms)

Glutamate

PO-Dentate gyms 85 ± 8 2.8 ± 0.7 12 ± 2.4 6.0 ±1.934 ± 5 % 24 ± 2 % 42 ± 6 %

PO-Stiiatum 55 ± 5 2.3 ±0.6 11 ±0.7 4.5 ± 0.644 ± 4 % 21 ± 7% 35 ± 7%

NM DA

PO-Dentate gyms 82 ± 4 4.2 ± 0.3 13 ± 0.7 6.4 ± 0.83 2± 5% 29 ± 2% 39± 4%

PO-Striatum 61 ± 3 1.9 ±0.3 11 ±0.5 6.5 ± 0.431 ± 3% 13 ± 4% 5 6 ± 3 %

157

T able 5.33 Comparison of mean burst Popen between dentate gyrus and striatum

jPopen

Glutamate

PO-Dentate Gyrus 0.98 ± 0.01

PO-Striatum 0.95 ± 0.02

NM DA



5.4.3.4 Clusters

Clusters of openings from PO-dentate gyrus NMDA receptors were also compared with data

obtained from PO-striatum NMDA receptors. Only clusters obtained from patches with a high

conductance pattern of single-channel activity were compared. For glutamate, mean tait values were

30 ± 5.4 ms and 16 ± 1.7 ms for dentate gyrus and striatum, respectively. For NMDA, mean ten

values were 13 ± 0.5 ms and 14 ± 3.5 ms for dentate gyrus and striatum, respectively.

Glutamate-activated clusters showed a larger variation in mean cluster length in PO-dentate

gyrus than in PO-striatum; in contrast, no apparent differences in mean cluster lengths were found

between NMDA-activated clusters in dentate gyrus and striatum. A similar degree of variation in the

number of exponential components that could be fitted to cluster length distributions was observed

between dentate gyrus and striatum (Table 5.32). For NMDA, the main difference was the

absence in clusters from PO-dentate gyrus of a 2"* exponential component with a mean time

constant ~2 ms. For distributions of total open time per cluster a similar situation was observed. No

apparent differences in mean total open time per cluster were evident (Table 5.3^. Mean Popen values

were lower for glutamate than for NMDA. This was consistent between in NMDA receptors from

striatum and dentate gyrus (Table 5.36).

158

T able 5.34 Comparison of cluster lengths between dentate gyrus and striatum

Xi I , T, T. Tc mean

W (ms) (ms) (ms) (ms) (ms)

GlutamatePO-Dentate gyms 110 ±10

31 + 4%2.4 ± 0.319 ± 7 %

12 ± 2 32 ± 4 %

+42 ± 1 15 ± 13 %

*1363%

14.8 ± 6.2

PO-Striatum 56 ± 6 45 ± 5 %

1.2 ± 0.2 9 ± 3 %

11± 3 33 ± 10 %

+30 ± 2 10 ± 5 %

+92 ± 7 2 ± 1 %

9.1 ±0.9

NMDA

PO-Dentate gyms 120 ± 30 33 + 8 %

8 ± 1 45 ± 5 %

36 ± 4 22 ± 8 %

10.4 ± 1.7

PO-Striatum 80 ±19 32 ± 2 %

n .2 ± 0.7 9 ± 4 %

15 ± 4 48 ± 4 %

^45 ± 5 11 ± 5%

12.2 ± 0.6

+2 out of 4 patches; *1 out of 4 patches, '•'3 out of 5 patches

T a b l e 5.35 Comparison o f total open time per cluster between dentate gyrus and striatum

Xi 2 4 5 mean


Glutamate

PO-Dentate gyms 120 ± 14 32 ± 4 %

2.1 ±0.2 19 ± 8 %

9.5 ± 1.2 34 ± 4 %

+33 ± 2 13 ± 13 %

*1172%

10.3 ± 4.3

PO-Striatum 57 ± 3 46 ± 4 %

1.1 ± 0.2 11 ±4%

10 ±2.1 32 ± 9 %

+25 ± 0.9 9 ± 4 %

+82 ± 9 2 ± 1 %

6.9 ± 0.8

NMDA

PO-Dentate gyms 120 ± 20 32 ± 9 %

6.1 ±1.1 43 ± 6 %

27 ± 3 25 ± 10 %

8.8 ± 1.5

PO-Striatum 67 ±10 33 ± 2 %

n . 6 ± 0 . 4 10 ± 3 %

13 ±2.1 50 ± 3 %

»37±38 ± 4 %

9.7 ± 0.4

+2 out of 4 patches; *1 out of 4 patches, '*'3 out of 5 patches

159

T able 5.36 Comparison of mean cluster Popen between dentate gyrus and striatum

Pcopen

(ms)

Glutamate



NM DA




The characteristics of super-clusters produced by activation of PO-dentate gyrus NMDA

receptors were also compared with those of PO-striatum NMDA receptors. Super-clusters obtained

from patches containing only high conductance single-channel activity were compared. For

glutamate, a mean W value of 1704 ± 660 ms (2 out of 4 patches) was obtained for PO-dentate gyms

NMDA receptors but no tait values could be obtained for PO-striatum. For NMDA, mean tcri values

were 589 ± 303 ms and 139 ± 10 ms for dentate gyrus and striatum, respectively. Large variations in

the critical gap lengths (w ) calculated to separate super-clusters were observed between patches.

For glutamate, distributions of super-cluster lengths could not be compared. For NMDA,

the mean super-cluster length was larger in PO-dentate gyrus than in PO-striatum NMDA receptors.

In contrast with the large difference observed in mean super-cluster length between dentate gyms

and striatum, no differences in mean total open time per super-cluster were observed between

dentate gyrus and striatum. For NMDA, the mean super-cluster Popen was larger in PO-striatum than

in PO-dentate gyrus.

160

T ab le 5.37 Comparison o f super-cluster lengths between dentate gyrus and striatum

Tl ' 2 ' 3 ^4 mean

W (ms) (ms) (ms) (ms)

Glutamate

PO-Dentate gyrus 60 ±13 10 ± 1 %

5.4 ± 3 11 ± 3%

45 ±21 7 ± 1

2150 ± 735 71 ± 6 %

1452 ± 407

PO-Striatum - - - - -

N M DA

PO-Dentate gyrus93 ± 8

21 + 3 %6 ± 0.6

20 ± 2 %525 ± 187 59 ± 4 %

278 ± 123

PO-Striatum 66+12 25 + 2 %

1.2 ± 0.4 8 ± 2 %

14 ± 3 25 ± 3 %

233 ± 88 42 ± 4 %

116.2 ±51

T ab le 5.38 Comparison of total open time per super-cluster between dentate gyrus and

striatum

:2 3 mean

W (ms) (ms) (ms) (ms)

Glutamate

PO-Dentate gyrus 118 + 43 11 + 2%

14 ± 2 22 ± 1 %

66 ± 17 60 ± 8 %

*2776%

57.2 ± 13.5

PO-Striatum - - - -

NM DA

PO-Dentate gyrus 135 + 30 25 + 6 %

7.9 ± 2.9 30 ± 6 %

33 ± 5 45 ± 1 %

17.6 ± 2.6

PO-Striatum 89 ±18 26 ± 1 %

5.8 ± 2 28 ± 8 %

40 ± 9 46 ± 8 %

18.1 ±2.2

* 1 out of 2 patches

161

T able 5.39 Comparison of mean super-cluster Popen between dentate gyrus and striatum

jPopen

(ms)

Glutamate


PO-Striatum

NM DA



Although lack of information about the properties of bursts, clusters and super-clusters

produced by recombinant NMDA receptors, precludes a more thorough and probably more

informative comparison of the single-channel properties of NMDA receptors in PO-dentate gyms.

The functional evidence presented in this chapter suggests that at the single channel level high

conductance single-channel currents and activations of PO-dentate gyrus NMDA receptors share

many of the functional properties of those from PO-striatum. This finding agrees with evidence

suggesting the presence of NMDA receptors containing NR2B subunits in PO-dentate gyrus as well

as in PO-striatum. As in PO-striatum, use of sub-unit selective drugs will be useful in order to have a

more clear idea about the potential subunit composition of PO-dentate gyrus NMDA receptors.

162

CHAPTER 6

Effects of Ifenprodil on the Single-Channel Properties of

NMDA Receptors from PO-Hippocampal Granule Cells

6.1 Su m m a r y

i. The effects of ifenprodil, an antagonist of NMDA receptors containing NR2B subunits, were

studied on single-channel activity produced by activation of NMDA receptors in outside-out

patches from hippocampal granule cells in slices from 0 -day-old rats.

ii. Ifenprodil produced a selective reduction of single-channel activity mediated by high

conductance NMDA channels in both patches with only high conductance and patches with

high and low conductance single-channel activity. Ifenprodil had no effect on the single-channel

current amplitudes.

iii. In the presence of ifenprodil mean shut times were larger than under control conditions in all

patches. The effects of ifenprodil on the shut time distribution were more evident in patches

with only high conductance single-channel activity in which considerable increases in the mean

time constants of the 4 th 5 th and 6 ^ exponential components were observed.

iv. Ifenprodil reduced the overall mean open time and opening frequency. The mean open time of

currents to the 17 pS level remained constant while those to 33, 42 and 50 pS levels became

smaller in the presence of ifenprodil. The most clear effects were on currents to the 50 pS level;

the number of openings to the 50 pS level was reduced by —70 % and their mean open times

were reduced by —45 %.

V . In patches containing only high conductance single-channel activity, ifenprodil produced a 50 -

70 % reduction in the number of bursts together with a 50 % reduction in mean burst length

while the mean burst Fopen was only slightly reduced. In clusters, it produced a 50 - 85 %

reduction in the number of clusters, a 50 % reduction in mean total open time per cluster and 28

- 54 % reduction in mean cluster Popen- In contrast, ifenprodil had apparently no significant

effects on mean cluster length. Ifenprodil also had no apparent effects on the mean decay time-

course of ensemble averages built with clusters of openings from single-NMDA receptor-

channel activations.

vi. In summary, the inhibitory effects of ifenprodil were apparently selective for single-channel

activity mediated by high conductance NMDA receptors. These findings strongly suggested that

163

in PO-hippocampal granule cells high conductance single-channel activity is quite probably

mediated by NMDA receptors containing the NR2B subunit.


The functional evidence described in the previous chapter (Chapter 5) seems to suggest that

in PO-hippocampal granule cells high and low conductance single-channel activity are mediated by

two functionally different NMDA receptor populations. Low conductance single-channel activity

showed striking similarities with single-channel activity mediated by recombinant NMDA receptors

containing NR2D subunits (Wyllie et oL, 1996) while high conductance single-channel activity was

similar to that mediated by recombinant NMDA receptors containing NR2B or NR2A subunits

{Stem et al., 1992).

Whether NMDA receptors containing NR2B subunits contributed to high conductance

single-channel activity was investigated by using ifenprodil, a non-competitive antagonist selective

for NMDA receptors containing NR2B subunits. Ifenprodil has been shown to inhibit macroscopic

responses mediated by NR1/NR2B recombinant NMDA receptors with an IC5 0 of 0.34 pM

(Williams et oL, 1993). In contrast, a 300-fold higher IC5 0 (145 pM) is reported for ifenprodil

inhibition of NR1/NR2A recombinant NMDA receptors (Williams etoL, 1993). Ifenprodil does not

inhibit macroscopic responses mediated by heteromeric NR1/NR2D recombinant NMDA

receptors (Williams, 1995) which produce low conductance single-channel activity (Wyllie et al,

1996) similar to that observed in PO-hippocampal granule cells. The mechanism and molecular

determinants responsible for the subunit-specificity of ifenprodil antagonism are not yet known

(Gallagher et <ï/., 1996).

6.3 R e su l t s

6 .3.1 Effects o f ifenprodil on single-channel a c tiv ity

Under steady state conditions, ifenprodil (1 pM) reduced the single-channel activity

produced by NMDA (1 -5 pM) and glycine (3 - 10 pM) in outside-out patches containing high and

low conductance single-channel activity (n = 2 ) and in patches containing only high conductance

single-channel activity (n = 2). Figure 6.1 shows characteristic single-channel activity under control

conditions (Panel 6.1 A) and during application of ifenprodil (1 pM) (Panel 6 .1C) in a patch

containing high and low conductance single-channel activity. Figure 6.1 also shows characteristic

single-channel activity under control conditions (Panel 6 . IB) and during application of ifenprodil ( 1

pM) (Panel 6 . ID) to a patch containing only high conductance single-channel activity.

164

6.3.2 Effects on single-channel current amplitudes

Ifenprodil had no apparent effects on the mean amplitude of the single-channel currents

(Table 6.1). In patches with only high conductance single-channel activity, ifenprodil reduced the

number of currents to both amplitude levels with no apparent effects on their mean relative area. In

contrast, in patches with high and low conductance single-channel activity, ifenprodil reduced the

relative area occupied by currents to the largest amplitude level (4 Gaussian component) (Figure

6.2).

T a b le 6.1 Distribution of single channel current amplitudes

Gaussian Components

%st 2. à 4 th

(pA) (pA) (pA) (pA) n

Only High

Control 2.48 ±0.06 3.32 ±0.09 731515 ± 1 % 85 ± 1 %

Ifenprodil 1 pM 2.37 ±0.05 3.15 ±0.02 197412 ± 0.3 % 88 ± 0.3 %

Ifenprodil 3 pM 2.58 ± 0.06 3.31 ± 0.05 95913 ± 1 % 87 ± 1 %

High & Low

Control 1.08 ±0.05 1.94 ±0.04 2.66 ±0.04 3.34 ±0.04 59105 ± 0.3 % 5 ± 1 % 34 ± 5 % 56 ± 6 %

Ifenprodil 1 pM 0.99 ± 0.05 1.93 ±0.03 2.71 ±0.04 3.33 ±0.01 447714± 9% 7 ± 2 % 53 ± 3% 27± 10%

165

High & Low Conductance Only High Conductance

Control B Control

' 4 U -...... ' • 1

11 T i 1 i ■' 1' L 1 viwiilJL. • <¥ 11 . ' " 11'^ L' t 1,p-.

l T l l_ r W T " 1 tlU ' s 'J. r r -

i '1 ' T 1 i “ ' i l l u 4, n ' ' u

Hr iFT f T ' 1 1 ' 1- i rLri 1

L, 1 jr_ L '

i; Ti '1 j L ! r L T f fl ! I" ,

' , r y l ' "1 "'ij' '"'1 rU. I f

' -

r100 ms

1

jT'l'

i r 1100 ms

1 1

ifenprodil 1 pM D Ifenprodil 1 pM

1 ' t M f u i , g 1 ' Ik

’ 1 "11 ] * n ' ' " — 1

" ' " ' " ' 1” ' I I I ' I' '

. Î 111 fl"“ T i ' I l 1

1 ..... H i T

■ ' 1 II!' u 1--------------- Yf

1 'U 1 ' 1

........1 1 1 1 1 f .---------

r I100 ms

s r 1100 ms

!

Figure 6.1 Effects of ifenprodil on single-channel activity m ediated by N M D A receptors in PO- hippocam pal granule cells. Characteristic NM DA receptor-channel activity in outside-out patches from dentate gyrus granule cells under control conditions (A,B) and during application of 1 |iM ifenprodil (C,D). Holding potential was -60 mV. Openings are downwards. Each panel contains ten contiguous 500 ms sweeps of single channel recording. Single channel activity was recorded in the continuos presence of NM DA (5 pM) and glycine (10 pM).

166


Control Control400 1250

<pA)1.162.002.713.39

area <%) 0.9 6.6

27.764.8

(pA)2.573.45

area <%) 13.4 86.6

1000

^ 750

200CO 500

250

■5

Ifenprodil 1 jxM D Ifenprodil 1 p.M400

y . <pA) area <%> 0.919

1.90 2.77 3.35

1 9.548.040.80

« 2001

Current (pA)

&S'® 500

I250

y (pA) area (%) 2.44 11.63.18 88.4

-2 -3

Current (pA)

Figure 6.2 Effects of ifenprodil on the distribution of single-channel current amplitudes in patches with high and low conductance (A,C) and only high conductance (B,D) single-channel activity mediated by NMDA receptors. Histograms are frequency distributions containing current amplitudes longer than 2 filter rise-times (0.332 ms). Distributions were fitted with the sum of 4 (A,C) and 2 (B,D) Gaussian components. The mean amplitude and relative area of each Gaussian component are shown. Data records with the same length were analysed. Length of the records were 405 s (A,C) and 295 s (B,D). Holding potential was -60 mV.

167


Ifenprodil (1 produced an overall increase in mean shut time in all patches. This

increase was considerably larger in patches with onty' high conductance than in patches with high and

low conductance single-channel activity, A further increase in mean shut was observed in patches

with only high conductance single-channel activity, when a higher (3 pM) ifenprodil concentration

was used. Shut time distributions from patches with only high conductance and patches with high

and low conductance single-channel activity were fitted with 5 and 6 exponential components,

respectively (Figure 6.3, Table 6.2).

6.3.3.1 In patches w ith only high conductance currents

For the exponential component, mean time constants remained apparently unchanged

but relative areas became smaller. For the 2" exponential component, mean time constants also

remained apparently unchanged while mean relative areas increased instead. For the 3* exponential

component, a -100 % increase in mean time constants was observed with mean relative areas being

smaller only in 3 pM ifenprodil. For the 4^ exponential component, mean time constants were also

—100 % longer with mean relative areas being larger only in 3 pM ifenprodil. For the ejqponential

component, mean time constants became increasingly longer when the ifenprodil concentration was

increased; mean relative areas became larger only in 3 pM ifenprodil. For the 6 exponential

component, mean time constants also became increasingly longer with ifenprodil concentration but

mean relative areas remained apparently constant (Table 6.2).

6.3.3.2 In patches w ith high and low conductance currents

For the 1 exponential component, mean time constants became larger while mean relative

areas were reduced by ~50 %. For the exponential component, no changes in mean time

constant or relative area were observed. For the 3* exponential component, a —100 % increase in

mean time constants was observed while mean relative areas remained constant. For the 4**

exponential component, no changes in mean time constant or relative area were observed. For the

5 ' exponential component, mean time constants remained apparent^ unchanged while mean

relative areas showed a clear increase (Table 6.2).

6.3.4 Effects on individual open times

Distributions of all individual open times were built and fitted with the sum of 3 exponential

components (Figure 6.4). Table 6.3 shows mean time constants and relative areas for each one of the

fitted exponential components.

168


Control

2 160

40E

BT (ms) area (%)

360

0.0335 30.0 ¥0.232 10.4

1.72 11.916.7 26.3 2 160

64.5 21.4

tE 40Ez

Control

ifl I 111 mil1 10 100

Stiut time (ms) (log scale)

T (ms) a re a (%)0.0216 60.40.124 3.00.692 16.24.96 6.129.4 3.1

129.5 12.2


Ifenprodil 1 nM Ifenprodil 1

T (ms) area (%)0.06240.334

0> 40


T (m s) a r e a (%) 0.0191 54.30.209 8.3

1.55 8.813.9 4.845.1 7.3

179.8 16.5

O 160

0> 40


Figure 6.3 Distribution of the duration of shut times under control conditions (A,B) and after 1 |iM ifenprodil (C, D) in patches containing high and low conductance (A,C) and only high conductance (B,D) NMDA receptor single-channel activity. Histograms are frequency distributions containing shut times longer than 75 |is (A,C) and 50 ps (B,D). Histograms were fitted with 5 (A,C) and 6 (B,D) exponential components. Time constants and relative areas for each exponential component are shown. Predicted overall mean shut time and number of events were: (A) 15.6 ms, 5340; (C) 20.3 ms, 4339; (B) 17.1 ms, 4803 and (D) 33.9 ms, 2595. NMDA (5 pM) and glycine (10 pM) were used as agonists.

169


Control B Control640

T (mi) area <%) 0.0687 25.4

1.02 47.23.58

« 36027.4

^160

40

0.01 0.1 100Open time (ms) (log scale)

360

T (mi) area (%) 0.0845

1.35 3.85

9.336.354.4g 160

40

0,0.01 1

Open time (ms) (log scale)0.1 10010

Ifenprodil 1 pM D Ifenprodil 1 pM

Sw 360

E 40 Z

- T (mi) area (%)0.0616 20.8

0.777 43.91.95 35.2

1 1 1 1 1 1 1 1 m l 1 1 1 111 III ^ ^ ' I I I m l0.1 1 10


360

T (mi) area (%) 0.0661

1.62 3.80

24.551.6 24.0O 160

40

00.01 0.1 1 10

Open time (ms) (log scale)100

Figure 6.4 Distribution of individual open times under control conditions (A,B) and after 1 pM ifenprodil (C, D) in patches containing high and low conductance (A,C) and only high conductance (B,D) NMDA receptor single-channel activity. Histograms are frequency distributions of individual open times. Histograms were fitted with 3 exponential components. Time constants and relative areas for each exponential component are shown. Predicted overall mean open time and number of events were: 1.48 ms, 6878 (A); 1.04 ms, 6659 (C); 2.59 ms, 3765 (B) and 1.76 ms, 2074 (D). NMDA (5 pM) and glycine (10 pM) were used as agonists

170

'Cl

T a b le 6 . 2 Distribution of shut times

T2 T3 T4 'Cs 'Ce Mean

(ps) (ms) (ms) (ms) (ms) (ms) (ms)

O nly H igh

Control 2 1 ± 1 0 . 2 ± 0 . 0 2 0.8 ± 0.04 6 . 6 ± 1 . 2 102 ± 52 241 ± 79 25 ± 6

(n= 2 patches ) 61 ± 0.3 % 5 + 1% 14 ± 1 % 5 ± 1 % 7 ± 3 % 8 ± 3 % (????)

Ifenprodil 1 pM 23 ± 3 0 . 2 + 0 . 0 2 1 . 6 + 0 . 0 1 14 ± 0.2 368 ± 228 1478 ±918 232 ± 140(n= 2 patches) 40 ± 10 % 15 ± 5 % 1 2 + 2 % 7 ± 2 % 12 ± 3 % 14 ± 2 % (3277)

Ifenprodil 3 pM 2 1 + 2 0.3 + 0.001 1.8 ±0.5 13 ± 1.5 911 ±503 5654 ± 3595 595 ± 366(n= 2 patches) 46 + 2 % 8 + 2 % 9 ± 0.2 % 13 ± 0.03 % 16 ± 5 % 7 ± 1 % (1961)

H igh & Low

Control 31 + 2 0.3 + 0.02 1.6 ± 0.3 15 ±0.1 503 ± 320 92 ± 54(n= 2 patches ) 36 + 3 % 14 + 2 % 13 ± 1 % 17 ± 4 % 21 ± 3 % (8492)

Ifenprodil 1 pM 55 ± 16 0.3 ± 0.02 3.3 ±0.1 19 ± 5 660 ± 435 236 ± 153(n= 2 patches ) 16 ± 5 % 13 + 1 % 13 ± 3 % 23 ± 7 % 36 ± 0.4 % (5250)

171

T a b le 6.3 Distribution of individual open times for currents to all amplitude levels

( is) (ms) (ms)

mean

(ms)

O nly H igh

Control

Ifenprodil 1 pM

Ifenprodil 3 pM

H igh & Low

Control

Ifenprodil 1 pM

6 7 ± 14 11.7 + 2%

55 ± 8 19 + 4 %

52 ± 2 22 + 5 %

49 + 14 24 ± 1 %

57 + 318.6 + 1 %

1.2 ± 0.0434.6 ± 0.02 %

1.2 ± 0.3 54 + 1 %

0.8 ± 0.1 31 + 0.4%

1.1 + 0.02 37 + 7 %

0.8 ± 0.0351.6 + 7%

3.6 ± 0 .153.6 ± 2 %

2.8 ± 0.7 27 ± 3 %

1.7 ± 0 .1 47 ± 5 %

3.5 ± 0 .1 39 ± 8 %

1.9 ± 0 .729.9 ± 5 %

2.4 ± 0.2 (9790)

1.4 ± 0.3 (3064)

1.1 ± 0.02 (1672)

1.8 ± 0.2 (10391)

1.0 ± 0.04 (7622)

172

In the presence of ifenprodil mean open times and number of openings were reduced in

both patches with only high conductance and patches with high and low conductance single-channel

activity (Table 6.3). These effects were considerably larger in patches with only high conductance

than in patches with high and low conductance single-channel activity.

6.3.4.1 In patches w ith only high conductance currents

In patches with only high conductance single-channel activity, the reduction in mean open time was

the result of changes in mean time constants and/or mean relative areas of the 2" and 3'"

exponential components. For the (fastest) exponential component, no apparent changes in mean

time constants and relative areas were observed. For the 2"< component, mean time constants

became apparently smaller only in 3 pM ifenprodil while mean relative areas were larger in 1 pM

ifenprodil but similar to control conditions in 3 pM ifenprodil. For the 3' exponential, mean time

constants became significantly smaller only in 3 pM ifenprodil while mean relative areas were

considerably smaller in 1 pM but similar to control conditions in 3 pM ifenprodil (Table 6.3).

6.3.4.2 In patches w ith high and low conductance currents

In patches with high and low conductance single-channel activity, the reduction in mean

open time was apparently the result of changes in mean time constants and/or mean relative areas of

all three exponential components. For the (fastest) exponential component, no apparent changes

in mean time constants were observed but its mean relative area became smaller in the presence of

ifenprodil. For the 2"* component, the mean time constant was smaller but its mean relative area

became larger. For the 3^ exponential, the mean time constant became smaller with apparently no

significant change in its mean relative area (Table 6.3).

6.3.5 Effects on open times conditional on amplitude level

Before building open time distributions conditional on amplitude, critical amplitude values (Acrk)

between pairs of adjacent Gaussian components fitted to amplitude distribution histograms were

calculated to separate individual currents based on their amplitude. Acii values giving an equal

percentage of misclassified events were calculated. In patches with only high conductance single

channel activity, mean Acrit values between the two Gaussian components: 2.90 ± 0.08 pA (control),

2.74 ± 0.03 pA (1 pM ifenprodil) and 2.83 ± 0.03 (3 pM ifenprodil). In patches with high and low

conductance single-channel activity, mean Aait values between the four Gaussian components were:

1.57 ± 0.06 pA (1 & 2"( , 2.27 ± 0.09 pA (2"< & 3*‘‘ and 3.00 ± 0.03 pA (3'‘‘ & 4 ) under control

conditions and 1.56 ± 0.05 pA (1 & 2"« , 2.29 ± 0.04 pA (2"‘ & 3*‘‘ and 3.01 ± 0.03 pA (3'‘‘ &4^) in

1 pM ifenprodil.

173

17 pS currents 33 pS currents

AControl90 T (ms) area (%)

1.49 100.003

gI 40

2

Ez

10


BControl90 T (ms) area (%)

0.881 100.00)

I 40

£3S’


S(O 90

3 40

E 10

Ifenprodil IpM

0.1

T (ms) area (%) 1.70 100.0

j ' I I 1111____I__I_I I I 111 _i 1 I I 11111 10


D Ifenprodil IpM90 T (ms) area (%) 0.561 100.0

I 40

£

œ 10"Ei

1000.1 1 10 Open time (ms) (log scale)

Figure 6.5 Distribution of the duration of individual openings to the 17 and 33 pS conductance levels under control conditions (A,B) and after 1 pM ifenprodil(C, D). Histograms are distributions of individual open times longer than 300 ps. Data obtained from a single outside-out patch containing high and low conductance NMDA receptor single-channel activity. Histograms were fitted with 1 exponential component. Time constants and relative areas are shown. Predicted number of events were: (A) 427; (C) 1194; (B) 204 and (D) 314. NMDA (5 ph^ and glycine (10 pM) were used as agonists.

174

6.3.5.1 Effects on 17 and 33 pS currents

Open time distributions containing openings to the 17 pS conductance level were fitted with

a single exponential component (Figure 6,5). Open time distributions containing openings to the 33

pS conductance level were also fitted with a single exponential component (Figure 6.5). In the

presence of ifenprodÜ (1 pM), the mean open time of 17 pS currents remained apparently constant

while that of 33 pS currents became smaller (Table 6.4).

T able 6.4 Distribution of open times to the 17 and 33 pS level

(ms)

17 pS

Control 1.1 ± 0.3 (464)Ifenprodil 1 pM 1.3 ± 0.3 (1213)

33 pS

Control 0.9 ±0.03 (255)Ifenprodil 1 pM 0.6 ± 0.01 (425)

6.3.5.2 Effects on 42 pS currents

In both patches with only high conductance and patches with high and low conductance single

channel activity, currents to the 42 pS conductance level had 30 to 50 % smaller mean open times in

the presence of ifenprodil than under control conditions. For the 1 esqjonential component, the

mean time constant was smaller in 1 pM ifenprodil in all patches; in contrast, only in patches with

high and low conductance single-channel activity its mean relative area became notably smaller. For

the 2"( exponential component, mean time constants became smaller but its relative area was larger

in the presence of ifenprodil than under control conditions. In patches with only high conductance

single-channel activity that were exposed to 3 pM ifenprodil only a single exponential component

with a mean time constant of 0.7 ± 0.06 ms could be detected (Table 6.5).

175

42 pS Currents


/K Control160 T (ms) area <%) 1.29 94.55.13I 5.5

ie

Iz1000.1


B Control160 T (ms) area (%) 0.727

2.8378.321.7s

o22III


C Ifenprodil 1pM160 T (ms) area (%) 0.665 67.3

1.39 32.7Io22gST

IE


D Ifenprodil 1pM160 T (ms) area (%) 0.262 54.80.967a) 45.2

II

90

2gS’

E

1001 10 Open time (ms) (log scale)

0.1

Figure 6.6 Distribution of the duration of individual open times for the 42 pS conductance level under control conditions (A,B) and after 1 pM ifenprodil (C,D). Histograms are distributions of individual open times longer than 300 ps. A and C are from a patch with high and low conductance NMDA channels. B and D are from a patch with only high conductance NMDA channels. Histograms were fitted with 2 exponential components. Time constants and relative areas for each exponential component are shown. The predicted overall mean open time and number of events were: (A) 1.50 ms, 619; (C) 0.90 ms, 475; (B) 1.18 ms, 768 and (D) 0.58 ms, 385. NMDA (5 pM) and glycine (10 ph^ were used as agonists.

176

50 pS Currents


kControl Control180 3 60T (m*> area <%)

1.91 3.93

T (m*) area {%) 2.19 4.04

52.447.6

64.236.8I

I 2 16080

2

_g 20

Iz

40

1 10

Open time (ms) (log scale)1000.1 0.1 100


C Ifenprodil 1pM Ifenprodil 1pM180 360T (ms) area <%) 0.630 40.4

2.46

T (ms) 0.892

1.82

(%)65.334.7I 59.6

i O 160

2gS'

Iz

1001 10 Open time (ms) (log scale)

0.1 0.1 1 10 Open time (ms) (log scale)

Figure 6.7 Distribution of the duration of individual open times for the 50 pS conductance level under control conditions (A,B) and after 1 pM ifenprodil (C,D). Histograms are distributions of individual open times longer than 300 ps. A and C are from a patch with high and low conductance NMDA channels. B and D are from a patch with only high conductance NMDA channels. Histograms were fitted with 2 exponential components. Time constants and relative areas for each exponential component are shown. The predicted overall mean open time and number of events are: (A) 2.87 ms, 2174; (C) 1.72 ms, 742; (B) 2.85 ms, 4216 and (D) 1.22 ms, 741. NMDA (5 pM) and glycine (10 pM) were used as agonists.

177

T able 6.5 Distribution of individual open times for the 42 pS level

mean

Only High

Control

Ifenprodil 1 |uM

Ifenprodil 3 |nM

High & Low

Control

Ifenprodil 1 pM

(ms)

0.7 ± 0.03 83 + 4 %

0.3 ± 0.05 73 ± 13 %

0.7 ± 0.06 100 %

1.1 ± 0.290 + 3 %

0.5 ±0.1 47 ± 14 %

(ms)

2.5 ± 0.3 17 + 4 %

1.2 ± 0.2 27 ± 13 %

4.4 + 0.510 + 3 %

1.3 + 0.04 53 ± 14 %

(ms)

1.0 ± 0.1 (1769)

0.5 ± 0.03 (540)

0.7 ± 0.06 (220)

1.4 ±0.1 (3043)

1.0 ± 0.05 (3588)

Table 6.6 Distribution of individual open times for the 50 pS level

Only High

Control

Ifenprodil 1 pM

Ifenprodil 3 pM

High & Low

Control

Ifenprodil 1 pM

(ms)

2.2 ± 0.01 53 ± 8 %

1.5 ± 0.5 74 ± 6 %

1.5 ±0.1 100 %

1.7 ± 0.2 29 ± 17 %

0.7 ± 0.09 36 ± 3 %

(ms)

4.0 ± 0.002 47 ± 8 %

3.2 ± 1.0 26 ± 6 %

3.8 ±0.1 71 ± 17 %

2.1 ± 0.3 64 ± 3 %

mean

(ms)

3.1 ±0.2 (6901)

1.9 ± 0.5 (2054)

1.5 ± 0.1 (1010)

3.2 ± 0.2 (3445)

1.6 ± 0.1 (1092)

178

6.3.S.3 Effects on 50 pS currents

Currents to the 50 pS conductance level had 40 to 50 % smaller mean open times in the

presence of ifenprodil than under control conditions. A considerabty' reduction (70 - 85 %) in the

number of openings to the 50 pS level was also observed in the presence of ifenprodil.

The effects of ifenprodil on each one of the two exponential components fitted to distributions

of open times to the 50 pS level were as follows: for the exponential component, mean time

constants became smaller in all patches while mean relative areas were notably latter only in patches

with only high conductance single-channel activity. For the 2 exponential component, mean time

constants in the presence of ifenprodil were smaller in patches with high and low conductance while

in patches with only high conductance single-channel activity it showed no apparent changes. In

contrast, mean relative areas became smaller in all patches. In patches with only high conductance

single-channel activity that were exposed to 3 pM ifenprodil, only a single exponential component

with a mean time constant of 1.5 ± 0.1 ms could be detected (Table 6.6)

6.3.6 Effects on hursts

The effects of ifenprodil on bursts of openings from patches with only high conductance

single-channel activity were analysed. In the presence of ifenprodil, a considerable reduction (50-70

%) in number of bursts, mean burst length and mean total open time per burst was observed. In

contrast, only a small reduction in mean burst/open was observed (Tables 6.7, 6.8, 6.9).

6.3.6.1 Effects on hurst lengths

In the presence of ifenprodil three rather than four exponential components were enough to

obtain a satisfactory fit of the distribution of burst lengths. Mean fcm values were 1.1 ± 0.1 ms

(control), 1.8 ± 0.2 ms (1 pM ifenprodil) and 2.4 ± 0.6 ms (3 pM ifenprodil). The mean burst length

was —50 % smaller in the presence of ifenprodil than under control conditions.

For the 1 exponential component, mean time constants showed no apparent changes while

mean relative areas were larger in the presence of ifenprodil. For the 2“* exponential component,

mean time constant and relative area were considerably larger. For the 3''' exponential component,

mean time constants were similar but its relative area became increasingly smaller as the ifenprodil

concentration was increased. In the presence ifenprodil, a 4* longest) exponential component

observed under control conditions could not be detected (Table 6.7).

179

Only High Conductance

ControlT (ms) area (%)0.04380.685

0.1 1 10 100Burst length (ms) (log scale)

BControl160 T (ms) area (%)

37.90.441 6.6

6.53 45.118.0

0.0417

10.4

40

0,0.01 10.1 10 100 1000


C Ifenprodil 1pM160 T (ms) area (%) 63.4

1.96 30.510.3 16.1

0.0628

2i23 40w"

ÈEZ

0.01 1000.1 1000Burst length (ms) (log scale)

D Ifenprodil IpM160 T (ms) area (%) 52.8

1.69 29.89.00 17.4

0.0540

40

00.01 100.1 1

Open time per burst (ms) (log scale)100 1000

Figure 6.8 Distribution of burst lengths and total open time per burst under control conditions (A,B) and after 1 pM ifenprodil (C, D). Data was obtained from a patch with only high conductance singe-channel activity. Histograms were fitted with 3 (C,D) and 4 (A,B) exponential components. Time constants and relative areas for each exponential component are shown. The predicted overall mean burst length (A,C), mean total open time per burst (B,D) and total number of bursts were: (A) 5.15 ms, 2443; (C) 2.28 ms, 653; (B) 4.86 ms, 2435 and (D) 2.13 ms, 433. NMDA (5 pM) and glycine (10 pM) were used as agonists.

180

T able 6.7 Distribution o f burst lengths

mean


Control 60 ±11 34 + 3 %

0.86 ±0.12 6 ± 0.3 %

7.9 ± 0.6 50 ± 2 %

20.6 ± 1.2 10 ± 0.01 %

6.0 ± 0.6 (3693)

Ifenprodil 1 pM 57 ± 3 50 + 3 %

2.70 ± 0.5 31 ± 1 %

10.5 ±0.1 19 ± 2 %

2.9 ± 0.4 (1712)

Ifenprodil 3 pM 71 ±10 44 ± 1 %

3.43 ± 0.5 50 ± 1 %

12.0 ± 0.9 6 ± 1 %

2.5 ± 0.4 (961)

6.3.6.2 Effects on total open time per burst

As in burst length distributions, three exponential components could be fitted to distributions of

total open time per burst in the presence of ifenprodil (Figure 6.8). The mean total open time per

burst was also reduced (~50 %) in the presence of ifenprodil (Table 6.8)

For the (fastest) exponential component, mean relative areas increased in the presence of

ifenprodil but mean time constants remained apparent^ unchanged. For the 2 exponential

component, mean time constants and mean relative areas increased considerably in the presence of

ifenprodil. For the exponential component, mean time constants remained apparently unchanged

while mean relative areas became increasingly smaller as the ifenprodil concentration increased. In

the presence of ifenprodil, a 4^ (longest) exponential component observed under control conditions

could not be detected (Table 6.8).

T able 6.8 Distribution of total open time per burst

' 4 mean


Control 60 ±13 34 ± 3 %

0.67 ±0.16 7 ± 0.04 %

7.5 ± 0.7 49 ± 3 %

19.8 ± 1.3 10 ± 0.1 %

5.8 ± 0.7

Ifenprodil 1 pM 56 ± 6 50 ± 3 %

2.36 ± 0.8 29 ± 4 %

9.5 ± 0.8 21 ± 1 %

2.7 ± 0.4

Ifenprodil 3 pM 74 ±12 44 ± 1 %

3.03 ± 0.3 50 ± 1 %

10.4 ±0.1 6 ± 1 %

2.2 ± 0.4

181

6 .3 .6 .3 Effects on hurst Popen

Ifenprodil produced a small reduction in mean burst Popen (Table 6.9).


Mean open time per Mean burst length Mean burst Popenburst

(ms) (ms)

Control 5.8 ± 0.6 6.0 ± 0.6 0.97 ± 0.01

Ifenprodil 1 pM 2.7 ± 0.4 2.9 ± 0.4 0.95 ± 0.01

Ifenprodil 3 pM 2.2 ± 0.3 2.5 ± 0.4 0.90 ± 0.03

6 .3 .7 Effects on clusters

Ifenprodil produced a reduction in number of clusters (55 - 87 %), mean total open time per

cluster ( ~ 60 %) and mean cluster Popen (28 - 54 %) with apparently relative^ small effects on mean

cluster length. Clusters were separated by tmt values giving equal number of misclassihed events

between the 4‘ and 5* component of the shut time distributions. Mean tcm values became larger in

the presence of ifenprodil with values of 12.6 ± 0.04 ms (control), 23.4 ± 3.3 ms (1 pM ifenprodil)

and 38.4 ± 13.0 ms (3 pM ifenprodil).

6.3 .7 .1 Effects on cluster lengths

Ifenprodil (1 pM) produced a slight reduction in mean cluster length while 3 pM ifenprodil had

apparently no effects. In both cases, control and ifenprodil, distributions of cluster lengths could be

fitted with 4 exponential components (Figure 6.9A,B). In the presence of ifenprodil, the 1*

exponential component showed no change in mean time constants but mean relative areas increased

considerably. In contrast, the 2“‘ exponential component had slightly faster mean time constant in 1Lre

pM but not in 3 pM ifenprodil while mean relative areas slightly larger. The 3>’ exponential

component showed no apparent changes in its mean time constant but its relative area became

increasingly smaller as the ifenprodil concentration was increased. The 4* exponential component

had larger mean time constant and smaller mean relative area in 1 pM ifenprodil. In contrast, in 3

pM ifenprodil, it had a mean time constant similar to that in control conditions but its mean relative

area became considerably larger (Table 6.10).

182

Only High Conductance

A Control160 T (ms) area <%) 36.6

1.62 11.510.0 40.433.8 11.6

0.0488

SI2

Ez

1 10

Cluster lengtti (ms) (log scale)100 10000.01 0.1

B Control160 T (ms) area (%) 36.1

1.43 10.97.74 39.726.7 13.3

0.0509

I£

0.01 0.1 1 10 Open time per cluster (ms) (log scale)

100 1000

E 4

Ifenprodil 1pMT (ms) area (%) 0.0415 60.0

0.693 16.213.0 22.3

78.7 2.6

\■■■■I I I I 11 lil I

0.1 1 10 100 Cluster length (ms) (log scale)

D Ifenprodil 1pM64 T (ms) 0.0408

0.623 6.62 73.7

(%)60.016.421.9I« 36

1£

I "Iz

0.01 1000.1

Open time per cluster (ms) (log scale)1000

Figure 6.9 Effects of ifenprodil on cluster length and total open time per cluster. Histograms are distributions of cluster lengths and total open time per cluster under control conditions (A,B) and after 1 pM ifenprodil (C, D). Data was obtained from a patch in which activation of NMDA receptors produced only high conductance single-channel activity. Histograms were fitted'Texponential components. Time constants and relative areas for each exponential component are shown. The predicted overall mean cluster length (A,C), mean total open time per cluster (B,D) and total number of clusters were: (A) 8.16 ms, 1656; (C) 4.99 ms, 340; (B) 6.80 ms, 1640 and (D) 2.76 ms, 335. NMDA (5 pM) and glycine (10 pM) were used as agonists

183

Table 6.10 Distribution of cluster lengths

Tl ' 2 ' 3 mean


Control 65 ±11 35 ± 1 %

1.95 ± 0.2 10 + 1 %

13 ±2.2 45 ± 4 %

44 ± 7 10 ± 1 %

10.3 ± 1.5 (2356)

Ifenprodil 1 pM 49 ± 3 52 + 6 %

0.84 + 0.2 14 + 1 %

13 ±0.1 33 ± 7 %

110 ±22 2 ± 1 %

5.9 ± 0.6 (1059)

Ifenprodil 3 pM 62 ± 7 48 + 2 %

1.43 ± 0.07 18 + 3 %

*12713%

37 ± 0.4 22 ± 11 %

9.7 ±2.6 (312)

detected in 1 out of 2 patches

6.3.Z.2 Effects on total open time per cluster

In contrast to cluster lengths, ifenprodil produced a considerable reduction (~60 %) in mean

total open time per cluster. In 1 pM ifenprodil four exponential components could be fitted (Figure

6.8) while in 3 pM ifenprodil only three exponential component were detected.

For the 1* (fastest) exponential component, no apparent changes in mean time constant were

observed but its mean relative area became larger. The 2"* and exponential components in 1 mM

ifenprodil had smaller mean time constant and no apparent change in their mean relative area was

observed. The 4^ component had a larger mean time constant but a smaller relative area. In 3 mM

ifenprodil only three exponential components could be fitted with the (fastest) and 2*“*

(intermediate) components having the largest relative areas (Table 6.11).

Table 6.11 Distribution o f total open time per cluster

Tl ' 2 3 mean


Control 67 ±11 35 ± 1 %

1.84 ± 0.3 12 ± 1 %

11 ±2.3 43 ± 2 %

38 ± 8 10 ± 2 %

8.5 ± 1.2

Ifenprodil 1 pM 51 ± 6 51 ± 6 %

0.59 ± 0.03 14 ± 2 %

7 ±0.3 33 ± 7 %

97 ±25 1 ± 0.5 %

3.6 ± 0.6

Ifenprodil 3 pM 67 ± 5 48 ± 3 %

3.9 ± 0.8 44 ± 3 %

23 ± 4 7 ± 0.2%

3.4 ± 0.5

184

6.3.7.3 Effects on cluster Popen

The mean cluster Popen became increasingly smaller as the ifenprodil concentration was increased

(Table 6.12).

T a b le 6.12 Cluster Popen

Mean open time/cluster Mean cluster length Mean cluster P<open

(ms) (ms)

Control 8.5 ± 1.2 10.3 ± 1.5 0.83 ± 0.01

Ifenprodil 1 |nM 3.6 ± 0.6 5.9 ± 0.6 0.60 ± 0.03

Ifenprodil 3 pM 3.4 ± 0.5 9.7 ± 2.6 0.38 ± 0.05

6.3.8 Effects on the decay time-course o f aligned clusters

Ifenprodil had apparent^ no effect on the mean decay time course

built after aligning clusters of openings from receptor activations (Table 6.13).

of ensemble averages

T a b le 6.13 Decay time-course o f aligned clusters

'Cl 'Cz 'C3mean

(ms) (ms) (ms) (ms)

Control 1.3 ±0.1 0.9 ±0.1

9.1 ± 2.5 39 ± 9

31.7 ±6.5 60 ± 9

21.9 ± 2.9

Ifenprodil 1 |uM 1.8 ± 0.5 21 ± 7

10.4 ± 0.04 33 ± 8

46.6 ± 19 46 ± 1

24.9 ± 8.6

Ifenprodil 3 pM 1.6 ± 0.004 19 ± 1

*8.719

30.7 ± 3 61 ±15

20.0 ± 1.9

6.4 D i s c u s s i o n

Ifenprodil produced an overall reduction in single-channel activity mediated by NMDA

receptors in outside-out patches from PO-hippocampal granule cells. This effect was produced

through a selective reduction of high conductance single-channel activity with no apparent effects on

low conductance single-channel activity. Unfortunately, no single-channel data on the effects of

ifenprodil on recombinant NMDA receptors containing any of the NR2 subunits is currently

available for comparison.

185

6.4.1 Effects on single-channel current amplitudes

Ifenprodil had apparently no effect on the mean amplitude of any of the four single-channel

current levels that were identified. A lack of effect of ifenprodil on the single-channel conductance

of NM DA receptors in cultured hippocampal neurons have been previously described by Legendre

& Westbrook (1991). This lack of effect of ifenprodil on the mean amplitude of the single-channel

currents was particularly important because single-channel activity in patches was classified based on

the type of conductance levels observed.


In the presence of ifenprodil the mean time constants of the 4*’, and 6 e3q)onential

components became considerably larger in patches with only high conductance single-channel

activity. This suggested that ifenprodil had effects not only between but within individual single

channel activations. The 3^ and 4* exponential components have been shown to occur within burst

and clusters, respectively, contained within individual single-channel activations. The effects on

bursts and clusters are discussed in sections 6.4.4 and 6.4.5 of this discussion.

6.4.3 Effects on individual open times

The effects of ifenprodil on the distribution of open times was characterised by a reduction

in the mean open time and frequency of channel opening. A reduction in the mean open time and

number of channel openings has also been previously reported by Legendre & Westbrook (1991).

Ifenprodil produced a reduction in the mean open time of openings to 33, 42 and 50 pS

levels; while the mean open time of openings to the 17 pS level remained constant. Because 17 pS

currents have only been observed in NR1/NR2C and NR1/NR2D recombinant NMDA receptors

which have been shown to be insensitive to ifenprodil (Williams, 1995), this finding suggests that

low conductance single-channel activity was probably the result of the activation of native NMDA

receptors containing NR2C or NR2D subunits. So far, protein or mRNA signals encoding NR2C

subunits have been shown not to be present in PO-hippocampus, so the apparent lack of effect of

ifenprodil on 17 pS current may suggest that low conductance single-channel activity were produced

by native NMDA receptors containing NR2D rather than NR2C subunits. In contrast, the dramatic

effects of ifenprodil on openings to the 50 pS level suggested that high conductance single-channel

activity was produced by activation of NMDA receptors containing NR2B subunits. Ifenprodil

produced a 70 - 80 % reduction in the number of openings to the 50 pS level together with a 40 -50

% reduction in their mean open time.

6.4.4 Effects on hursts

In patches containing only high conductance single-channel activity, the most significant

effects of ifenprodil were a 50 - 70 % reduction in the number of bursts together with a 50 %

186

reduction in mean burst length. In the presence of ifenprodil, bursts with the longest duration (21 ±

1 ms) became absent while the frequency of bursts having intermediate duration (8 ± 0.6 ms) were

less frequent with their relative area going from 50 % under control conditions to 6 % in 3 pM

ifenprodil.

Even when the mean time constant of the 3*" exponential component, which was classified

as being within bursts, became larger such lengthening did not seem to produce a proportional

increase in mean burst length. This could be explained by the considerable reduction in mean open

time per burst which was characterised by the absence of the longest (4 ) exponential component of

the distribution of total open time per burst in the presence of ifenprodil. Surprisingly, the mean

burst Popen was only slightly reduced by ifenprodil.

6.4.5 Effects on clusters

In patches containing only high conductance single-channel activity, ifenprodil produced a

50 - 85 % reduction in the number of clusters. Surprisingly, ifenprodil had no apparent effect on

mean cluster length. This suggested that the effects of ifenprodil on clusters were different from

those observed in bursts. In the presence of ifenprodil, long clusters were apparently always present

although their mean total open time was reduced by —50 %. In addition, the mean tcû. values, which

were calculated between the 4^ and 5* component of the shut time distribution and were used to

separated clusters, increased by 88 % and 200 % in the presence of 1 pM and 3 pM ifenprodil,

respectively. The increase in mean tcm values can be probably explained by the -100 % increase in

mean time constant of the 4 exponential component which was classified as being within clusters.

In summary, these findings suggest that in PO-hippocampal granule cells high conductance

single-channel activity is mediated by an ifenprodil-sensitive NMDA receptor population, a property

only found in NMDA receptors containing NR2B subunits. In contrast, the ifenprodil insensitivity

and characteristic single-channel properties (Chapter 5) suggest that low conductance single-channel

activity is mediated by a NMDA receptor population containing NR2D subunits.

187

CHAPTER 7

G e n e r a l D i s c u s s i o n

The aim of the work discussed in this thesis was to describe, at the single-channel

level, the functional and pharmacological properties characterising NM DA receptors in the rat

hippocampal dentate gyrus and striatum at birth (PO). At PO, hippocampal and striatal neurons

show a much simpler pattern of expression of NM DA receptor subunits than that found in

adult brain. These native NM DA receptors were studied at the single-channel level because of

the very high resolution single-channel measurements provide which is useful in detecting

contributions made by specific receptor subunits to the functioning of recombinant and native

N M D A receptors (Stern et a i, 1992; 1994; Wyllie et al., 1996; F arrant é t a l , 1994; Momiyama

et ai , 1996). Little is known about the functional properties of native NM D A receptors in PO

hippocampal dentate gyrus and striatum. In this thesis, the single-channel data obtained from

these native receptors has been correlated with current knowledge about the pattern of

expression of NM DA receptor subunits in these structures and knowledge of the functional

properties of recombinant NM DA receptors.

7.1 NM D A receptors in PO striatal neurons

In PO striatal neurons, analysis of NM DA receptor single-channel properties revealed

firstly, that activation of NM DA receptors produced a high conductance pattern of single

channel activity characterised by 44 pS and 54 pS single-channel currents; and secondly, that

in the presence of a saturating glycine concentration, spermine was able to produce an increase

in receptor activity, a property found only in recombinant NM DA receptors containing both

N R la and NR2B subunits (reviewed by Williams, 1997). These findings were consistent with

evidence showing that m RNA (Monyer et a l, 1994; Riva et a l, 1994; Laurie & Seeburg, 1994)

and protein (Portera-Cailliau et a l, 1996; Wenzel et a l, 1997) encoding N R l-la and NRT2a

splice variants and NR2B subunits are highly expressed in new-born striatal neurons.

7.1.1 Can striatal NM DA receptors he considered a homogeneous population of

N R la /N R 2 B native NM DA receptors^

Currently, the answer is not. The possibility that these receptors contain other

subunits should not be excluded. Although spermine is apparently useful in revealing the

presence of N R la and NR2B subunits, it does not provide information about the presence of

other subunits. Zhang et a l, (1994) reported that glycine-independent spermine potentiation

188

can still be observed in recombinant NM DA receptors produced after co-expressing N R la,

NR2B and NR2A subunits; so, the possibility that NM DA receptors showing glycine-

independent potentiation by spermine also contain NR2A subunits should not be discarded.

Wenzel et al., (1997) have recently described very low levels of NR2A protein in PO-striatum,

which is in sharp contrast with previous evidence reporting an absence of NR2A m RNA

(Monyer e ta l , 1994) and protein (Portera-Cailliau e ta l , 1996) in striatum before postnatal day

16 (P16). Flint et a l, (1997) have recently shown, using single cell RT-PCR, that expression of

N R 2 A m RNA just above background levels (> 1 0 %) is apparently enough to produce a fast

decay in N M D A receptor-mediated excitatory postsynaptic currents (EPSCs), a property only

observed in N M D A receptors containing NR2A subunits. In addition to the biochemical

evidence describing absence of NR2A subunits in striatum before P16 (Monyer et at., 1994;

Portera-Cailliau et ai,, 1996), Gotz et al., (1997) have recently found that N M D A receptor-

mediated macroscopic currents in patches from P10-P15 striatal neurons have a slow mean

deactivation time constant characteristic of NR2B-containing NM DA receptors. They did not

find functional evidence of a heterogeneous population of NM DA receptors in P10-P15

striatal neurons.

Even when currently available single-channel data (Stern et a l, 1992; 1993; 1994)

shows no apparent functional differences between single-channel activity produced by

N R 1/N R 2A and NR1/NR2B recombinant NM DA receptors, functional differences have

been found in the mean deactivation time constant of currents produced by N R 1/N R 2A and

N R 1/N R 2B recombinant NM DA receptors (See sections 1.8.1.1.1 and 1.8.1.1.2; Monyer et

a l, 1994; Vicini et a l, 1998). Because the activation-inactivation kinetics of N M D A receptors

are determined by the agonist-receptor interaction (reviewed by Lester et a l , 1994), this

suggests that functional differences between receptors do exist but may not be easily detected

by looking at individual single-channel events but to groups of events such as bursts, clusters

and/or super-clusters. Analysis of bursts, clusters and super-clusters produced by NM DA

receptors obtained after co-expression of different subunit combinations may prove useful in

detecting how NR2A subunits contribute to the fast mean deactivation time constants

associated with recombinant (Monyer et a l, 1994; Vicini et a l, 1998; Wyllie et a l, 1998) and

native (Flint e ta l , 1997) NM DA receptors containing this particular subunit.

Use of a pharmacological approach may also prove useful in detecting contributions

made by NR2A subunits to the phenotype of NM DA receptors in PO striatum. In initial

experiments carried out during this study, it was assumed that because the glycine equilibrium

dissociation constant for native NM DA receptors was reported to be around 0.13 pM

189

(reviewed by Ascher & Johnson, 1994), an extracellular concentration of 3 |iM glycine should

have been enough to saturate all NM DA receptors present in the patch. After Wenzel et al.,

(1997) reported very low expression of NR2A subunits, which have low glycine affinity, a

higher glycine concentration (10 pM) was used. In recombinant NM DA receptors, EC 5 0

values for glycine range between 1 and 3 pM for N R 1/N R 2A (Williams, 1996; Zhong et a l,

1996; Stern et a l, 1992) and between 0.2 and 0.8 pM for NR1/NR2B (Kutsuwada et a l, 1992;

Laube e ta l , 1993; Williams, 1996; 7hon% etal, 1996; Kashiwagi e ta l , 1996; Hirai e ta l , 1996/

If a sub-population of low glycine sensitivity NR2A-containing NM DA receptors is present in

PO striatal neurons then an increase in single-channel activity would have been observed after

the extracellular glycine concentration was increased from 3 to 10 pM. However, mean

values were 0.035 ± 0.010 (n= 8 ) and 0.035 ± 0.012 (n=4) in 3 pM and 10 pM glycine,

respectively. In cultured hippocampal neurons, Kendrick et a l, (1998) have recently shown

evidence of a low glycine sensitive NR2A-like population of NM DA receptors. Even though

these findings are apparently consistent with an absence of NR2A-containing NM DA

receptors in PO striatum, further experiments will be needed in order to identify any potential

contribution made by NR2A or any other subunits to NM DA receptors in this brain

structure.

7.1.2 Future experiments

It has recently been shown that high affinity Zn^ inhibition of NR2A-containing

N M D A receptors (Chen et a l, 1997; Paoletti et a l, 1997) can apparently be removed by

chelating extracellular contaminating Zn^ with TPEN (N ,N ,N ’,N ’-tetrakis-(2 -pyridilmethyl)-

ethylenediamine), a non-reducing metal chelating agent (Paoletti et a l, 1997). The effect of

TPEN is described as a potentiation of macroscopic currents produced by N M D A receptors

in cultured cells and by N R la/N R 2A recombinant N M D A receptors expressed in HEK 293

cells and Xenopus oocytes (Paoletti et a l, 1997) but not by N R la/N R 2A receptors expressed

in Chinese hamster ovary (CHO) cells (Brimecombe et a l, 1997). If a sub-population of

NR2A-containing NM DA receptors with high Zn^ sensitivity is present in PO striatal

neurons then an increase in single-channel activity will be expected after contaminating Zn^^

is removed by adding TPEN; although, evidence from N R la /N R 2 A receptors expressed in

CH O cells suggests that may not always be the case (Brimecombe et a l, 1997).

A different approach has been recently used by Brimecombe et a l , (1997) to describe

the presence of NR2A and NR2B subunits in recombinant NM DA receptors produced after

co-expressing N R l, NR2A and NR2B subunits in CHO cells. They used the sulfydryl redox

reagents DTT (dithiothreitol) and DTNB (5,5'-dithio-6 zj[2 -nitrobenzoic acid]), and CP-

190

101,606, an ifenprodil analogue (Chenard et al. 1995). DTT and DTNB selectively increase

and reduce, respectively, the mean open time of N R 1 /N R 2 A but not NR1/NR2B

recombinant NM DA receptors. They found that some NM DA receptors having a

NR l/N R 2B-like behaviour (insensitive to DTT and DTNB) also had a N R l/N R 2 A-like

behaviour (insensitive to CP-101,606). This evidence was interpreted as an indication of

NR2A and NR2B subunits being present in a single receptor-channel complex. Similar

approaches could be used to assess the potential contribution of NR2A subunits to NM DA

receptors in PO striatal neurons.

7.2 N M D A receptors in PO dentate gyrus

Analysis of the single-channel properties of NM DA receptors present in neurons from

the granule cell layer of PO hippocampal dentate gyrus produced a number of new findings,

including some not yet clearly understood. First, it was found that PO dentate gyrus NM DA

receptors were able to mediate two different patterns of single-channel activity. One pattern of

activity was similar to that produced by PO striatum NM DA receptors in which only high

conductance (42 and 51 pS) single-channel activity was observed and a second pattern of single

channel activity which, in addition to high conductance (42 and 49 pS) single-channel activity,

showed low conductance (17 and 42 pS) single-channel activity. Second, in the high and low

conductance pattern of single-channel activity, receptor activations were not evenly

distributed but segregated into periods of either high or low conductance. The reason for this

apparent segregation was not known and will need further investigation. Third, the functional

behaviour of the NM DA receptors producing the high conductance pattern of single-channel

activity was apparently independent of whether they were present in patches with either only

high conductance or high and low conductance single-channel activity and fourth, in patches

with either only high conductance or high and low conductance single-channel activity, high

conductance single-channel activity was selectively inhibited by ifenprodil, a non-competitive

antagonist of NM DA receptors containing NR2B subunits (Williams, 1993). The presence of

high conductance ifenprodil-sensitive and low conductance ifenprodil-insensitive single

channel activity is consistent with evidence showing m RNA (Monyer et al. 1994; Laurie &

Seeburg, 1994) and protein (Wenzel et al. 1997) encoding NR2B and NR2D subunits in the

granule cell layer of the hippocampal dentate gyrus.

7.2.1 Is the high and low conductance pattern o f single-channel ac tiv ity the result o f the

activation o f two different NM DA channels or a single NM DA channel species w ith two

different single-channel behaviours^.

191

Before trying to answer this question it is important to discuss the reasons why the

analysis of single-channel conductances has proved to be an useful criteria in identifying the

source of multiple single-channel conductance levels. This is explained by the fact that being

an intrinsic property of the channel, for any particular ionic condition the singe-channel

conductance depends on the structure of the channel. The structure of the channel is

determined by the type of subunits that take part in its assembly and, in some cases, by the

position each subunit occupies within the channel structure. It is possible to find large

differences in conductance between channels permeable to the same ion species and even

between channels belonging to the same family (Hille, 1992). Based on this current

knowledge, the observation of single-channel currents with different conductance and

pharmacology is interpreted as an indication of a heterogeneous population of NM DA

channels rather than as a single NM DA channel population with functional and

pharmacologically different single-channel behaviours.

Because of their different functional and pharmacological properties and striking

similarities with NR1/NR2B and N R 1/N R 2D recombinant N M D A receptors, it was

concluded that high and low conductance single-channel activity were the result of the

activation of two functionally and pharmacologically different N M D A receptor-channel

populations: high conductance ifenprodil-sensitive NR2B-like NM D A receptors and low

conductance ifenprodil-insensitive NR2D-like NM DA receptors.

7.2.2 Is it possible from the data obtained to suggest which subunits are taking p a rt in the

assembly o f these two functionally and pharmacologically different NM D A receptor

populations?.

Although the selective effect of ifenprodil suggested that high conductance single

channel activity was apparently produced by NR2B-containing N M D A receptors, it provided

no information about the presence in high conductance NM DA channels of NR2D or indeed

any other subunits. Unfortunately, no selective antagonist for NM DA receptors containing

NR2D-subunits is yet available. Immunoprécipitation studies by Dunah et al. (1998) have

recently shown evidence of binary (N R l/2D), ternary (N R 1/2A /2D , N R 1/2B /2D ) and

probably quaternary (NR1/2A/2B/2D) complexes of NM DA receptor subunits in rat brain.

In addition, Buller & Monaghan (1997) have recently reported the existence of a

pharmacologically distinct population of recombinant NM DA receptor after co-expressing

N R l, N R 2 B and N R 2 D subunits in Xenopus oocytes. Taken together, this data suggests that

in the brain NR2B and NR2D subunits may assemble to produce an N M D A receptor species

with a distinct pharmacology.

192

If high conductance ifenprodil-sensitive NR2B-like single channel activity is produced

by N M D A receptors containing both NR2D and NR2B subunits, then the effect that NR2D

subunits may be having on single-channel conductance, kinetics or ifenprodil-sensitivity is

probably either very subtle or undetectable by using single-channel measurements. An

alternative explanation would be that a majority of the high conductance ifenprodil-sensitive

NR2B-like single-channel activity is mediated by NM D A receptors lacking NR2D subunits

and those containing NR2D subunits probably represent a very small fraction of the receptor

population. A similar argument would help to explain the presence of low conductance

ifenprodil-insensitive NR2D-like single channel activity.

An alternative explanation would be that high and low conductance NM D A channels

have actually the same subunit composition (NR1/NR2B/NR2D) but their single-channel

properties and ifenprodil-sensitivity is determined by the order in which subunits are

assembled within the receptor. The order in which subunits assemble has been shown to

determine the conductance of cyclic nucleotide-gated ion channels (Liu et al., 1996). That

might also explain the differences in ifenprodil sensitivity. The high affinity ifenprodil

binding site is associated with the N R l subunit (WiUiams, 1993) and assembly with N R 2

subunits other than NR2B seems to disrupt it (Williams, 1993; WilUams, 1995; Avenet et al.,

1996; Whittemore et al., 1997). Further experiments will be needed in order to identify the

functional and pharmacological contributions of both NR2B and NR2D when present in a

single native NM DA receptor.

7.2.3 Future experiments

In order to make valid comparisons between the functional and pharmacological

properties of native and recombinant NM DA receptors, it will be necessary to carry out

similar single channel studies using recombinant receptors produced by co-expressing two,

three and even four different NM DA receptor subunits in a mammalian cell line.

7.3 Physiological role o f NM DA receptors in the immature brain

In embryonic and early postnatal stages of development, fast glutamatergic synaptic

transmission at hippocampal (Durand et al., 1996), thalamocortical (Isaac et al., 1997), retino-

tectal (Wu et a i, 1997) and spinal (Li & Zhuo, 1998) synapses is mediated mainly by NM DA

receptors. Throughout postnatal development fast synaptic transmission mediated by non-

N M D A receptors (mainly AMP A receptors) develops and become the main form of

glutamatergic synaptic transmission in the adult brain. An initial requirement for the

development of AMP A receptor-mediated synaptic transmission is the activation of NM DA

193

receptor-channels which produce postsynaptic influx. This increase in intracellular Ca

triggers a cascade of intracellular events thought to involve activation of the Ca^^-calmodulin-

dependent protein kinase II or CaMKJI which mediates through a mechanism not yet known

the appearance of AMP A receptor-mediated synaptic transmission (Cline e ta l, 1996).

If this is the case, an important question to answer will be what properties have NM DA

receptor-channels in embryonic and early postnatal brain that allow them to play such role?

First, functional NM DA receptors appear early in the brain: neurons in the embryonic brain

exhibit functional NM DA receptors soon after neuronal differentiation and before

synaptogenesis (Blanton e ta l, 1990; Lo Turco e ta l, 1991). In the embryonic and early postnatal

brain, N R l, NR2B and NR 2 D subunits are the only NM DA receptor subunits that can be

found in the brain (reviewed by Watanabe, 1997). A vital role for N R l and N R 2 B subunits in

brain functioning and survival has been recently demonstrated by studies showing that mice

lacking N R l or NB2B subunits die soon after birth (Li et a l, 1994; Forrest et a l , 1994;

Kutsuwada et a l, 1996) while mice lacking the NR2D (e4) subunit show normal growth but

reduced spontaneous behavioural activity (Ikeda e ta l , 1995).

If the functional properties of NM DA receptors are determined by which receptor

subunits take part in the assembly of the receptor-channel complex (reviewed by Feldmeyer &

Cull-Candy, 1996; Stern & Colquhoun, 1998) then what properties can N R l, NR2B and NR 2 D

subunits confer to NM DA receptors in the immature brain?

N R l subunits are indispensable for assembly, expression and functioning of NM DA

receptors. N R l subunits contain the binding site for glycine (Hirai et a l, 1996) while NR2

subunits contain that for glutamate (Laube et a l, 1997; Anson et a l, 1998) two absolute

requirements for NM DA receptor activation (Kleckner & Dingledine, 1988). NM DA

receptors containing NR2D subunits have a very high apparent affinity for glutamate (EC5 q= 0.3

- 0.4 pM, Ikeda et a l, 1992; Hess et a l, 1998) and glycine (ECso= 0.09 - 0.16 pM, Ikeda et a l,

1992; Buller et a l, 1997) while NM DA receptors containing NR2B subunits have intermediate

affinities for glutamate (ECgo= 0.8 - 2.8 pM; Kutsuwada et a l, 1992; Hess et a l , 1998; Laube et

a l, 1993; Kashiwagi e ta l , 1996; Hirai e ta l , 1996) and glycine (EC^= 0 . 2 - 0.8 pM, Kutsuwada

e ta l , 1992; Laube e ta l , 1993; WilHams, 1996; Zhong e ta l , 1996; Kashiwagi et a l, 1996; Hirai

et a l, 1996y). NM DA receptors with a high affinity for glutamate and glycine are expected to

produce synaptic currents with a slow decay time course (Lester & Jahr, 1992). Indeed, the

deactivation time-course of macroscopic currents produced by recombinant NM DA receptors

containing NR2D or NR2B subunits have very slow mean deactivation time constants: 400 ms

for NR 1/N R2B and an incredibly long 4500 - 4800 ms for N R 1/N R 2D (Monyer et a l, 1994;

Wyllie et a l, 1997; Vicini et a l, 1998). These subunit attributes are consistent with NM DA

194

receptor-mediated EPSCs having 2 to 5 times slower mean decay time constants in young

animals than in adults (Hestrin, 1992; Carmignoto & Vicini, 1992). The high affinity for

agonists conferred by their characteristic subunit composition, suggests that N M D A receptors

in embryonic and early postnatal brain have a high probability of being active given estimates

of the extracellular concentration of glutamate (0,6 pM) in brain tissue (Bouvier et al., 1992).

7.4 Concluding remarks

Although recombinant NM DA receptors produced by co-expression of N R l and

NR2 subunits can reproduce rather accurately many of the functional and pharmacological

properties observed in native NM DA receptors (Feldmeyer & Cull-Candy, 1996; Monaghan et

at., 1997; Stern & Colquhoun, 1998), the possibility that additional related subunits or a

family of subunits (Das et ai^ 1998; Aistrup et al.y 1996) may also be contributing to these

properties should not be excluded.

Even though combinations of only two types of NM DA receptor subunits (N R l and

NR2) can apparently reproduce qualitatively and quantitatively many of the single-channel

properties of native NM DA receptors, there is so far no experimental evidence to suggest that

more than two types of subunits can not assemble; on the contrary, an increasing body of

evidence suggest that native NM DA receptors can contain not only two but three or even

more different subunits (Sheng et al., 1994; Luo et al., 1997; Chazot & Stephenson, 1997;

Dunah et al., 1998). Currently, what seems to be a source of controversy is the proportion in

which native N M D A receptors containing two, three and probably four different subunits are

present in the brain (Discussed by Chazot & Stephenson, 1997).

If more than two different types of subunits can assemble to form native NM DA

receptors, it is somehow unexpected to find at the single channel level and apparently at a

more macroscopic level (Gotz et al., 1997) that NM DA channels behave either like high

(NR2A- or NR2B-like) or low (NR2C- or NR2D-like) conductance N M D A receptors. An

explanation for this behaviour could depend on the stoichiometry of the native receptor,

which is currently unknown.

Through identification and localisation of NM D A receptor subtypes it should be

possible to improve our understanding of how NM DA receptor-mediated signalling is

regulated within different brain regions and how subtype-selective drugs could be more

efficient in targeting specific brain areas affected by particular neuropathological conditions,

reducing at the same time unwanted side effects.

195

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discovery.ucl.ac.uk · University of Lo ndo n Abstract A Patch-Clamp Study Of The Single Channel Properties Of N ative N -M eth y l-d -A spar tate (NM DA) Receptors In N eurons From