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DEVELOPMENTAL CONSEQUENCES OF N-METHYL-D-ASPARTATE RECEPTOR
HYPOFUNCTION
by
Marija Milenkovic
A thesis submitted in conformity with the requirements for the degree of Master of Science
Department of Pharmacology and Toxicology University of Toronto
© Copyright by Marija Milenkovic 2011
ii
Developmental Consequences of
N-methyl-D-aspartate Receptor Hypofunction
Marija Milenkovic
Master of Science
Department of Pharmacology and Toxicology
University of Toronto
2011
Abstract
NMDA receptor signaling is required for proper synapse formation, maintenance, plasticity and
function. Dysregulation of the NMDA receptor has been implicated in pathophysiology of
schizophrenia, which has an adult onset of symptoms. NMDA receptor deficient mice were
utilized to assess the developmental consequences of NMDA receptor hypofunction. Locomotor
activity was elevated throughout development; however, deficits in social interaction and
working memory only manifest in adulthood and did not progress with age. Age-dependent
deficits in neuron synapse biology were also detected; postsynaptic spine number was normal in
juveniles, decreased post-adolescence, and progressively declined in adulthood. To investigate
possible molecular mechanisms underlying the observed changes in spine number, protein levels
of RhoGTPases and their downstream effectors were examined. Significant changes in Rac1 and
downstream effectors were detected at different developmental stages. These studies provide
clarification of the temporal sequence of events and mechanisms by which NMDA receptor
dysfunction affects neurodevelopment.
iii
Acknowledgments
I would like to thank those who have made completion of this thesis possible:
First and foremost I offer my sincere gratitude to my supervisor and mentor, Dr. Amy
Ramsey, for her encouragement, guidance and support throughout my thesis.
I would like to thank Dr. Rachel Tyndale (advisor) and Dr. Ali Salahpour for their
valuable comments and suggestions.
I would also like to thank members of the Ramsey and Salahpour lab for their support
and thoughtful discussions during my graduate study.
Lastly, special thanks to my family and friends for their continued support and
encouragement throughout the entire period of my graduate education.
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Table of Contents
Acknowledgments .......................................................................................................................... iii
Table of Contents ........................................................................................................................... iv
List of Figures ................................................................................................................................ vi
Abbreviations ................................................................................................................................ vii
Chapter 1 INTRODUCTION .......................................................................................................... 1
1 Specific Aims and Working Hypotheses ................................................................................... 1
2 NMDA Receptor Biology .......................................................................................................... 3
2.1 Structure .............................................................................................................................. 3
2.2 Synaptic Regulation ............................................................................................................ 5
2.3 Synaptic Function ............................................................................................................... 6
2.4 Dysregulation and Neurological Disorders ......................................................................... 8
3 Spine Dynamics ....................................................................................................................... 11
3.1 Spine Structure .................................................................................................................. 11
3.2 Spine Regulation ............................................................................................................... 12
3.2.1 Actin cytoskeleton ................................................................................................ 12
3.2.2 Rho family of small GTPases ............................................................................... 13
3.2.3 NMDA receptors ................................................................................................... 15
3.3 Spine Morphology and Neurological Disorders ............................................................... 16
4 Neurodevelopmental Hypothesis of Schizophrenia ................................................................. 17
5 Modeling Behavioral Endophenotypes of Schizophrenia in Mice .......................................... 19
6 NR1 Knock-down Mice ........................................................................................................... 20
6.1 Generation ......................................................................................................................... 20
6.2 Behavioral Phenotypes ...................................................................................................... 22
v
6.3 Cellular and Molecular Phenotypes .................................................................................. 23
6.4 Impact on Dopaminergic System ...................................................................................... 24
Chapter 2 MATERIALS AND METHODS ................................................................................. 26
1 Animals .................................................................................................................................... 26
2 Locomotor Activity Assay ....................................................................................................... 26
3 Sociability Assay ...................................................................................................................... 27
4 Working Memory (Y-maze) Assay .......................................................................................... 27
5 Subchronic MK-801 Treatment ............................................................................................... 28
6 Diolistic Labeling and Imaging of Neurons ............................................................................. 28
7 Western Blotting ...................................................................................................................... 29
Chapter 3 RESULTS ..................................................................................................................... 31
1 Elevated locomotor activity in juvenile, post-adolescent and adult NR1-KD mice ................ 31
2 Adult-onset social deficits in NR1-KD mice ........................................................................... 34
3 Impaired spatial working memory detected in adult NR1-KD mice ....................................... 36
4 Age-dependent deficits in MSN spine density ......................................................................... 38
5 Age-dependent alterations in Rac1 in NR1-KD mice .............................................................. 45
6 Reduced Wave1 and N-Wasp protein levels in NR1-KD mice ............................................... 48
7 Elevated phosphorylated cofilin levels detected in adult NR1-KD mice ................................ 50
Chapter 4 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS ............................. 53
1 Discussion ................................................................................................................................ 53
2 Conclusions .............................................................................................................................. 60
3 Recommendations .................................................................................................................... 63
References ..................................................................................................................................... 65
List of Publications and Abstracts ................................................................................................ 79
vi
List of Figures
Figure 1……………………………………………………………………………………….4
Figure 2………………………………………………………………………………………11
Figure 3………………………………………………………………………………………14
Figure 4………………………………………………………………………………………18
Figure 5………………………………………………………………………………………20
Figure 6………………………………………………………………………………………21
Figure 7………………………………………………………………………………………33
Figure 8………………………………………………………………………………………35
Figure 9………………………………………………………………………………………37
Figure 10……………………………………………………………………………………..40
Figure 11……………………………………………………………………………………..41
Figure 12……………………………………………………………………………………..42
Figure 13……………………………………………………………………………………..43
Figure 14……………………………………………………………………………………..44
Figure 15……………………………………………………………………………………..46
Figure 16……………………………………………………………………………………..47
Figure 17……………………………………………………………………………………..48
Figure 18……………………………………………………………………………………..51
Figure 19……………………………………………………………………………………..62
vii
Abbreviations
AD Alzheimer’s disease
ADF Actin-depolymerizing factor
ADHD Attention-deficit hyperactivity disorder
ANOVA Analysis of variance
AMPA A-amino-3-hydroxy-5-methyl-4-isoxazole propionate
Arp2/3 Actin-related protein 2/3
CaMKII Calcium/calmodulin-dependent protein kinase II
cAMP cyclic adenosine monophosphate
Cdc42 Cell division cycle 42
Cdk5 Cyclin-dependent kinase 5
CNS Central nervous system
CREB cAMP-response element-binding protein
C-terminal Carboxyl-terminal
DAAO D-amino acid oxidase
D1, D2 Dopamine receptor 1, 2
DAT Dopamine transporter
DiI 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
DISC1 Disrupted-in-schizophrenia 1
viii
DOPAC 3,4-Dihydroxyphenylacetic acid
EPSC Excitatory postsynaptic current
ER Endoplasmic reticulum
ErbB4 V-erb-a erythroblastic leukemia viral oncogene homolog 4
G72 D-amino acid oxidase activator
GABA Gamma-aminobutyric acid
GAP GTPase –activating protein
GDP Guanosine diphosphate
GEF Guanine nucleotide exchange factor
GluR Glutamate receptor
G-protein Guanine nucleotide-binding protein
Grin1 NR1 subunit gene
GTP Guanosine triphosphate
GTPase Guanosine triphosphate-ase
HD Huntington’s disease
HFS High-frequency stimulation
HVA Homovanillic acid
L-DOPA L-3,4-dihydroxyphenylalanine
LIMK Lens intrinsic membrane kinase
LTD Long term depression
ix
LTP Long term potentiation
MK-801 Dizocilpine
MLCK Myosin light chain kinase
MLCP Myosin light chain phosphatase
MRLC Myosin regulatory light-chain
MSN Medium spiny neuron
NDEL1 Nuclear distribution protein nude-like 1
NMDA N-methyl-D-aspartate
NR1,2 or 3 NMDA receptor 1,2 or 3 subunit
NR1-KD NMDA receptor 1 subunit knock-down
N-terminal Amino-terminal
N-WASP Neuronal Wiskott-Aldrich syndrome protein
6-OHDA 6-hydroxydopamine
PCP Phencyclidine
PD Parkinson’s disease
PKA, PKC Protein kinase A, C
PP2B Protein phosphatase 2B
PSD-95 Postsynaptic density-95
Rac1 Ras-related C3 botulinum toxin substrate
RGS4 Regulator of G protein signaling 4
x
RhoA Ras homolog gene family member A
ROCK Rho associated coiled-coil containing kinase
SAP-102 Synapse-associated protein – 102
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Wave1 WASP-family verprolin homology protein-1
WT Wild-type
1
Chapter 1 INTRODUCTION
1 Specific Aims and Working Hypotheses
Proper glutamatergic neuron transmission is crucial for synaptogenesis, synapse
maintenance, synaptic plasticity and neuron physiology (Mattson, 2008). Changes in synapse
number and synaptic communication that result from impaired glutamatergic signaling have been
correlated with neuronal, cognitive and behavioral dysfunction (Penzes et al., 2011).
Furthermore, the dysregulation of N-methyl-D-aspartate (NMDA) receptors has been implicated
in the pathophysiology of a number of neuropsychiatric disorders including schizophrenia (Cull-
Candy et al., 2001). A previously generated genetic model of NMDA receptor hypofunction,
NR1-KD mice (Mohn et al., 1999), will be used to study the developmental consequences of
NMDA receptor dysfunction and its impact on the molecular pathways that regulate synapse
biology. NR1-KD mice have a global reduction in functional NMDA receptors, and show
schizophrenia-related behavioral phenotypes similar to those detected following pharmacological
inhibition of NMDA receptors.
Specific Aim 1: To determine the onset and severity of schizophrenia-related behaviors observed
in the NR1-KD mice.
Rationale: The neuropsychiatric symptoms of schizophrenia manifest in adulthood; however,
many of the environmental and genetic risk factors for schizophrenia play a role in
neurodevelopment (Rapoport et al., 2005). We hypothesize that impaired NMDA receptor
function through the course of brain development will lead to alterations in neuron physiology,
but that the behavioral consequences may not be fully evident until adulthood, in a manner
similar to what is observed for schizophrenia. While there are several behavioral abnormalities
observed in adult NMDA receptor deficient mice, the developmental onset of these behaviors is
unknown. We want to determine if the onset and severity of schizophrenia-relevant behaviors
are age-dependent. Locomotor activity, sociability and working memory will be examined in
three week (juvenile), six week (post-adolescent), twelve week (adult) and twenty-four week old
2
NR1-KD mice. These ages represent developmental periods in mice that correspond to periods
in humans before symptoms of schizophrenia are fully evident (juvenile), when the first onset of
symptoms begins to emerge (post-adolescence), and when symptoms are fully evident and
treated with antipsychotic medications (adulthood).
Specific Aim 2: To investigate the developmental effects of NMDA receptor hypofunction on
spine density of striatal medium spiny neurons (MSNs).
Rationale: Dendritic spines are major sites for information processing and storage and regulate
synapse function and plasticity (Nimchinsky et al., 2002). Changes in dendritic spine
morphology and density have been detected in postmortem brains of individuals with
neuropsychiatric disorders including schizophrenia (Penzes et al., 2011). Deficits in MSN
dendritic spines of the striatum were previously detected in six week old NR1-KD mice (Ramsey
et al., 2011). We want to investigate whether or not the deficits in the spine density remain
stable or worsen with age. We hypothesize that the temporal trajectory of spine number will
provide an indication of the role of NMDA receptors in regulating spine number. If spine
density remains stable after the spine loss at six weeks, this would indicate that the major effect
of NMDA receptor hypofunction is seen during the process of synapse refinement, which occurs
between weeks 5-7 in the mouse (Adriani and Laviola, 2004). If spine loss progresses beyond
six weeks of age, this would indicate that NMDA receptors also play a role in the maintenance of
spines throughout adulthood. Therefore spine density will be analyzed in three, six, twelve, and
twenty-four week old NR1-KD and WT mice.
Specific Aim 3: To examine the developmental effects of NMDA receptor hypofunction on actin
regulating proteins.
Rationale: Dendritic spine formation, maturation and plasticity are processes that depend on the
proper regulation of actin cytoskeletal rearrangements (Saneyoshi et al., 2010; Sekino et al.,
2007). Actin dynamics are regulated in part by RhoGTPase-mediated signaling cascades
(Hotulainen et al., 2009). We hypothesize that the observed changes in spine density reflect
changes in the biochemical signaling cascades regulated by RhoGTPases. There are several
effector proteins downstream of RhoGTPases that influence actin dynamics (Cingolani and
Goda, 2008; Saneyoshi et al., 2010). Cortactin, N-Wasp and Wave1 mediate actin
polymerization through modulation of the actin-binding protein complex Arp2/3. LIMK
3
mediates actin disassembly through regulation of the activity of cofilin, an actin depolymerizing
protein. To investigate the possibility that these signaling cascades underlie spine loss in NR1-
KD mice, their protein levels will be assessed in NR1-KD mice at three different developmental
stages (three, six and twelve weeks of age).
2 NMDA Receptor Biology
The transfer of information in the central nervous system (CNS) occurs primarily at
chemical synapses. The three major components of a chemical synapse are the presynaptic
neuron (axon terminal), the synaptic cleft, and the postsynaptic neuron (dendrite).
Neurotransmitters are synthesized, stored and released from presynaptic neurons. Release of
neurotransmitter into the synaptic cleft is triggered by an action potential and subsequent
depolarization of the terminal membrane. Once released, neurotransmitters act on the
postsynaptic neuron by binding to specific receptors expressed on the surface of the postsynaptic
membrane.
The major neurotransmitter systems of the CNS are cholinergic, monoaminergic,
GABAergic and glutamatergic. Glutamatergic neurons are activated by the excitatory
neurotransmitter glutamate. Glutamate receptors are divided into two main groups:
metabotropic (G-protein coupled receptors) and ionotropic (transmitter-gated ion channels)
(Javitt, 2007). Ionotropic receptors are further subdivided based on their chemical agonist
specificity: a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), N-methyl-D-aspartate
(NMDA) and kainite receptors. While the function of the kainite receptors is not well
understood, AMPA and NMDA receptors have been shown to play a crucial role in mediating
the majority of the CNS’s fast excitatory synaptic transmission (Javitt, 2007).
2.1 Structure
The NMDA receptor is a glutamate-gated ion channel. Three NMDA receptor subunits
have been identified: the NR1 subunit, four NR2 subunits (A, B, C and D), and two NR3
subunits (A and B) (Ciabarra et al., 1995; Laurie and Seeburg, 1994; Monyer et al., 1994; Sucher
et al., 1995). NMDA receptor subunits structure consists of an extracellular N-terminal domain,
4
three transmembrane domains including a re-entry loop, and an intracellular C-terminal domain
(Javitt, 2007). Multiple NR1 subunits assemble with at least one NR2 and or NR3 subunit to
form a heteromeric receptor complex (Cull-Candy et al., 2001). NMDA receptor subunits have
distinct regional and developmental expression patterns in the brain. In rat studies, NR1, NR2B
and 2D subunit expression was shown to occur during prenatal development, while expression of
NR2A and 2C was delayed and only detected postnatally (Monyer et al., 1994). The functional
properties of the NMDA receptor are greatly influenced by different regional expression patterns
of NR1 splice variant isoforms and developmental expression patterns of the NR2 subunits
(Monyer et al., 1994). NR1 subunit isoforms have varying sensitivity to changes in cellular pH
levels (Cull-Candy et al., 2001). NR2B subunit-containing receptors are predominant in
immature neurons and have a higher affinity for the ligand, slower decay time of excitatory
postsynaptic currents (EPSCs), and lower channel opening probability (Monyer et al., 1994). In
contrast, NR2A-containing receptors are more predominant in mature neurons and have lower
ligand affinity, faster decay time of
EPSCs, and higher channel opening
probability (Monyer et al., 1994).
While the presence of NR1 subunit is
essential for proper receptor
functioning, the presence of the NR3
subunit has an inhibitory effect on
receptor activity (Lau and Zukin,
2007). Modifications of NMDA
receptor kinetics, as a result of the
developmental shift in NMDA
receptor subunit composition, play a
significant role in proper synapse
formation, pruning and maturation
(Cull-Candy et al., 2001).
NMDA-gated channels are permeable to Na+, Ca
2+, and K
+ ions. The NMDA receptor
inward ion current is ligand and voltage-dependent. At resting membrane potential, the channel
is closed. Upon glutamate and glycine binding the ion channel changes to an open conformation,
Figure 1. Schematic diagram of the ionotropic NMDA
receptor. (Adapted from homepage.psy.utexas.edu)
5
but the pore is blocked by an extracellular Mg2+
ion. When the membrane potential depolarizes
to become less negatively charged, the Mg2+
ion is released from the channel pore; this allows
for Na+ and Ca
2+ ions to enter the cell and K
+ ions to leave the cell. In addition to the glutamate
binding site located on the NR2 subunit, the NMDA ion channel also contains modulatory and
regulatory sites (Javitt, 2007) (Figure 1). Amino acids glycine and D-serine bind to the
modulatory site present on the NR1 subunit and act as co-activators of the receptor containing
the NR2 subunit. On receptors containing the NR3 subunit, glycine and D-serine have different
effects; glycine still acts as an activator while D-serine exhibits an inhibitory effect (Chatterton et
al., 2002). As co-activators they are able to modulate the time the channel remains in the open
state as well as its desensitization rate (Javitt, 2007). Agents such as polyamines, Zn2+
, and
protons are able to exert their influence on the channel by binding to the regulatory sites present
on the subunits (Javitt, 2007). Phencyclidine (PCP), dizocilpine (MK-801) and ketamine are
well known NMDA receptor antagonists. These pharmacological agents bind to a site located on
the inner part of the channel and therefore inhibit the receptor in a noncompetitive matter
(Javitt, 2007).
2.2 Synaptic Regulation
NMDA receptor targeting and insertion at the synapse (trafficking) is a tightly regulated
process that influences synaptic plasticity. Alterations in NMDA receptor trafficking have been
implicated in a number of neuropsychiatric disorders including Huntington’s, Alzheimer’s,
Parkinson’s, and schizophrenia (Lau and Zukin, 2007). Synaptic localization of NMDA
receptors is highly regulated by synaptic activity as discussed below.
Within few hours of glutamatergic synapse formation, NMDA receptors along with the
AMPA receptors appear on the postsynaptic neuron (Lau and Zukin, 2007). In the first step of
NMDA receptor trafficking, NMDA receptor assembly occurs in the endoplasmic reticulum
(ER). Following assembly, the receptor becomes part of a large molecular complex that contains
a transport vesicle, scaffolding protein (SAP-102), adaptor proteins and a motor protein (Lau and
Zukin, 2007). The newly formed complex allows for the NMDA receptor to be transported from
the cell body to the dendritic spines along the microtubules. Once in dendritic spines, the
receptor is inserted into the postsynaptic membrane. The delivery time of the assembled
complex to the synapse is regulated by protein kinases PKA and PKC through phosphorylation
6
of the NR1 subunit (Lau and Zukin, 2007). Activity of other synaptic receptors, such as AMPA-
type GluR1 (Lan et al., 2001) and D1 (Dunah and Standaert, 2001; Hallett et al., 2006), can also
influence NMDA receptor trafficking to the postsynaptic membrane.
There are a number of synaptic scaffolding proteins, in addition to SAP-102, that play a
crucial role in proper synaptic targeting and functioning of the NMDA receptor. One of these
proteins is the postsynaptic density-95 (PSD-95) protein, a PDZ domain containing protein,
which is able to directly bind to the NMDA receptor through its NR2 subunit PDZ binding site
(Lin et al., 2004). PSD-95 protein has the ability to enhance synaptic clustering of the receptor
(El-Husseini et al., 2000), stabilize surface expression of the receptor through attenuation of
receptor internalization (Roche et al., 2001), regulate specific NR2 subunit targeting to the
synapse (Losi et al., 2003), increase channel opening (Lin et al., 2004), and decrease receptor
desensitization (Li et al., 2003). Similarly, it serves as a linker protein between synaptic NMDA
receptors and downstream signaling cascades.
NMDA receptor internalization is dependent on synaptic activity and phosphorylation,
and is subunit specific (Snyder et al., 2005). The internalization signal is located on the C-
terminal domain of the NR2 subunit. NR2A and 2B subunits have unique internalization signals
(Lavezzari et al., 2004). The first step in NMDA receptor endocytosis is lateral diffusion from
the synapse to the extrasynaptic site known as endocytic zone (Blanpied et al., 2002). Once in
the endocytic zone, NMDA receptor endocytosis is mediated through the clathrin/dynamin
pathway. NR2B subunit containing receptors have a faster rate of internalization than NR2A
(Lavezzari et al., 2004). Once internalized, NMDA receptors are either targeted for recycling to
the membrane or degradation.
2.3 Synaptic Function
Due to its kinetic properties (slow but lasting activation) and its ability to transmit Ca2+
ions into the postsynaptic neuron, NMDA receptor plays a crucial role in synapse remodeling
and plasticity. Long-term potentiation (LTP) and long-term depression (LTD) are major cellular
mechanisms underlying synaptic plasticity (Debanne et al., 2003). While LTP is associated with
learning and memory (Bliss and Collingridge, 1993), LTD on the other hand is more associated
with memory storage (Massey and Bashir, 2007). NMDA receptors have been implicated in
7
LTP and LTD induction through regulation of the AMPA receptor surface expression and
activation.
NMDA receptor activation results in Ca2+
entry into the postsynaptic neuron. Ca2+
ion is
a known activator of a multitude of signaling cascades including protein Ca2+
/calmodulin-
dependent protein kinase II (CaMKII). NMDA receptor-mediated activation of CaMKII triggers
a number of downstream effects. First of which is CaMKII autophosphorylation, followed by
further activation and recruitment of other CaMKII proteins to the postsynaptic density (PSD),
CaMKII direct binding to the NMDA receptors and subsequent phosphorylation of the AMPA
receptors (Barria and Malinow, 2005; Lau and Zukin, 2007). Phosphorylation of the AMPA
receptor increases AMPA receptor surface expression and channel activity, resulting in
strengthening of synaptic transmission. Similarly, activated CaMKII has the ability to in turn
enhance NMDA receptor subunit-specific surface expression as well as interaction with other
scaffolding and signal transducing proteins of PSD (Lau and Zukin, 2007).
NMDA receptor induced Ca2+
influx is also able to activate protein kinases (PKA and
PKC) through activation of the cyclic adenosine monophosphate (cAMP)-pathway. Activated
PKA can positively modulate NMDA ion channel activity and targeting to the postsynaptic
membrane through subunit phosphorylation (Scott et al., 2003). PKA has also the ability to
activate cAMP-response element-binding protein (CREB) resulting in induction of gene
transcription and subsequent new protein synthesis (Waltereit and Weller, 2003). Therefore,
NMDA receptor-mediated activation of the cAMP/PKA molecular pathway plays a significant
role in synapse efficiency and remodeling. NMDA receptor subunits can also be phosphorylated
by PKC. In some studies, PKC promoted NMDA receptor movement away from the synapse
(Fong et al., 2002; Tingley et al., 1997) while in others it enhanced receptor activity (Chen and
Huang, 1992; Xiong et al., 1998) and receptor targeting to the synapse (Lan et al., 2001).
Phosphorylation of the NMDA receptor by both PKC and PKA promotes NMDA receptor
removal from the ER and subsequent delivery to the postsynaptic membrane (Scott et al., 2003).
High-frequency stimulation (HFS) that induces LTP of the adult rat CA1 neurons of the
hippocampus also causes activation of PKC and tyrosine kinase Src. This results in the induction
of NMDA receptor synaptic expression and enhancement of NMDA excitatory postsynaptic
currents thus triggering LTP of NMDA receptors (Grosshans et al., 2002). Activation of the
8
mGluR1/5 by HFS can also contribute to the promotion of LTP of NMDA receptors (O'Connor
et al., 1994). Conversely, low-frequency stimulation (LFS) that induces LTD of the CA1
neurons induces mGluR1/5-mediated NMDA receptor internalization (Snyder et al., 2001) and
NMDA receptor-mediated NMDA receptor lateral diffusion to the extrasynaptic sites and actin
depolymerization (Morishita et al., 2005), resulting in promotion of LTD of NMDA EPSCs.
2.4 Dysregulation and Neurological Disorders
Changes in NMDA receptor subunit composition, synaptic expression, internalization
and phosphorylation can all contribute to NMDA receptor dysfunction. Changes in synaptic
transmission and efficacy as a result of NMDA receptor dysfunction can have devastating
neurological consequences. While the research goals of this thesis have focused on the role of
NMDA receptors in schizophrenia, altered NMDA receptor function has been implicated in the
pathophysiology of a number of neuropsychiatric disorders including Huntington’s disease,
Alzheimer’s disease, and Parkinson’s, as discussed below.
Huntington’s disease (HD) is a neurodegenerative genetic disorder with motor and
behavioral deficits caused by excessive number of CAG repeats in the gene coding for huntingtin
(htt) protein (Landles and Bates, 2004). Disease progression leads to striatal and cortical cell
death (Vonsattel and DiFiglia, 1998). NMDA receptor-mediated neurotoxicity has been
implicated in the pathology of HD. NMDA, GABA, and muscarinic cholinergic receptor
binding was examined in the striatum of HD patients (Young et al., 1988). Radio-ligand binding
to all three receptors was reduced in HD brains when compared to the control brains. The most
dramatic reduction (93%) was observed with binding to the NMDA receptor, suggesting that
NMDA receptors play a crucial role in neuronal cell death evident in HD patients. In non-human
primates, striatal administration of a NMDA receptor agonist caused neuronal cell death and HD
resembling behavioral phenotypes (Hantraye et al., 1990). NMDA receptors have been
implicated in HD neuropathology even in the absence of neuronal cell death.
Electrophysiological properties of NMDA receptors are altered in striatal medium spiny neurons
in a mouse model of HD (Starling et al., 2005). In this model, NMDA receptors were more
sensitive to NMDA and less sensitive to Mg2+
. In addition, NMDA receptor phosphorylation,
synaptic expression (mediated by PSD-95), and downstream signaling was shown to be altered in
HD mouse models (Fan and Raymond, 2007).
9
Alzheimer’s disease (AD) is a degenerative disease of the brain which worsens with
time. It is characterized by deficits in memory, thought, and behavior (Selkoe, 2002). The
postmortem hallmarks of the disease are the presence of amyloid plaques and neurofibrillary
tangles that are believed to result in synaptic dysfunction and neuronal degeneration (Selkoe,
2002). Dysregulation of glutamatergic synapses has been implicated in the neuropathology of
AD. Deficits in dendritic spines and synapses (Davies et al., 1987) and NMDA receptor
expression levels (Hynd et al., 2001) in brain regions most susceptible to the disease were
reported. -amyloid-induced spine and synapse loss in rat hippocampus was shown to be
dependent on NMDA receptor-mediated signaling pathways (Shankar et al., 2007). In addition,
-amyloid has the potential to modify NMDA receptor trafficking through decreasing receptor
synaptic targeting and increasing receptor internalization (Snyder et al., 2005). As described in
section 1.2, NMDA receptor endocytosis is regulated by receptor phosphorylation. -amyloid
activates tyrosine phosphatase responsible for NMDA receptor regulation resulting in enhanced
NMDA receptor internalization. The NMDA receptor antagonist, memantine, is used
therapeutically to treat Alzheimer’s disease (Lipton, 2005).
Parkinson’s disease (PD) is another progressive neurodegenerative disorder in which
NMDA receptors function has been implicated. PD is characterized by dopaminergic
nigrostriatal neuron degeneration resulting in dysfunctioning of the motor system (Samii et al.,
2004). The strongest evidence of NMDA receptor involvement in PD pathophysiology comes
from studies done with NMDA receptor antagonists. In animal models of PD (Loschmann et al.,
2004; Nash et al., 2000) and humans (Uitti et al., 1996) NMDA receptor antagonists attenuated
parkinsonian symptoms. Alterations in NMDA receptor synapse localization and function were
observed in animal models of PD (Dunah et al., 2000; Hallett et al., 2005). A decrease in
NMDA receptor synaptic expression and NMDA receptor phosphorylation in the synaptosomal
membrane fractions was observed in the PD 6-hydroxydopamine (6-OHDA) lesion rat model
(Dunah et al., 2000). Chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment reversed the
NMDA receptor synaptic levels and phosphorylation deficits. Furthermore, dopamine D1
agonist was shown to be able to enhance NMDA receptor levels and tyrosine phosphorylation in
the synaptosomal membrane fractions (Dunah and Standaert, 2001). Thus, alterations in NMDA
receptor trafficking may play a role in molecular mechanisms underlying PD neuropathology.
10
Schizophrenia is a debilitating mental disorder with unknown etiology. It is clinically
diagnosed based on behavioral symptoms. Schizophrenia symptoms manifest post-adolescence
and are managed in adulthood. Because etiology of schizophrenia is unknown, there are a
number of valid neurotransmitter based hypotheses that have been proposed to explain the
disorder. One of these hypotheses focuses on the role of glutamate (Javitt, 2007). The glutamate
(NMDA receptor) hypothesis posits that either the primary etiology, or the pathophysiology, of
schizophrenia is due to reduced levels or activity of NMDA receptors, or a dysregulation of
glutamatergic signaling. This hypothesis arises from observations that NMDA receptor
antagonists induce schizophrenia-like symptoms (positive and negative) in healthy individuals
(Javitt and Zukin, 1991; Krystal et al., 1994; Lahti et al., 2001) and such symptoms are
exacerbated in schizophrenic individuals (Lahti et al., 1995; Malhotra et al., 1997).
A number of additional studies findings support the NMDA receptor hypofunction
hypothesis of schizophrenia. In an in vivo study, NMDA receptor binding was reported to be
significantly reduced in hippocampus of medication-free schizophrenic patients when compared
to the controls (Pilowsky et al., 2006). Analysis of postmortem hippocampal tissue revealed a
decrease in mRNA levels of NR1 subunit in schizophrenic individuals (Gao et al., 2000).
Recently, reductions in NMDA receptor expression were detected in neurons generated from
induced pluripotent stem cells of schizophrenic patients (Brennand et al., 2011).
There are a number of schizophrenia susceptibility genes that have been implicated in
glutamatergic neurotransmission, including neuregulin1 gene (Stefansson et al., 2002) and PP2B
gene (Gerber et al., 2003). Neuregulin 1 is a growth factor that plays a role in regulating
NMDA receptor trafficking through activation of the ErbB4 receptor present on the postsynaptic
membrane (Lau and Zukin, 2007). In both animal (Gu et al., 2005) and postmortem study (Hahn
et al., 2006) of the prefrontal cortex, neuregulin-induced ErbB4 activation potentiated NMDA
receptor internalization and attenuated NMDA receptor activity. Neuregulin-mediated
alterations in NMDA receptor function were more pronounced in schizophrenic subject than the
compared normal controls (Hahn et al., 2006). Similarly, protein phosphatase 2B (PP2B)
regulates NMDA receptor trafficking through activation of a tyrosine phosphatase responsible
for suppressing NMDA receptor phosphorylation (Lau and Zukin, 2007). Subsequent
dephosphorylation of the NMDA receptor promotes receptor internalization. Thus, alterations in
NMDA receptor trafficking resulting in NMDA receptor hypofunction may contribute to the
11
pathophysiology of schizophrenia. Other susceptibility genes include serine racimase (Labrie et
al., 2009) and D-amino acid oxidase (DAAO), which are responsible for metabolism of D-serine,
a co-agonist of NMDA receptors, and G72, which regulates the activity of DAAO (Chumakov et
al., 2002). In addition, the metabotropic glutamate receptor mGluR3 (Fujii et al., 2003) and
dysbindin (Talbot et al., 2004; Weickert et al., 2004), which modulate glutamate release, and
proline oxidase (Liu et al., 2002), enzyme that converts proline to glutamate in mitochondria,
have been identified as susceptibility genes. Some association studies have shown positive
correlation between NR2B subunit of the NMDA receptor and schizophrenia (Martucci et al.,
2006; Ohtsuki et al., 2001), while others have shown negative correlation (Fallin et al., 2005;
Hokyo et al., 2010).
3 Spine Dynamics
3.1 Spine Structure
Postsynaptic dendritic spines play a crucial role in neuronal communication, information
processing and storage, and synaptic plasticity (Nimchinsky et al., 2002). Spine formation,
elimination and morphology are tightly regulated by
the conformation of the actin cytoskeleton. Spines are
made up of three regions: spine base, spine neck and
spine head (Hotulainen and Hoogenraad, 2010). Spine
length can vary from 0.2-2 microns while spine
density varies anywhere from 1-10 spines per micron
of a dendrite depending on the neuron type and the
activity of the neuron (Sorra and Harris, 2000). Based
on their morphology spines can be categorized into
three types: thin (long filopodia-like), stubby (short
without a distinct neck) and mushroom (defined neck
with large head) shaped (Bourne and Harris, 2008).
As neuronal development progresses, spine
morphology changes from thin filopodia-like structure
Figure 2. Schematic diagram of a
mature dendritic spine. Adapted from
(Hotulainen and Hoogenraad, 2010).
12
to a more mature structure with a distinct neck and head (Oray et al., 2006). The molecular
components of a mature dendritic spine include adhesion molecules, glutamate receptors, a
postsynaptic density containing scaffolding and signaling molecules, and an actin cytoskeleton
(Figure 2). Actin polymers, linear and branched, make up the backbone of dendritic spines
(Landis and Reese, 1983). The spine neck is mainly composed of linear actin filaments while the
head consists of short highly branched filaments which aid in PSD organization and stabilization.
3.2 Spine Regulation
Spine morphology is continuously changing as a result of alterations in neuronal circuitry
or experience resulting in strengthening or weakening of synapses. Changes in actin
polymerization and reorganization have a profound effect on dendritic spine morphology and
dynamics (Cingolani and Goda, 2008). Sustained synaptic activation, modeled through the
generation of long term potentiation, causes the actin equilibrium to shift toward actin
polymerization; this results in spine volume increase (Fukazawa et al., 2003). Conversely,
manipulations of synaptic strength that lead to synaptic depression, modeled through long-term
depression, cause the equilibrium to shift toward actin depolymerization, resulting in spine
volume decrease (Okamoto et al., 2004).
3.2.1 Actin cytoskeleton
Actin can be found in cells in two forms: globular (G-actin) and filamentous (F-actin).
Actin filaments undergo a process called treadmilling; at one end of the filament G-actin
monomers are added on (barbed end, +), while at the opposite end (pointed, -) G-actin is released
(Cingolani and Goda, 2008). At a steady state, equilibrium is reached and the filament remains
unchanged in length and structure. A shift in the equilibrium leads to either actin filament
polymerization (+ end) or depolymerization (- end).
Actin polymerization and branching is mainly regulated by the actin-related proteins
(Arp2/3) complex. Arp2/3 complex potentiates actin polymerization by binding to the side of an
existing filament and creating a nucleation core for a new filament, resulting in filament
branching (Goley and Welch, 2006). Major regulators of the Arp2/3 complex are cortactin,
neural Wiskott-Aldrich syndrome protein (N-Wasp), and WASP-family verprolin homology
protein-1 (Wave1) (Hotulainen and Hoogenraad, 2010). Knock-down of endogenous cortactin in
13
hippocampal neurons induces a decrease in spine density (Hering and Sheng, 2003). Similarly,
hippocampal loss of N-Wasp and Arp3 caused a reduction in spine and excitatory synapse
number (Wegner et al., 2008). Genetic knockout of Wave1 leads to hippocampal and cortical
spine density deficits and alterations in synaptic transmission (Soderling et al., 2007). In
addition, Wave1 knockout mice have reduced anxiety, sensorimotor function, and deficits in
learning and memory (Soderling et al., 2003).
Actin depolymerization is regulated by the actin-depolymerizing factor (ADF)/cofilin
family of proteins. ADF/cofilin regulates actin depolymerization in two ways: through binding
and severing of the actin filaments, and through enhancing G-actin removal from the pointed end
(Sekino et al., 2007). ADF/cofilin activity is regulated by lens intrinsic membrane kinase 1
(LIMK1). LIMK1 phosphorylation of ADF/cofilin results in ADF/cofilin inactivation
(Hotulainen et al., 2009). In neurons, cofilin plays a role in regulating spine head and synapse
morphology during an LTP (Chen et al., 2007). Genetic deletion of cofilin kinase (LIMK1)
causes alterations in spine morphology, hippocampal LTP, locomotor activity, and associative
and spatial learning (Meng et al., 2002).
In addition to modulating actin assembly and disassembly, actin-binding proteins also
regulate actin filament motility. Actin contractility is mediated in an ATP-dependent matter by a
motor protein myosin (Cingolani and Goda, 2008). Loss of myosin II in hippocampal neurons
promotes alterations in spine structure and motility and in synaptic transmission (Ryu et al.,
2006). Myosin II activity is regulated by myosin regulatory light-chain (MRLC) (Cingolani and
Goda, 2008).
3.2.2 Rho family of small GTPases
The Rho family of small GTPases are major regulators of actin-binding and actin-
modulating proteins. RhoGTPases play a crucial role in modulating actin cytoskeleton in
numerous cell types including neurons (Etienne-Manneville and Hall, 2002). They alternate
between guanosine triphosphate (GTP)-bound, active and guanosine diphosphate (GDP)-bound,
inactive state. RhoGTPase activation is regulated by guanine nucleotide exchange factors
(GEFs) while inactivation is potentiated by GTPase-activating proteins (GAPs). RhoGTPases
14
play central role in regulating dendritic spine morphogenesis and dynamics through their ability
to modulate the actin cytoskeleton (Saneyoshi et al., 2010). The most extensively studied
members of Rho family of GTPases are ras homolog gene family member A (RhoA), ras-related
C3 botulinum toxin substrate 1 (Rac1) and cell division cycle 42 (Cdc42) proteins.
Rac1 and Cdc42 signaling pathways promote actin polymerization and stabilization
through mediating Arp2/3 complex activation and cofilin inactivation (Figure 3). RhoA
signaling pathway plays a major role in mediating actomyosin contraction and cell retraction
Figure 3. Simplified schematic diagram of RhoGTPase signaling pathways.
Adapted from (van Galen and Ramakers, 2005). ADF, actin-depolymerizing factor; Arp2/3,
actin-related protein 2/3; Cdc42, cell division cycle 42; LIMK, lens intrinsic membrane kinase;
MLCK, myosin light chain kinase; MLCP, myosin light chain phosphotase; MRLC, myosin
regulatory light-chain; N-WASP, neuronal Wiskott-Aldrich syndrome protein; PAK, p21-
activated kinase; Rac, Ras-related C3 botulinum toxin substrate; RhoA, Ras homolog gene
family member A; ROCK, Rho associated coiled-coil containing kinase; WAVE, WASP-family
verprolin homology protein-1.
15
through regulation of myosin II motor proteins. In dendritic spines, Rac1 activation generally
promotes spine formation and maintenance, while RhoA activation generally potentiates spine
retraction and shrinkage. In neurons, transient Rac1 activation causes spine head growth while
constitutively active Rac1 can induce spine loss (Hayashi-Takagi et al., 2010) or cause an
increase in spine number (Luo et al., 1996) depending on the study. Constitutively active RhoA
induces a decrease in spine density and spine length (Tashiro et al., 2000). The role of Cdc42 in
modulation of spine morphology has not been well characterized. The role of RhoGTPases in
regulation of the actin cytoskeleton is not as simple as it might seem due to cross-talking
between pathways (Li et al., 2002). Integrated activity of the three pathways is likely required
for proper spine dynamics in the developing spine and in response to neuronal activation.
3.2.3 NMDA receptors
Postsynaptic receptors, including the NMDA receptor, are able to alter spine dynamics
through regulation of actin-modulating proteins. Receptors can regulate the activity of actin
modulators either through direct association (Tolias et al., 2005) or through regulation of the
intracellular Ca2+
levels required for their proper activation (Fischer et al., 2000). A number of
studies have shown that deficits in NMDA receptors result in a decrease in spine density
(Brigman et al., 2010; Ramsey et al., 2011; Ultanir et al., 2007) as well as deficits in synaptic
plasticity (LTD) and cognitive function (Brigman et al., 2010). Enhancement of the NMDA
receptor function through removal of the inhibitory NR3 subunit causes an increase in NMDA
receptor activity and spine density (Das et al., 1998). One way that NMDA receptor activity
may modulate actin dynamics is through the regulation of RhoGTPases.
An example of NMDA receptor-mediated regulation of actin dynamics is seen in the
biology of Kalirin-7. Kalirin-7 is a guanine-nucleotide exchange factor (GEF) specifically
expressed in the brain. It interacts with a number of postsynaptic scaffolding proteins, including
PSD-95, and is an activator of Rac1 (Xie et al., 2007). Through modulation of Rac1, Kalirin-7
plays a significant role in the regulation of the actin cytoskeleton and spine morphology. In
cultured pyramidal neurons, NMDA receptor activation induces CaMKII activity, Rac1
activation, and spine enlargement (Xie et al., 2007). This NMDA receptor mediated Rac1
activation is dependent on CaMKII activation of Kalirin-7. In addition to Rac1 regulation,
Kalirin-7 interacts with AMPA receptors and regulates AMPA receptor surface expression and
16
activity. Changes in spine morphology correlated with an increase in synaptic transmission
(AMPA-mediated). Kalirin-7 knockout mice showed deficits in hippocampal pyramidal neuron
spine density, LTP and passive avoidance task (Ma et al., 2008).
Disrupted-in-schizophrenia 1 (DISC1) has been identified as a schizophrenia
susceptibility gene. More recently, DISC1 was shown to play a role in regulating glutamatergic
spine structure and function through interaction with Kalirin-7 (Hayashi-Takagi et al., 2010).
DISC1 sequesters Kalirin7 thus preventing it from interacting with and activating Rac1. Short-
term DISC1 knockdown in primary cortical neurons caused an increase in spine size and number
as well as an increase in AMPA-mediated mEPSCs frequency. Conversely, long-term DISC1
knockdown caused a reduction in spine size and mEPSCs frequency. Both in vivo and in
primary neurons, NMDA receptor activation induced Kalirin-7 dissociation from DISC1 and
subsequent activation of Rac1. DISC1/Kalirin-7/Rac1 signaling cascade is a potential
mechanism by which glutamatergic dendritic spine dynamics can be regulated.
In addition to GEFs, RhoGTPase activity can also be modulated by GAPs. p250GAP
binds to and enhances intrinsic GTPase activity of RhoGTPases including RhoA (Nakazawa et
al., 2003; Wayman et al., 2008). p250GAP was shown to be present in the PSD and to have the
ability to associate with NMDA receptor. Furthermore, activation of the NMDA receptor was
shown to attenuate p250GAP activity (Nakazawa et al., 2003). In cultured hippocampal neurons,
p250GAP knock-down was shown to cause an increase in spine width while p250GAP
overexpression caused a decrease (Nakazawa et al., 2008).
3.3 Spine Morphology and Neurological Disorders
Dysregulation of spine morphology and density has been implicated in the
pathophysiology of a number of neuropsychiatric disorders with prominent cognitive deficits
including autism spectrum disorder, Alzheimer’s disease and schizophrenia (Penzes et al., 2011).
In these disorders, postmortem studies revealed significant changes in the spine number of
cortical and/or hippocampal neurons. For example, autistic human brain tissues showed an
increase in cortical neuron spine density (Hutsler and Zhang, 2010). Enhanced spine number
supports the autism spectrum disorder hypothesis of neuronal circuit hyperactivity (Geschwind
and Levitt, 2007) resulting in synaptic dysfunction. Alzheimer’s human brain tissues revealed
deficits in spine density in both cortex (DeKosky and Scheff, 1990) and hippocampus (Walsh
17
and Selkoe, 2004) resulting in synapse loss. Alterations in the synapses as a result of changes in
spine density correlated with the cognitive deficits associated with the affected brain regions
(DeKosky and Scheff, 1990; Walsh and Selkoe, 2004).
The dysfunction of the prefrontal cortex has been associated with the cognitive deficits of
schizophrenia. Examination of the schizophrenic postmortem brains revealed a significant
decrease in cortical pyramidal neuron spine density when compared to the normal controls
resulting in weakening of the excitatory input and synapse connectivity (Garey et al., 1998;
Glantz and Lewis, 2000). In order to further investigate potential molecular mechanisms
underlying changes in cortical dendritic spine number, expression levels of molecules involved
in the RhoGTPase signaling cascades were analyzed in schizophrenic postmortem brains. Cdc42
mRNA levels were significantly lower in the prefrontal cortex of schizophrenic individuals (Hill
et al., 2006; Ide and Lewis, 2010). Similarly, a decrease in Kalirin-7 (Rac-GEF) mRNA
expression was detected in the cortex of schizophrenic patients and it correlated with the detected
spine loss (Hill et al., 2006). Genetic animal models which exhibit behaviors relevant to
schizophrenia show similar deficits in spine density as those observed in postmortem studies.
Neuregulin 1 deficient mice showed a decrease in hippocampal spine density (Chen et al., 2008).
Similarly, mice deficient in ErbB2 and ErbB4 receptors showed a decrease in both hippocampal
and cortical spine number (Barros et al., 2009). Mice lacking Kalirin exhibited deficits in
cortical spine number and morphology (Cahill et al., 2009). While cortical neuron spine density
has yet to be determined, NMDA receptor deficient mice do display reductions in striatal spine
density (Ramsey et al., 2011).
4 Neurodevelopmental Hypothesis of Schizophrenia
The neurodevelopmental hypothesis of schizophrenia postulates that schizophrenia is a
disorder of altered connectivity between brain regions as a result of impairments in synaptic
function and plasticity (Rapoport et al., 2005). Synaptic dysconnectivity is believed to be caused
by genetic and environmental factors during prenatal and postnatal stages of development
(Karlsgodt et al., 2008). Improper development and formation of the neural circuits during
childhood and adolescence could then result in behavioral dysfunction in adulthood.
18
Figure 4. Proposed role of NMDA receptor in the
developmental hypothesis of schizophrenia (Kantrowitz
and Javitt, 2010).
In early childhood, behavioral schizophrenia symptoms are undetected. Although
retrospective and prospective studies have identified mild developmental abnormalities,
including motor and cognitive, as possible schizophrenia risk factors, these abnormalities cannot
be used to predict the disease (Messias et al., 2007). The prodromal symptoms of schizophrenia
appear post-adolescence, one to two years before the onset of first psychotic episode, and are
usually identified in retrospect; they include social withdrawal, lack of concentration and
attention, difficulties with decision making, and mild psychosis. The adult onset of
schizophrenia generally occurs, in both males and females, between ages of 15 and 25 (Messias
et al., 2007). Clinical diagnosis occurs after the initial psychotic episode and is followed by
treatment with antipsychotics. Schizophrenia is clinically diagnosed based on behavioral
symptoms: positive (delusions, hallucinations, paranoia, and psychosis), negative (flattened
affect, impaired attention and social withdrawal) and cognitive (intellectual deterioration and
impaired working memory) (Association, 2000).
Identification of a number of susceptibility genes with a role in regulation of synaptic
function, including synapsin and RGS4, led to the hypothesis that schizophrenia is a “disease of
the synapse” (Mirnics et al.,
2001). The hypothesis states that
impairments in synaptic
transmission cause alterations in
synapse formation and refinement
during childhood and adolescence
leading to adult behavioral
dysfunction. Neuroimaging
studies showing gray matter loss
in adolescent schizophrenics
with progression into adulthood
provide further support for the
developmental hypothesis
(Thompson et al., 2001).
Genetic and environmental
insults, which occur during the early stages of brain development, are thought to converge on the
19
glutamatergic system resulting in NMDA receptors dysfunction (for example see Figure 4). Loss
of NMDA receptor function leads to altered synaptic connectivity and subsequent development
of negative, positive, and cognitive schizophrenia symptoms (Kantrowitz and Javitt, 2010).
5 Modeling Behavioral Endophenotypes of Schizophrenia in Mice
A number of behavioral endophenotypes of schizophrenia, mainly negative and
cognitive, can be modeled in mice (Amann et al., 2010; Kellendonk et al., 2009). Schizophrenic
individuals show deficits in working and explicit memory; in humans this can be measured with
the N-back test (Kellendonk et al., 2009). In mice, working memory can be assessed through
maze exploration tasks such as radial, Y, and Morris water maze. Such tasks require the animal
to remember their last place of visit so that they can make a decision regarding where to explore
next. Explicit memory in mice can be investigated with the novel object recognition test (Amann
et al., 2010). The object recognition task relies on a mouse’s preference for exploration of a
novel object over a familiar one. Schizophrenia patients also have impairments in their ability to
shift cognitive strategies, termed cognitive flexibility; in humans this is quantified with the
Wisconsin Card Sort test (Kellendonk et al., 2009). Cognitive flexibility in mice can be assessed
with the latent inhibition test and the Morris water maze test. Latent inhibition measures a
mouse’s ability to recondition to a previously exposed stimulus, while Morris water maze
assesses their ability to locate a platform after it is moved from a learned to a new location.
Another negative symptom of schizophrenia is social withdrawal (Amann et al., 2010).
Sociability in mice can be measured through behavioral monitoring of the mouse when exposed
to a novel, non-aggressive mouse.
Some positive symptoms of schizophrenia have also been modeled in mice. For example,
sensorimotor gating deficits have been observed in schizophrenic patients and related to
psychotic symptoms. Measurement of the pre-pulse inhibition (PPI) of acoustic startle response
test has been used to demonstrate these sensorimotor gating deficits in schizophrenic patients.
The same test can be used to assess alterations in sensorimotor gating in mice. However, a
number of positive symptoms (psychosis, hallucinations and delusions) cannot be modeled in
mice with the same degree of face validity. An indirect way of assessing psychosis in mice is
20
through analysis of their locomotor activity, which does have construct and predictive validity
(Amann et al., 2010). Locomotor activity in rodents has been used as a behavioral correlate of
positive symptoms of psychosis for a number of reasons. This assay has predictive validity,
because psychosis-inducing drugs such as amphetamine and phencyclidine have been shown to
induce hyperactivity in rodents (Marcotte et al., 2001). Furthermore, antipsychotic drugs that
ameliorate positive symptoms also reduce locomotor activity (Marcotte et al., 2001). This assay
also has construct validity to some extent, because the brain structures that regulate locomotor
activity in rodents (cortex and striatum) are thought to be the same neural structures that play a
role in the generation of psychotic behaviors. Therefore, locomotor activity assay has become a
commonly used behavioral assay in rodents used to model schizophrenia related symptoms.
6 NR1 Knock-down Mice
6.1 Generation
For many years pharmacological antagonists of NMDA receptors have been used to
model NMDA receptor hypofunction, particularly to generate animal models for schizophrenia
research (Jentsch and Roth, 1999). NMDA
receptor NR1 subunit knockdown mice
(NR1-KD) were generated to complement
those studies, and represent a genetic
correlate of NMDA receptor hypofunction
(Mohn et al., 1999; Ramsey, 2009). NR1-
KD mice were generated by the targeted
insertion of an approximately 2kb foreign
DNA (neomycin selectable marker) into
intron 17 of the gene encoding for the NR1
subunit, Grin1, using homologous
recombination in embryonic stem cells
(Mohn et al., 1999). Northern and western
analyses reveal drastic reductions in full-
Figure 5. Protein expression levels of
NMDA (NR1, NR2A, NR2B) and AMPA
(GluR1, GluR2/3) receptor subunits in the
hippocampus of NR1-KD mice. (Ramsey et
al., 2008).
21
Figure 6. Regional deficits of NMDA receptor based on radio-ligand binding
study. Top: Illustration showing regional differences between wild type and NR1-KD
mice. Bottom: NMDA receptor level (maximal binding, Bmax) calculated from
radio-ligand competition binding with 3HMK801 (Mohn et al., 1999).
length mRNA and protein levels of the NR1 subunit, and were estimated in NR1-KD mice to be
at 5-10% of wild-type levels. Both NR1 and NR2 subunits are required for proper assembly and
functioning of the receptor. In addition to the NR1 deficits, reduction in the NR2 subunit levels
are detected, leading to significant reductions in receptor levels (Ramsey et al., 2008) (Figure 5).
Protein expression levels of the AMPA receptor subunits GluR1 and GluR2/3 were unaltered in
the striatum, prefrontal cortex, and hippocampus of the NR1-KD mice (Ramsey et al., 2008).
Radio-ligand binding assay with NMDA receptor antagonist (3H
MK-801) reveals global
reduction of the functional NMDA receptor (Mohn et al., 1999) (Figure 6). The most dramatic
reduction was observed in the brain regions with the highest expression levels of the receptor.
Regional brain deficits were as follows: 90% reduction in hippocampus, 86% in frontal cortex,
79% in striatum, and 60% reduction in brainstem (Mohn et al., 1999). Complete knockdown
22
of the NR1 subunit results in neonatal death as a result of improper functioning of the respiratory
system (Forrest et al., 1994; Li et al., 1994). It has been proposed that the moderate reduction of
the NMDA receptor in the brainstem of the NR1-KD mice is still sufficient for suckling and
breathing and therefore neonatal survival (Ramsey, 2009).
6.2 Behavioral Phenotypes
As discussed in section 5, there are a limited number of behavioral endophenotypes
relevant to schizophrenia that can be investigated in rodents. The psychotic symptoms are more
difficult to model in animals. Based on the ability of amphetamine to induce purely positive
symptoms in humans and to induce locomotor activity in rodents (Segal and Mandell, 1974), we
can use locomotor activity as a readout for positive symptoms. Conversely, some negative
(social withdrawal) and cognitive (impaired working memory) schizophrenia related symptoms
can be investigated.
Adult NR1-KD mice possess a number of behavioral phenotypes relevant to
schizophrenia. These mice showed an increase in locomotor activity, as a result of inability to
habituate to a novel environment, and in stereotypic behavior (Mohn et al., 1999). Low doses of
PCP and MK-801 do not exacerbate hyperactivity in NR1-KD mice. Other behavioral
alterations include deficits in spatial and working memory (Dzirasa et al., 2009), sensorimotor
gating (Duncan et al., 2004), and reduced sociability (Duncan et al., 2004; Mohn et al., 1999;
Ramsey et al., 2011). In addition, NR1-KD mice lack the normal social aggressive behavior
during resident-intruder test (Duncan et al., 2004; Mohn et al., 1999) and showed reduced
anxiety-like behavior in an elevated zero maze (Halene et al., 2009). Antipsychotics were able to
attenuate hyperactivity (Duncan et al., 2006; Mohn et al., 1999) and improve sociability (Mohn
et al., 1999) and sensorimotor gating (Duncan et al., 2006).
The developmental trajectory of the behavioral symptoms observed in NR1-KD mice was
not determined, because all of the previously described studies were performed with adult mice.
The studies described in this thesis were performed in order to map out the age onset of the
behavioral phenotypes detected in the genetic mouse model of NMDA receptor hypofunction.
23
6.3 Cellular and Molecular Phenotypes
A genetic model allows for investigation of the developmental consequences of NMDA
receptor hypofunction at both cellular and molecular level. Changes in synaptic transmission as
a result of NMDA receptor hypofunction were examined by Jocoy et al. NMDA receptor-
mediated currents (EPSCs) were drastically reduced in adult NR1-KD mice following electrical
stimulation of the cortex (Jocoy et al., 2011). In contrast, AMPA receptor-mediated EPSCs were
elevated in NMDA receptor deficient mice. Changes in the synapse were investigated through
analysis of medium spiny neurons (MSNs) of the striatum (Ramsey et al., 2011). MSNs spine
morphology and density was examined in juvenile and post-adolescent NR1-KD mice (Ramsey
et al., 2011). MSNs spine number and type was unaltered in juvenile, two-week old NR1-KD
mice when compared to the WT controls. In contrast, MSNs spine density was significantly
decreased (18%) in post-adolescent, six-week old NMDA receptor deficient mice. The spine
reductions appeared to be due to loss of mushroom shaped spines, which are thought to reflect
mature synapses (Yoshihara et al., 2009). The number of NR1-KD stubby and thin shaped
spines was comparable to those detected in the WT mice. The spine deficits become apparent
post-adolescence, but it was not determined in that study whether or not progressive spine loss
occurred beyond six weeks of age. One of the goals of this thesis is to determine the
developmental onset of synapse loss in NR1-KD mice, and determine whether or not spine loss
was progressive.
The potential molecular mechanism underlying spine loss in the NR1-KD mice was
investigated by examining two proteins implicated in regulation of spine dynamics and
schizophrenia pathology, 14-3-3 and DISC1. It was first determined that the synaptic levels of
14-3-3 were reduced in NR1-KD striatum by an unbiased proteomic approach (Ramsey et al.,
2011). Protein 14-3-3 is a member of the 14-3-3 family of proteins, which interact with
phosphorylated proteins and regulate their activity and stability (Aitken, 2006). Furthermore,
14-3-3 genes have been implicated as schizophrenia susceptibility genes (Ikeda et al., 2008;
Wong et al., 2003) and reductions in 14-3-3 gene products have been detected in post-mortem
schizophrenic brains (Middleton et al., 2005). One of the indirect targets of 14-3-3 , through its
interaction with NDEL1, is the scaffolding protein DISC1 (Taya et al., 2007). DISC1 is one of
the most widely reported schizophrenia susceptibility genes, and has multiple cellular functions
including the regulation of spine morphology and number (Hayashi-Takagi et al., 2010; Lee et
24
al., 2011). In adult NR1-KD mice, significant reductions in both 14-3-3 and DISC1 synaptic
protein levels were detected in the striatum (Ramsey et al., 2011). Striatal synaptic DISC1
deficits were further confirmed with immunogold labeling and electron microscopy. More
modest reductions in synaptic DISC1 levels were observed in frontal cortex and hippocampus.
Chronic administration of NMDA receptor inhibitor (MK-801) resulted in striatal synaptic
deficits in DISC1 but not 14-3-3 levels. In juvenile two-week old NR1-KD mice, subtle
decreases in striatal synaptic DISC1 levels were detected and 14-3-3 levels were unaltered
when compared to the age-matched WT mice. Because modest reductions in DISC1 were
detected at two weeks of age, when spine density was normal, changes in the synaptic DISC1
levels appeared to precede the NR1-KD deficits in the spine density. Alterations in DISC1 as a
result of NMDA receptor hypofunction suggest that the DISC1 signaling pathway requires
NMDA receptor activity and may contribute to the observed synaptic structural deficits.
There is additional evidence that NMDA receptor mediated transmission affects DISC1
biology, and ultimately alters spine morphology. Hayashi-Takagi et al demonstrated that one of
the functions of DISC1 is to act as a negative regulator of Rac1 (Hayashi-Takagi et al., 2010),
which controls spine morphology through regulation of actin dynamics (Hotulainen et al., 2009).
DISC1 normally interacts with the Rac1-regulatory protein Kalirin, a guanine nucleotide
exchange factor that activates Rac1. When DISC1 is associated with Kalirin, it prevents Kalirin
from activating Rac1; hence, DISC1 is a negative regulator of Rac1. Hayashi-Takagi et al
demonstrated that mutations or reductions in DISC1 led to more Rac1 activation, which caused a
transient increase in spine size, but ultimately led to reduction in spine size. Thus, reductions in
DISC1 can lead to loss of spines or spine diminishment in part through its regulation of Rac1.
6.4 Impact on Dopaminergic System
Hyperactivity and stereotypic behavior are usually associated with changes in the
dopaminergic system. Some parameters of dopamine neurotransmission were examined in adult
NR1-KD mice (Mohn et al., 1999). Tissue content of dopamine and its metabolites (DOPAC
and HVA) was measured in the striatum and no significant differences were detected when
compared to control mice. Similarly, extracellular levels of dopamine in NR1-KD mice
measured using microdialysis were comparable to those detected in the wild-type mice (Mohn et
al., 1999). Furthermore, striatal dopamine transporter and postsynaptic D1 and D2 receptor
25
levels and activity were unchanged in NR1-KD mice (Ramsey et al., 2008). Interestingly, even
though dopaminergic system appeared to be intact in NR1-KD mice, the psychostimulant
cocaine did not further enhance the hyperactivity observed in NR1-KD mice. Conversely,
amphetamine had similar stimulatory effects on the locomotor activity of both NR1-KD and
wild-type mice (Ramsey et al., 2008). The dopaminergic tone and system appear to be fairly
unaltered in the adult NR1-KD mice, suggesting that the pronounced motor behaviors are mainly
due to dysregulation of the glutamatergic system.
26
Chapter 2 MATERIALS AND METHODS
1 Animals
Knockdown of NR1 subunit of the NMDA receptor (NR1-KD) was achieved by targeted
insertion of a neomycin cassette into intron 17 of the Grin1 gene, which encodes the NR1
subunit (Mohn et al., 1999). The mutation was backcrossed onto two genetic backgrounds,
C57Bl/6J and 129X1/SvJ, for over 20 generations. Because congenic C57Bl/6J Grin1 -/- (NR1-
KD) mice were not viable, experimental mice were generated from the following breeding
strategy: intercross of heterozygous C57B1/6J Grin1+/- and 129X1/SvJ Grin1+/- mice to
produce F1 hybrids. The resulting F1 progeny share the same genetic background, which is 50%
C57Bl/6J and 50% 129X1/SvJ, and all three genotypes are generated from this intercross (Grin1
+/+, +/-, and -/-).
Animals were genotyped at two weeks of age by PCR analysis of tail biopsy DNA. The
following primers were used to amplify the native, wild-type allele: 5' TGA GGG GAA GCT
CTT CCT GT 3' and 5' AAG CGA TTA GAC AAC TAA GGG T 3'. Amplification of the
targeted allele was achieved using the primers 5' AAG CGA TTA GAC AAC TAA GGG T 3'
and 5' GCT TCC TCG TGC TTT ACG GTA T 3'.
Both male and female wild-type and NR1-KD mice were used in all of the experiments
performed. All mice were housed with a 12-h light/dark cycle (7am-7pm), and in accordance
with the institutional and federal guidelines for the care and use of animals (University of
Toronto Faculty of Medicine and Pharmacy Animal Care Committee and Canadian Council on
Animal Care).
2 Locomotor Activity Assay
Locomotor activity and habituation to a novel environment were measured using Accuscan
digital activity monitors (Columbus Instruments). Animals were placed in 20 cm x 20 cm x 45
27
cm Plexiglas chambers, and their activity was recorded for a total of two hours. Infrared light
beam sensors within the monitor were used to track the animal’s movement. Multiple chambers
were used to simultaneously monitor up to twelve mice at a time. Several parameters of
locomotor activity, including total distance travelled, were digitally monitored and collected in 5-
minute bins. Locomotor testing was performed during the light cycle, between the hours of 10
am and 4 pm, with indirect lighting of 25 lux. Collected locomotor data was analyzed using
Excel and GraphPad Prism statistical software. Statistical significance was determined using
Student’s t-test or ANOVA.
3 Sociability Assay
The sociability test was an adaptation of the test performed by Moy et al (Moy et al., 2004) and
as described in Ramsey et al ((Ramsey et al., 2011). The test mice (WT or NR1-KD) were
placed in an opaque-white walled Plexiglas arena (62 cm x 40.5 cm x 23 cm) with two inverted
wire cups: one empty (non-social, NS) and one containing a novel gender-matched none-
aggressive C3H/HeJ mouse (social, S). A circle was drawn around each cup to define zones (5
cm from the wired cup). Over a ten-minute period, the test mouse was video-recorded and its
movements were tracked and analyzed by the Biobserve Viewer2 software. Centre of the body
mass of the mouse was used as the reference point for the software. Number of entries to the
zones, total time spent in each zone, and total distance travelled during the 10 minute period was
recorded by the software. Sociability was measured as the time spent in the social zone around
the novel C3H mouse. Collected data was analyzed using GraphPad Prism statistical software.
Statistical significance was determined using two-tailed Student’s t-test.
4 Working Memory (Y-maze) Assay
Y-maze and Biobserve Viewer2 tracking software were used to measure working memory as
previously described in Mandillo et al (Mandillo et al., 2008). Y-maze was constructed out of
opaque white Plexiglas. It consisted of three equivalent arms (30 cm x 5 cm x 15 cm) meeting at
the centre at an angle of 120º with paper flooring. Within the video tracking software, zones
28
around each arm were created (zone A, B and C), and for better zone separation, an open space
was left in the middle where the three arms meet. Mice were placed at the end of one arm and
allowed to freely explore the maze for 8 minutes. The floor surface was cleaned after each
mouse run. The number of spontaneous 3-arm alternations, defined as successive entries into
each of the tree zones (ABC, BCA, CAB), was recorded and collected by Viewer2 software.
Percent spontaneous 3-arm alterations = (number of three-arm alterations) / (total number of arm
entries - 2) x 100. Y-maze task requires mice to remember the arm that they just explored so that
they can select a different one for their next entry and exploration; thus working memory can be
measured using this task. Collected data was analyzed using GraphPad Prism statistical
software. Statistical significance was determined using two-tailed Student’s t-test.
5 Subchronic MK-801 Treatment
Adult (twelve week) F1 hybrid strain mice (C57Bl/6J:129X1/SvJ) generated as described in
section 2.1 were used in experiments. Mice were subchronically treated with either saline or
MK-801 for 7-10 days. Osmotic minipump (Alzet), subcutaneously implanted in the mice, was
used to deliver the drug at a constant rate of either 0.1mg/kg/hr or 0.2mg/kg/hr over the desired
time period. In order to verify that the drug was properly delivered during treatment, mice were
either assed for hyperactivity using locomotor assay, or were visually monitored for the presence
of ataxia. Animals that did not survive the surgery or removed the minipump during the
treatment period were excluded from the study.
6 Diolistic Labeling and Imaging of Neurons
Lipophilic dye bullet preparation
1 micron gold particles (BioRad) were coated with 1,1'-dilinoleyl-3,3,3',3'-
tetramethylindocarbocyanine perchlorate (Fast DiI, Invitrogen) as described in Ramsey et al and
O’Brien (O'Brien and Lummis, 2006; Ramsey et al., 2011). DiI dissolved in chloroform (1.25
mg/ml) was mixed with ~50 mg of gold particles, and the chloroform was allowed to evaporate
on a glass slide in a chemical hood. Using a tubing preparation station (BioRad), tubing
29
(ethylene tetrafluoroethylene, 3.175 mm outer and 2.36 mm inner diameter) was first coated with
polyvinylpyrollidone (PVP) and allowed to dry. Subsequently, the tubing was coated with the
DiI coated gold particles. DiI coated tubing was dried with nitrogen gas (0.3-0.4 L min-1
for 10
minutes) and cut to generate bullets. Generated bullets were protected from the light and stored
at 4ºC.
Brain tissue preparation and labeling
WT and NR1-KD mice were perfused with 4% paraformaldehyde and postfixed overnight at
4ºC. The following day, 100 micron coronal slices (of the striatum) were collected with a Leica
VT-1200 vibratome. Neurons from fixed slices were randomly labelled with DiI, delivered
using a BioRad Gene Gun. Labelled slices were protected from the light and left at room
temperature for one hour to allow for DiI diffusion. Slices were then transferred to the
microscope slides, covered with cover slips, and imaged.
Imaging and spine measurements
Neurons were selected on the basis of location and morphology. Confocal Z-stack images of
dendritic branches and spines were collected using an Olympus Fluoview FV1000 system with
IX81 microscope and 60X water objective. Nikon NIS Elements software was used to count the
dendritic spines and spine density was calculated as number of spines per 100 micron of
dendrite. Collected data was analyzed using Excel and GraphPad Prism statistical software.
Statistical significance was determined using two-tailed Student’s t-test or ANOVA.
7 Western Blotting
WT and NR1-KD mice were sacrificed by cervical dislocation and the brain was removed. The
striatum was rapidly dissected on ice and homogenized in PHEM lysis buffer (60mM Pipes,
25mM Hepes, 10mM EGTA, 2mM MgCl2, 0.5% Triton-X, 1.5 g/ml aprotinin, 10 g/ml
pepstatin A, 10 g/ml leupeptin, 0.25mM PMSF, 10mM NaF, 2.5mM sodium pyrophosphate,
1mM -glycerophosphate, 5mM sodium orthovanadate) with hand held homogenizer. BCA
protein assay (Pierce) was used to determine the total protein extract concentration. 15-20 g of
protein extracts were resolved on either 7.5% or 11.5% SDS-polyacrylamide gel electrophoresis.
30
Gels were transferred to polyvinylidene fluoride membrane (PVDF). Protein expression levels
were assessed by immunoblotting using commercially available antibodies (Cortactin H222, N-
WASP 30D10, LIMK1,Cofilin D3F9, and P-cofilin 77G2 from Cell Signaling Technology,
Wave1 K91/36 from Antibodies Incorporated, Actin JLA20 from Developmental Studies
Hybridoma Bank, and GAPDH from Sigma) GAPDH was used as a loading control.
Immunoreactivity was detected with a LiCor Odyssey infrared imaging system and densitometry
was measured using NIH ImageJ64 software. Statistical analysis was performed with Excel
software using two-tailed Student’s t-test.
31
Chapter 3 RESULTS
NR1 knock-down mice were used to investigate the developmental effects of NMDA
receptor deficiency. Developmental trajectory of behavioral phenotypes (hyperactivity, reduced
sociability and working memory) was examined in NR1-KD mice in order to determine the onset
and severity. In a number of neuropsychiatric disorders, cognitive and behavioral dysfunction
has been correlated with changes in spine morphology and number (Penzes et al., 2011).
Therefore, we examined the onset and progression of spine number loss previously detected in
the striatum of the NR1-KD mice by Ramsey et al (Ramsey et al., 2011). In addition, signaling
pathways associate with changes in spine dynamics through modulation of the actin cytoskeleton
were examined in order to better understand the molecular mechanism underlying NMDA
receptor-mediated spine loss.
1 Elevated locomotor activity in juvenile, post-adolescent and adult NR1-KD mice
Adult NR1-KD mice were previously shown to display hyperlocomotor activity (Mohn et
al., 1999). In order to determine the age-onset of hyperactivity in NR1-KD mice, locomotor
activity was assessed in three week (juvenile), six week (post-adolescent), twelve week (adult),
and 24 week (adult) wild-type and NR1-KD mice. Mice were digitally monitored for a two hour
period. Distance travelled was collected in five minute increments. Hyperactivity was detected
in all NR1-KD age groups when compared to the matched wild-type controls. At three weeks of
age, activity was 16X greater than wild-type. At six weeks of age, activity was 8X greater than
wild-type. At twelve weeks of age, activity was 10X greater, and at twenty-four weeks of age,
activity was 8X greater (Figure 7). Although we had hypothesized that behavioral abnormalities
would show an adult onset, as is seen in schizophrenia, surprisingly, the most pronounced
increase in locomotor activity was detected in juvenile NR1-KD mice. Juvenile (three week old)
NR1-KD mice showed clear hyperactivity in the initial 30 minutes (above 1200 cm per five
32
minute bin) followed by gradual decrease over the next 90 minutes as the mice start to habituate
to the environment. In contrast, post-adolescent and adult NR1-KD mice lacked the initial
dramatic increase in the locomotor activity and instead demonstrated a more constant
hyperactivity (around 500 cm per five minute bin) over the 120 minutes. Juvenile, post-
adolescent and adult NR1-KD mice all failed to completely habituate to the novel environment
over the two hour period when compared to the wild-type mice. The striking increase in
locomotor activity detected in the juvenile NR1-KD mice suggests that the dopaminergic system
might be altered more prominently at this stage of development.
33
Figure 7. NR1-KD mice display an increase in locomotor activity at 3, 6, 12 and 24 weeks.
Exploration of a novel (20 x 20 cm) environment was measured using Accuscan digital activity
monitors. Wild-type (white squares, white bars) and NR1-KD (black circles, black bars) animals
A
B
C
D
34
at [A] 3 weeks, [B] 6 weeks, [C] 12 weeks and [D] 24 weeks were simultaneously monitored for
a 2-hour period. Distance travelled was measured and collected in 5-minute increments. For
[A]-[C] one or two-way ANOVA of locomotor activity, p<0.0001 and two-tailed Student’s t test
of total distance, *** p<0.0001. For all groups n=10. Using Bonferroni’s multiple comparison
test, significant differences in NR1-KD mice were only detected between 3 weeks of age and the
other age groups, p<0.001.
2 Adult-onset social deficits in NR1-KD mice
Sociability in adult NR1-KD mice was previously investigated and NR1-KD mice
displayed reduced sociability (Mohn et al., 1999). Wild-type mice were photographed sleeping
piled in a nest while adult NR1-KD mice stayed physically distant from their cage mates during
sleep (Mohn et al., 1999). Similarly, the resident-intruder behavioral assay revealed that adult
NR1-KD mice demonstrated less social investigation of the intruder mouse and more escape
behavior when compared to the control wild-type mice (Mohn et al., 1999).
To assess the developmental onset of social withdrawal in the genetic model of NMDA
receptor hypofunction, mice were analyzed for social interaction at three, six, twelve, and
twenty-four weeks of age. Mice were placed in a rectangular arena containing two wire cups:
one empty (none social, NS) and one containing a non-aggressive, gender matched, social
stimulus mouse (social, S) (Figure 8A). A test mouse was placed in the arena and its movement
was video recorded and tracked over a ten-minute period. The time juvenile and post-adolescent
NR1-KD mice spent in the social and non-social zones were not significantly different from the
age matched wild-type mice (Figure 8B). Conversely, adult (twelve and twenty-four week)
NR1-KD mice spent significantly less time socializing with the novel mouse than the controls.
Even though the twelve week old wild-type mice were overall more social than those at twenty-
four weeks, twelve and twenty-four week old NR1-KD mice displayed similar levels of social
investigation with the novel mouse. In summary, social deficits observed in the NR1-KD mice
manifested in adulthood and did not worsen with age.
35
Figure 8. Adult NR1-KD mice display deficits in sociability.
A test mouse, wild-type or NR1-KD, was placed in an opaque-white walled (62x40.5x23cm)
arena with two wire cups. One cup was empty (non-social stimulus, NS) and one contained a
novel, gender-matched, C3H/HeJ mouse (social stimulus, S). Circular zones around each cup
were defined, and the time spent in each zone was recorded by the Biobserve Viewer software.
Over a ten-minute period, the test mouse was video-recorded and its movements tracked using
**
A
B
**
*
36
the software. Sociability was measured as the time spent in a zone around the novel mouse (S).
[A] A representative image of the video-recorder test arena and tracked movement of a WT
mouse. [B] WT and NR1-KD mice were tested at 3, 6, 12, and 24 weeks and zone durations
were recorded. For all ages and genotypes, n=10. Two-tailed Student’s t test *p=0.05,
**p<0.01.
3 Impaired spatial working memory detected in adult NR1-KD mice
Radial and Y-maze tasks are used widely to assess spatial working memory in rodents
(Kellendonk et al., 2009). Rodents prefer to explore a novel environment over a familiar one.
The maze task relies on the rodents’ ability to recall their last arm of entry in order to be able to
select a different arm for exploration on their next arm visit. In an 8-arm radial maze task, adult
NR1-KD mice were shown to display schizophrenia-related cognitive impairments in the form of
deficits in spatial working memory (Dzirasa et al., 2009).
The manifestation and progression of spatial working memory was further examined in
NR1-KD mice using Y-maze. Mice were placed at the end of one arm and their movement was
video recorded and tracked for eight minutes (Figure 9A). The number of spontaneous three-arm
alternations (ABC, BCA, CAB) was collected and analyzed. Spatial working memory in the
juvenile (three week) and post-adolescent (six week) NR1-KD mice was unaltered when
compared to the wild-type mice (Figure 9B). In contrast, significant reductions in the spatial
working memory were detected in adult NR1-KD mice (p<0.05). Twelve and twenty-four week
old NR1-KD mice displayed similar deficits in working memory indicating that the observed
behavioural phenotype does not further deteriorate with age.
37
Figure 9. Deficits in working memory in NR1-KD mice manifest in adulthood.
Spontaneous 3-arm alternation was assessed in a Y-maze composed of three equivalent opaque
white arms meeting at the centre and oriented at 120° angles, dimensions 30x5x15cm. [A]
Representative image of video-based tracking of the Y-maze exploration. [B] WT and NR1-KD
3 week, 6 week, 12 week and 24 week old mice were placed at the end of one arm and allowed
A
B
* *
38
to freely explore the maze for 8 minutes. The number of spontaneous 3-arm alternations (ABC,
BCA, CAB) were recorded and collected by Biobserve Viewer2 software. For all ages and
genotypes, n=10. Two-tailed Student’s t test, *p<0.05.
4 Age-dependent deficits in MSN spine density
Postsynaptic dendritic spines play an important role in synaptic structure, function and
plasticity (Nimchinsky et al., 2002). Alterations in spine morphology and density have been
implicated in the pathophysiology of a number of mental disorders including schizophrenia
(Penzes et al., 2011). Furthermore, changes in spine number were shown to correlate with
behavioral impairments (DeKosky and Scheff, 1990; Walsh and Selkoe, 2004). Medium spiny
neuron (MSN) spine density was previously examined in the striatum of juvenile and post-
adolescent NR1-KD mice (Ramsey et al., 2011). Spine density was shown to be unaltered in
two-week old, juvenile NR1-KD mice and reduced in six-week old, post-adolescent mice.
In order to investigate whether the deficits observed in MSNs of NR1-KD mice progress
with age, spine density was analyzed in adults (twelve and twenty-four weeks) in addition to
juvenile and post-adolescent mice. A diolistic neuron labeling technique was used to label
striatal neurons from fixed tissue (O'Brien and Lummis, 2006). Striatal MSNs were identified
based on tissue location and neuron morphology. Spine density was represented as spine
number/100 microns of dendrite. Spine density was unaltered in three week old NR1-KD mice
(NR1-KD 133.4+4.2, WT 134.6+5.3) (Figure 10). As expected from previous studies, spine
reduction of 10.4% was evident in six week old NR1-KD mice (NR1-KD 123.8+2.1, WT
138.2+1.5) (Figure 11). Further spine loss was detected in adult NR1-KD mice. Spine number
was reduced by 15.6% in twelve week old mice (NR1-KD 114.3+1.4, WT 135.5+1.7) (Figure
12) and 21.1% in twenty-four week old mice (NR1-KD 103.0+2.2, WT 130.4+2.7) (Figure 13A).
During development, spine density in wild-type mice remained constant while in NR1-KD mice
spine loss began post-adolescence and progressed with age (Figure 13B).
In order to confirm that the observed deficits in MSNs spine density were due to NMDA
receptor hypofunction, adult (twelve week old) wild-type mice were subchronically treated with
selective NMDA receptor antagonist, MK-801. Osmotic pump, implanted subcutaneously, was
39
used to deliver the drug at either 0.1mg/kg/h or 0.2mg/kg/h for 7-10 days. Following drug
treatment, neurons from fixed tissue were labeled and medium spiny neurons of the striatum
were analyzed. Dose-dependent decreases in spine number were detected in mice treated with
NMDA receptor antagonist (WT 139.1+5.0, 0.1 mg/kg MK-801 132.2+2.0, and 0.2 mg/kg MK-
801 123.7+1.4 p=0.0506) (Figure 14). Spine density deficits detected in twelve week old NR1-
KD mice (10.4%) were comparable to those observed following subchronic pharmacological
inhibition of the NMDA receptor (11.1%, 0.2mg/kg MK-801).
40
3 weeks
Figure 10. MSNs spine density is unaltered in 3 week old NR1-KD mice.
Three week old wild-type and NR1-KD mice were perfused with 4% paraformaldehyde, 100 m
thick striatal coronal sections were cut and collected, and neurons from fixed slices were
randomly labeled by diolistics with the lipophilic dye, DiI. Z-stack images of medium spiny
neuron (MSN) dendrites were collected using confocal microscopy (Scale bar: 10 m). Collected
images were analyzed using NIS Elements software (number of spines/100 micron dendrite).
For each group, mice n=3, dendrites n=15-16.
WT NR1-KD
41
6 weeks
Figure 11. 6wk NR1-KD mice show reduced MSNs spine density.
6 week old wild-type and NR1-KD mice were perfused with 4% paraformaldehyde, 100 m
thick striatal coronal sections were cut and collected, and neurons from fixed slices were
randomly labeled by diolistics with the lipophilic dye, DiI. Z-stack images of medium spiny
neuron (MSN) dendrites were collected using confocal microscopy (Scale bar: 10 m). Collected
images were analyzed using NIS Elements software (number of spines/100 micron dendrite).
For each group, mice n=3, dendrites n=18. Two-tailed Student’s t test **p<0.01.
WT NR1-KD
**
42
12 weeks
Figure 12. MSNs spine density is further decreased in 12 week NR1-KD mice.
12 week old wild-type and NR1-KD mice were perfused with 4% paraformaldehyde, 100 m
thick striatal coronal sections were cut and collected, and neurons from fixed slices were
randomly labeled by diolistics with the lipophilic dye, DiI. Z-stack images of medium spiny
neuron (MSN) dendrites were collected using confocal microscopy (Scale bar: 10 m). Collected
images were analyzed using NIS Elements software (number of spines/100 micron dendrite).
For each group, mice n=3, dendrites n=15. Two-tailed Student’s t test ***p<0.001
WT NR1-KD
***
43
24 weeks
Figure 13. Greater deficits in MSNs spine density detected in 24 week NR1-KD mice.
[A] 24 week old wild-type and NR1-KD mice were perfused with 4% paraformaldehyde, 100
m thick striatal coronal sections were cut and collected, and neurons from fixed slices were
randomly labeled by diolistics with the lipophilic dye, DiI. Z-stack images of medium spiny
neuron (MSN) dendrites were collected using confocal microscopy (Scale bar: 10 m). Collected
images were analyzed using NIS Elements software (number of spines/100 micron dendrite).
For each group, mice n=3, dendrites n=18-19. Two-tailed Student’s t test **p<0.01. [B]
A
WT NR1-KD
Age-dependent Spine Reductions B
**
44
Summary of the age-dependent deficits in MSNs spine density observed in NR1-KD mice (3, 6,
12, and 24 weeks). Using Bonferroni’s multiple comparison test, significant differences in the
NR1-KD density of spines were detected between 3 and 12 weeks, between 3 and 24 weeks, and
between 6 and 24 weeks, **p<0.01, ***p<0.001.
Subchronic drug treatment
Figure 14. Dose-dependent decreases in MSN spine number following MK-801.
Adult (12 week) wild-type mice were subchronically treated with saline or MK-801. Osmotic
pump, implanted subcutaneously, was used to deliver saline, 0.1mg/kg/h, or 0.2mg/kg/h MK-801
Saline MK-801 (0.2mg/kg)
*
45
over a 7-10 day period. Following drug treatment, mice were perfused with 4%
paraformaldehyde, coronal sections were cut, neurons were labelled with DiI, dendrite imaging
were taken and spine numbers were analysis (spines/100 micron dendrite). For each MK-801
treated group, mice n=3, dendrites n=16-18. Two-tailed Student’s t test, *p=0.0509.
5 Age-dependent alterations in Rac1 in NR1-KD mice
Spine morphology is highly regulated by the dynamic actin cytoskeleton (Hotulainen and
Hoogenraad, 2010). Changes in actin assembly, stability and reorganization can have strong
effects on spine structure and dynamics (Sekino et al., 2007). The Rho family of small GTPases
modulate actin cytoskeleton through the activation of actin-binding or actin-modulating proteins
(Cingolani and Goda, 2008). RhoA, Rac1 and Cdc42 are the best-characterized actin modulating
RhoGTPases. In order to investigate the possible role actin and RhoGTPases might play in the
age-dependent MSNs spine loss detected in NR1-KD mice, total protein expression levels of
actin, RhoA, Rac1 and Cdc42 were examined.
Total protein extracts from the striatum of three week, six week and twelve week wild-
type and NR1-KD mice were collected and analyzed by western blotting. Total actin levels
remained unchanged in the striatum of three, six and twelve week old NR1-KD mice (Figure 15).
Similarly, total RhoA levels do not appear to be affected by genetic knockdown of the NMDA
receptor (Figure 16). Cdc42 total protein levels were unaltered at three and twelve weeks but
were trending downward at six weeks; the decrease was not statistically significant. The most
dramatic changes were observed in Rac1 GTPase (Figure 16). In juvenile NR1-KD mice, total
Rac1 protein levels were elevated (25.8%, p<0.001). In contrast, total Rac1 levels were
significantly reduced in post-adolescent (p<0.05) and adult (p<0.01) NR1-KD mice (20-30%).
Of the RhoGTPases investigated, Rac1 appeared to be affected the most by genetic knockdown
of the NMDA receptor. NMDA receptor activity was shown to be able to modulate Rac1
activity (Hayashi-Takagi et al., 2010). Thus, Rac1-mediated signalling pathway could be a
potential molecular mechanism underlying age-dependent MSNs spine loss detected in NR1-KD
mice.
46
Figure 15. Actin expression is unaltered in NR1-KD mice.
Wild-type and NR1-KD mice were sacrificed by cervical dislocation and the striatum was
rapidly dissected. Tissue was homogenized in cold lysis buffer and 15-20 g of protein extracts
were used in each experiment. Protein expression levels at [A] 3 weeks, [B] 6 weeks and [C] 12
weeks were assessed by SDS-PAGE and western blotting for actin. GAPDH was used as a
loading control. Densitometry analysis was performed using NIH ImageJ software (proteins
were normalized to GAPDH). For all groups, n=4.
A
3 wks
B
6 wks
C
12 wks
47
Figure 16. Age-dependent changes in Rac1 protein expression in NR1-KD mice.
Wild-type and NR1-KD mice were sacrificed by cervical dislocation and striatum was rapidly
dissected. Tissue was homogenized in cold lysis buffer and 15-20 g of protein extracts was
A
3 wks
B
6 wks
C
12 wks
48
used in each experiment. Protein expression levels at [A] 3 weeks, [B] 6 weeks and [C] 12
weeks were assessed by SDS-PAGE and western blotting for RhoA, Rac1 and Cdc42. GAPDH
was used as a loading control. Densitometry analysis was performed using NIH ImageJ software
(proteins were normalized to GAPDH). Two-tailed Student’s t test, n=4, ***p<0.001, **p<0.01,
*p<0.05.
6 Reduced Wave1 and N-Wasp protein levels in NR1-KD mice
One way of modulating actin cytoskeleton dynamics is through the regulation of actin
filament polymerization and branching (Cingolani and Goda, 2008). The downstream effectors
of Rac1 and Cdc42 (cortactin, Wave1, and N-Wasp) regulate F-actin nucleation and assembly
through modulation of the activity of actin-binding complex, Arp2/3 (Cingolani and Goda,
2008). Neuronal changes in cortactin, Wave1, and N-Wasp were shown to induce alterations in
spine density and synaptic transmission (Hering and Sheng, 2003; Soderling et al., 2007; Wegner
et al., 2008). Therefore, protein expression levels of these molecules were assessed in the
striatum, the brain region where spine loss was detected in NR1-KD mice.
Striatal protein extracts from three week, six week and twelve week old mice were
assessed with SDS-PAGE and immunoblotting for cortactin, Wave1, N-Wasp and GAPDH
(Figure 17). Cortactin protein expression levels where unchanged in all age groups of NR1-KD
mice when compared to the wild-type controls. In contrast, deficits in Wave1 expression levels
in NR1-KD mice were detected at three, six and twelve weeks (20-35%, p<0.05). N-Wasp
changes in NR1-KD mice were age-dependent: at three weeks remained unchanged, at six weeks
were significantly reduced (28.2%, p<0.05) and at twelve weeks showed a subtle decrease
(p=0.07). NMDA receptor knockdown had the most profound effect on the actin-modulating
protein Wave1. Wave1 activity is generally modulated by Rac1 GTPase (Etienne-Manneville
and Hall, 2002), whose expression levels were also shown to be altered in NR1-KD mice.
Alterations in Wave1 levels further strengthen our hypothesis that the MSN spine loss in NR1-
KD mice is regulated through a Rac1-mediated signaling cascade.
49
Figure 17. Age-dependent reductions in Wave1 and N-Wasp protein expression in NR1-
KD mice. Wild-type and NR1-KD mice were sacrificed by cervical dislocation and striatum
was rapidly dissected. Tissue was homogenized in cold lysis buffer and 15-20 g of protein
A
3 wks
B
6 wks
C
12 wks
50
extracts were used in each experiment. Protein expression levels at [A] 3 weeks, [B] 6 weeks
and [C] 12 weeks were assessed by SDS-PAGE and western blotting for Cortactin, Wave1 and
N-Wasp. GAPDH was used as a loading control. Densitometry analysis was performed using
NIH ImageJ software (proteins were normalized to GAPDH). Two-tailed Student’s t test
*p<0.05.
7 Elevated phosphorylated cofilin levels detected in adult NR1-KD mice
Alterations in actin cytoskeleton could also result from modulation of actin
depolymerization and reorganization. These actin cytoskeleton processes are regulated by actin-
binding protein cofilin (Cingolani and Goda, 2008). One of the modulators of cofilin activity is
LIM kinase (Meng et al., 2003). Phosphorylation of cofilin by LIMK leads to cofilin
inactivation and stability of actin filament. GTPases Rac1 and RhoA have the ability to
modulate LIMK activity (Etienne-Manneville and Hall, 2002).
LIMK1 and cofilin (total and phosphorylated) protein levels were examined in the
striatum of three, six and twelve week old wild-type and NR1-KD mice (Figure 18). Protein
expression levels of LIMK1 remained unchanged in NR1-KD mice. Similarly, total cofilin
expression levels were not significantly altered in three, six or twelve week NR1-KD mice when
compared to the WT controls. Although not significant, total cofilin levels trended toward a
decrease in twelve week NR1-KD mice. Phosphorylated cofilin levels were not altered in three
or six week old NR1-KD mice but were elevated in twelve week mice when phosphorylated
levels were normalized to total cofilin (23.4%, p<0.05). Dampening of cofilin activity would
suggest that there is enhanced inhibition of actin depolymerization resulting in actin filament
stabilization.
51
Figure 18. Enhanced P-cofilin levels in adult NR1-KD mice.
Wild-type and NR1-KD mice were sacrificed by cervical dislocation and striatum was rapidly
dissected. Tissue was homogenized in cold lysis buffer and 15-20 g of protein extracts were
A
3 wks
B
6 wks
C
12 wks
52
used in each experiment. Protein expression levels at [A] 3 weeks, [B] 6 weeks and [C] 12
weeks were assessed by SDS-PAGE and western blotting for LIMK, Cofilin and P-cofilin.
GAPDH was used as a loading control. Densitometry analysis was performed using NIH ImageJ
software (proteins were normalized to GAPDH; in addition, P-cofilin was normalized to cofilin
(c)). Two-tailed Student’s t test *p<0.05.
53
Chapter 4 DISCUSSION, CONCLUSIONS AND
RECOMMENDATIONS
1 Discussion
Schizophrenia is a debilitating mental disease with unknown etiology that affects 1% of
world’s population. It is described as a neurodevelopmental disorder, highly influenced by
genetic and environmental factors (Rapoport et al., 2005). Symptoms of schizophrenia are
generally divided into three categories: positive (delusions, hallucinations and thought disorder),
negative (social withdrawal, flattened effect and impaired attention) and cognitive (intellectual
deterioration, impaired memory and executive function) (Association, 2000). Schizophrenia
symptoms begin to appear post-adolescence and are clinically treated with antipsychotics in
adulthood (Messias et al., 2007). Antipsychotics can alleviate the positive symptoms in patients
but have limited beneficial effects on disabling negative and cognitive symptoms (Javitt, 2007).
Although the etiology of schizophrenia is unknown, several neurotransmitter systems
have been implicated in the symptoms of the disease. In this thesis we have focused on the role
of glutamatergic NMDA receptors in the expression of schizophrenia symptoms. Previous
studies in adult NR1-KD mice demonstrated that deficits in NMDA receptor signaling can result
in many schizophrenia behavioral endophenotypes. NR1-KD mice showed deficits in working
memory (Dzirasa et al., 2009), sensorimotor gating (Duncan et al., 2004), and sociability
(Duncan et al., 2004; Mohn et al., 1999; Ramsey et al., 2011). Locomotor activity was highly
elevated in these mice (Mohn et al., 1999). Many of these behaviors were normalized with
antipsychotic drugs (Duncan et al., 2006; Halene et al., 2009; Mohn et al., 1999). However,
several cognitive domains such as explicit memory and cognitive flexibility have yet to be
examined. In this thesis, we examined the developmental trajectory of a few of the behavioral
endophenotypes, in order to determine whether the age onset observed in the animal model
correlates with the adult onset of symptoms in human schizophrenia patients.
54
Sociability, working memory, and locomotor activity were examined in juvenile, post-
adolescent, adult, and aged-adult NR1-KD mice. Reduced sociability was only detected in adult
NR1-KD mice. The deficits did not worsen with age as both of the adult age groups showed
similar reductions in social interaction. Similarly, adult-onset deficits in working memory were
detected in NR1-KD mice. Working memory did not appear to further deteriorate in aged-adult
mice. It is important to note that hyperactivity did not appear to play a confounding factor in the
sociability and working memory tasks. In the social arena, adult NR1-KD mice did not display
any hyperactivity when compared to the controls (data not shown). In the Y-maze test, twelve-
week old mice were slightly more active than wild-type, but substantial differences in activity
were not detected as in the locomotor assay. At 24-weeks of age, the activity of NR1-KD mice
in the Y-maze and the sociability assay was comparable to the wild-types. Furthermore, if
hyperactivity were to play an influential role in the social and Y-maze assays, it should have had
the largest effect in the juvenile mice, which had high levels of activity in the locomotor assay,
but performed similar to wild-type mice in the Y-maze and sociability assays. While delayed
onset of social withdrawal and impaired working memory in NR1-KD mice parallel with the
onset and progression of schizophrenia, the presence of extreme hyperactivity in juvenile mice
was unexpected.
The extreme increase in locomotor activity points to possible dysregulation of the
dopaminergic system. However, in adult NR1-KD mice, the integrity of the dopaminergic
system was evaluated and found to be generally unaltered. Both striatal tissue content and
extracellular dopamine levels in the NR1-KD mice were comparable to wild-type mice (Mohn et
al., 1999). In addition, dopamine transporter and postsynaptic D1 and D2 receptor levels and
activity were unchanged (Ramsey et al., 2008). Looking at the developmental trajectory of the
hyperactivity in the NR1-KD mice, there is a clear difference in the behavioral pattern between
juvenile and adult mice. Juvenile mice display a hyperactivity pattern similar to the one induced
in mice as a result of elevated striatal dopamine, suggesting that in juvenile mice the
dopaminergic tone could very likely be altered (Gainetdinov et al., 1999). Further experiments,
such as microdialysis and cyclic voltammetry, need to be performed to determine whether
dopamine tone is altered in the juvenile NR1-KD mice.
Possible differences in dopamine tone between juvenile and adult mice could be
explained in two ways. First, glutamatergic neurons have the ability to negatively modulate the
55
mesolimbic dopamine pathway by regulating the activity of inhibitory, GABAergic interneurons
(Stahl, 2007). Under normal conditions, glutamate has an excitatory effect on inhibitory
GABAergic interneurons, restraining the firing of dopamine neurons, and resulting in a
dampening of the mesolimbic dopamine tone (Stahl, 2007). Dysfunction of the glutamatergic
system, as a result of NMDA receptor knockdown, could lead to a disinhibition of the
dopaminergic neurons, resulting in an elevation of the mesolimbic dopamine tone. As a result, at
the earlier stage of development, dopamine levels could be elevated; as development proceeds
and mice age, the activation of unknown compensatory mechanisms could result in a dampening
of the dopamine tone. A second and related explanation for the developmental difference in
dopamine levels could be explained through unique developmental expression of the dopamine
transporter (DAT). The dopamine transporter is expressed on the presynaptic membrane, and is
responsible for dopamine reuptake/clearance from the synaptic cleft (Torres et al., 2003).
Developmental radioligand binding studies looking at postnatal rat striatal dopamine transporter
levels revealed that DAT levels were extremely low at birth; a slow increase was detected up to
week three, followed by a rapid increase between juvenile and adult stages of development
(Coulter et al., 1997). This would suggest that even though the dopamine system might be
altered in NR1-KD mice, the greatest effects would be observed in juvenile mice due to low
levels of dopamine transporter expression and subsequent inefficient clearance of the dopamine
from the synaptic cleft.
Because the NR1-KD mice could be thought to model a form of schizophrenia, and
because they exhibit a more extreme form of hyperactivity in juvenile stages of development, it
is tempting to draw a parallel between this hyperactivity and the juvenile psychiatric condition of
attention-deficit hyperactivity disorder (ADHD). A number of retrospective studies have been
done, examining co-morbidity of ADHD and schizophrenia, in order to determine if any parallels
exist between hyperactivity and schizophrenia patients (Marsh and Williams, 2006). In the more
atypical schizophrenia cases, adolescent studies suggest that ADHD-like symptoms appear in
children with schizophrenia premorbidly (Alaghband-Rad et al., 1995; Spencer and Campbell,
1994). In addition, a study by Elman and colleagues compared adolescent schizophrenic patients
with premorbid ADHD with schizophrenia-only patients. The study concluded that
schizophrenic individuals with childhood diagnoses of ADHD had earlier onset of psychosis and
poorer prognoses (Elman et al., 1998).
56
When considering the behavioral alterations that are observed in NR1-KD mice, it is
important to consider the brain regions that are thought to normally regulate these behaviors.
The prefrontal cortex is involved in regulating social interaction, executive function, problem
solving, and working memory (Shipp, 2007). The hippocampus plays a crucial role in the
execution of tasks that utilize spatial information, and is also necessary in many forms of
learning and memory formation (Eichenbaum, 2004). The basal ganglia (striatum in mice) was
initially only thought to be involved in regulating movement, emotions and consolidation of
sensory information, but is now understood to play a role in influencing cognitive behavior and
decision-making as well (Simpson et al., 2010). The striatum receives a large amount of
glutamatergic afferents from the cortex, and the cortex and striatum are anatomically and
functionally connected by these corticostriatal pathways (Simpson et al., 2010). Both striatal and
prefrontal cortex lesion studies cause deficits in working memory (Battig et al., 1960; Divac et
al., 1967). In non-humane primates, prefrontal cortex neurons found to be active following a
cognitive task, referred to as “memory cells”, were also discovered in the striatum (Hikosaka et
al., 1989). Similarly, the striatum was shown to be activated in healthy individuals during an
executive function task, Wisconsin Card Sorting Test (Rogers et al., 2000). Thus, the striatum,
in addition to the prefrontal cortex, can contribute to cognitive behaviors including working
memory and executive function.
The striatum is a relatively homogeneous brain region composed mainly of medium spiny
neurons (~95%) (Tepper and Bolam, 2004). This makes it particularly suitable for making
correlations between changes in synapse number and biochemistry. We first assessed changes in
striatal MSNs spine density in NR1-KD mice at the three crucial stages of synaptogenesis:
synapse formation (juvenile, three week mice), synapse elimination (post-adolescent, six week
mice) and synapse maturation (adult, twelve and 24 week mice) (Adriani and Laviola, 2004).
During the stage when the synapses are actively forming, spine density was unaltered in NR1-
KD mice, suggesting that NMDA receptors might not be crucial for this stage of synaptogenesis.
However, spine density was significantly altered in NR1-KD mice during the developmental
time periods that coincide with the processes of synapse elimination and maturation. Although
loss in spine density progressed in adult mice, the most rapid loss was detected following
synapse refinement, between the ages of six weeks and twelve weeks. This suggests that NMDA
receptors play an important role in mediating proper synapse refinement and stabilization.
57
NMDA receptor hypofunction resulting in altered synapse number is consistent with the
neurodevelopmental hypothesis of schizophrenia. The hypothesis states that schizophrenia is a
disorder of altered brain connectivity as a result of improper wiring of the brain during prenatal
and postnatal stages of development (Karlsgodt et al., 2008; Rapoport et al., 2005).
Developmental genetic and environmental insults are thought to contribute to the improper
assembly of neuronal circuits during development (Kantrowitz and Javitt, 2010).
The detected spine density deficits in the NR1-KD mice preceded the adult-onset
behavioral endophenotypes of schizophrenia that we examined: social withdrawal and impaired
working memory. The amount of spine loss detected during the developmental period when the
behavioral impairments were observed was ~15-20%. This raises a question whether such a loss
in spine number is sufficient enough to influence synaptic and behavioral function. In rodent
studies, changes in spine density of 10-20% were shown to correlate with changes in behavioral
function (Leuner et al., 2003; Rampon et al., 2000). In addition, dysregulation of spine density
has been implicated in the pathophysiology of a number of mental disorders including
schizophrenia and Alzheimer’s disease (Penzes et al., 2011). In a postmortem study of
schizophrenic brains, a 23% reduction in spine density was detected when compared to the
normal subjects and a 16% reduction was detected when compared to patients with other
psychiatric conditions; this level of decrease was suggested to result in weakening of the
excitatory input and synapse connectivity (Glantz and Lewis, 2000). Similarly, individuals with
Alzheimer’s disease were shown to have deficits in spine density which correlated with the brain
region specific cognitive impairments (DeKosky and Scheff, 1990; Walsh and Selkoe, 2004).
Based on the published findings discussed above, we would conclude that the percent loss in
spine density that we detected in adult NR1-KD mice is substantial and significant enough to
potentially affect synaptic and behavioral function.
As stated earlier, the prefrontal cortex plays an important role in regulating social
interactions and working memory. In our current study, changes in spine density were only
examined in the MSNs of the striatum. Future studies investigating developmental alterations in
the spine density of cortical pyramidal neurons need to be performed in NR1-KD mice. It is very
likely that developmental spine density deficits in both of the brain regions, striatum and
prefrontal cortex, are contributing to the behavioral impairments detected in NR1-KD mice.
58
As discussed, one of the advantages to studying synapse biology in the striatum is the
ability to correlate the loss of dendritic spines with biochemical processes that are implicated in
synapse biology due to the relative homogeneity of neuron type. Dendritic spine morphogenesis
and dynamics are highly regulated by the reorganization of the actin cytoskeleton, which in turn
is modulated by the Rho GTPases, RhoA, Rac1 and Cdc42 (Hotulainen and Hoogenraad, 2010).
While we did not detect substantial changes in RhoA and Cdc42 total protein levels, age-
dependent alteration in Rac1 protein levels were observed. Our findings suggest that
developmental alterations in the Rac1 signaling pathway could underlie the spine number loss
detected in NR1-KD mice. In the juvenile mice, when Rac1 protein expression was elevated,
spine density was unaltered. Conversely, at the developmental stages when spine deficits were
evident, Rac1 levels were significantly reduced. Rac1 activation generally promotes spine
formation and stabilization (Saneyoshi et al., 2010). At this time, we cannot say whether there is
a direct correlation between changes in Rac1 levels and their activity. Further studies examining
Rac1 activity at the different developmental stages in NR1-KD mice need to be completed. In
addition, the activity status of RhoA and Cdc42 should be analyzed. Even though their protein
levels were not drastically affected by NMDA receptor knockdown, their activity could be
altered considering that there is significant cross-talk between these three RhoGTPase signaling
cascades (Li et al., 2002).
There are several lines of investigation that have implicated Rac1 signaling in the
regulation of spine morphology and density. In cultured neurons, transient Rac1 activation was
shown to induce spine head growth, while constitutively active Rac1 was shown to induce spine
loss (Hayashi-Takagi et al., 2010) in some studies and spine enhancement in others (Luo et al.,
1996). Rac1 activity is modulated by guanine-nucleotide exchange factors (GEFs). GEFs
Kalirin-7 and Tiam1 are expressed in neurons and were shown to regulate Rac1 activity in an
NMDA receptor-mediated matter (Hayashi-Takagi et al., 2010; Tolias et al., 2005). Kalirin-7
knockout mice exhibit deficits in spine density, LTP and cognitive behavior (Ma et al., 2008).
NMDA receptor-mediated Kalirin-7 activation of Rac1 was shown to be modulated by DISC1, a
schizophrenia susceptibility factor (Hayashi-Takagi et al., 2010). DISC1 interaction with
Kalirin-7 prevents Kalirin-7 from activating Rac1. Thus, DISC1 negatively modulates Rac1
activity. NMDA receptor activity, in vivo and in primary neurons, promotes Kalirin-7
dissociation from DISC1 and subsequent Rac1 activation. Interestingly, in NR1-KD mice,
59
DISC1 was shown to be reduced in an age-dependent matter in total and synaptic extracts
(Ramsey et al., 2011). NMDA receptor-dependent changes in the DISC1-Kalirin7-Rac1
signaling cascade could lead to alteration in spine dynamics. Another Rac1 GEF shown to be
modulated by NMDA receptor activity is Tiam1 (Tolias et al., 2005). Tiam1 was shown to
interact with NMDA receptor; NMDA receptor activation induced activation of Tiam1, in a
calcium-dependent matter, and Rac1. Knockdown of Tiam1 expression in cultured neurons
results in reduced spine and synapse number. Thus, Rac1 activity can be regulated through
Kalirin-7 and Tiam1 signaling pathways following NMDA receptor activation and dysregulation
of either pathway could result in changes of glutamatergic dendritic spine dynamics.
RhoGTPase effector proteins were also examined in our biochemical studies. One of
these, Wave1, is a downstream effector of Rac1. Wave1 binds to the Arp2/3 complex and
induces a conformational change and subsequent activation of the complex (Rodal et al., 2005).
Activated Arp2/3 complex promotes branched actin polymerization. We detected significant
deficits in Wave1 protein expression levels at all stages of development in NR1-KD mice. Of all
the proteins investigated, Wave1 appeared to be most drastically affected by NMDA receptor
knockdown. In mice, Wave1 expression is brain-specific; expression is detected in most brain
regions including striatum, cortex and hippocampus (Soderling et al., 2003). Wave1 knockout
mice show hippocampal and cortical spine density deficits of ~20% and alterations in synaptic
plasticity (Soderling et al., 2007). Similarly, significant deficits in spine number were detected
in medium spiny neurons of the striatum (Kim et al., 2006). In addition, Wave1 knockout mice
show reduced anxiety, sensorimotor function, and deficits in learning and memory (Soderling et
al., 2003). Synaptic and behavioral changes detected in Wave1 knockout mice have resemblance
to those detected in NR1-KD mice. Therefore, developmental deficits in Wave1 likely
contribute to the synaptic and behavioral alterations detected in NR1-KD mice.
To further characterize Wave1 in NR1-KD mice, developmental changes in the activation
state of Wave1 need to be investigated. In addition to Rac1, Wave1 activity is regulated by
cyclin-dependent kinase 5 (cdk5). Cdk5 directly binds and phosphorylates Wave1, resulting in
Wave1 inactivation (Kim et al., 2006). Under resting condition, Wave1 is thought to be highly
phosphorylated and inactive; upon neurotransmitter stimulation and subsequent activation of
signaling pathways, Wave1 is believed to be dephosphorylated, activated, and able to promote
actin polymerization (Kim et al., 2006).
60
Similar to Wave1, N-Wasp is able to modulate actin polymerization in an Arp2/3
complex-dependent matter (Hotulainen and Hoogenraad, 2010). N-Wasp activity is generally
modulated by the activity of Cdc42 GTPase. In NR1-KD mice, significant reductions in N-Wasp
and subtle reductions in Cdc42 were detected in post-adolescent mice, the age-onset of spine
density deficits. In adult mice, which show further reductions in spine density, Cdc42 levels
were unaltered and N-Wasp levels showed subtle, non-significant decreases. Therefore,
Cdc42/N-Wasp signaling cascade may play a contributing role in the onset of spine density
reductions, but not in spine loss progression.
In addition to actin polymerization, spine dynamics can be modulated though
disassembly of the actin filament. The LIMK/cofilin signaling pathway regulates actin
depolymerization (Hotulainen and Hoogenraad, 2010; Meng et al., 2003). LIM kinase and
cofilin protein expression levels were fairly unaltered in NR1-KD mice; only subtle decreases in
total cofilin were detected in adult mice. Interestingly, cofilin in the adult NR1-KD striatum was
significantly more phosphorylated. Phosphorylation of cofilin results in cofilin inactivation and
subsequent inhibition of actin disassembly and F-actin stabilization (Hotulainen et al., 2009).
Increase in cofilin phosphorylation would suggest that the activity of LIM kinase might be
enhanced in adult NR1-KD mice. Multiple RhoGTPases have the ability to regulate
LIMK/cofilin signaling pathway, therefore it is difficult to speculate which pathway might be
responsible for the detected changes in cofilin phosphorylation state at this point. Taken
together, the data may suggest that cofilin phosphorylation is part of a compensatory mechanism
to stabilize or slow down the progression of spine loss through the stabilization of actin
filaments.
2 Conclusions
In these studies we aimed to trace the onset and progression of schizophrenia-related
behaviors through critical stages in development, and correlate this progression with changes in
synapse biology (Figure 19). Hyperactivity in NR1-KD mice was detected throughout the
developmental stages. However, deficits in sociability and working memory manifested in
adulthood and did not progress with age; this onset of behaviors in mice parallels the adult-onset
of behavioral symptoms in schizophrenia.
61
Examination of the developmental trajectory of medium spiny neuron spine density in
NR1-KD mice showed that the spine numbers were normal in juvenile mice, reduced in post-
adolescent, and progressively declined in adult mice. Age-dependent deficits in synapse
numbers preceded the schizophrenia-related behavioral endophenotypes, social withdrawal and
impaired working memory. This suggests that the NMDA receptor-mediated changes in synapse
number during the early stages of development could lead to cognitive and behavioral
dysfunction later on.
The molecular mechanism underlying the observed loss in spine number was further
investigated through the examination of protein expression levels of RhoGTPases and their
downstream effectors. In juvenile NR1-KD mice, when spine density is unaltered, Rac1 levels
were elevated while Wave1 levels were reduced. The Rac1/Wave1 signaling pathway promotes
filamentous actin stabilization and assembly. This would suggest that even though Wave1 was
significantly reduced, spine density remained unchanged because the increase in Rac1 levels
compensated for the loss of Wave 1. In post-adolescence, loss of spine density coincided with
deficits in protein levels of Rac1, Wave1, and N-Wasp, molecules involved in promoting
filamentous actin polymerization and stability. The most rapid decrease in spine number
occurred between post-adolescence and early adulthood. As mice aged, spine loss continued but
at a slower rate, suggesting that at this stage of development the system might be trying to
dampen the progression of spine loss. In twelve-week old adult mice, a significant increase in
phosphorylated cofilin was detected in addition to Rac1 and Wave1 deficits. Cofilin promotes
filamentous actin disassembly when active; phosphorylation of cofilin results in inactivation and
inhibition of filamentous actin depolymerization. Therefore, the system could be trying to slow
down the progression of spine loss through modulation of cofilin activity.
62
Figure 19. Proposed model of developmental consequences of NMDA receptor deficiency.
Top: behavioral changes (social interaction and working memory); Middle: synaptic changes
(spine number); Bottom: molecular changes (protein expression levels); Up arrow: increase;
Down arrow: decrease. In juvenile NR1-KD striatum, there are no detectable changes in spine
density, and the balance of actin assembly and disassembly is maintained because increases in
Rac1 offset decreases in Wave1. In post-adolescent NR1-KD striatum, spine loss is first
evident. At the molecular level, the balance of actin assembly and disassembly is shifted
towards disassembly by the combined reductions in Rac1, Wave1, and N-Wasp. In adult NR1-
KD striatum, there are slow, progressive reductions in spine density. At the molecular level, the
balance of actin assembly and disassembly is still weighted towards disassembly; however,
these effects are offset somewhat by increases in phospho-cofilin.
63
3 Recommendations
A number of studies can be done to further characterize the developmental consequences
of NMDA receptor hypofunction on biology of the synapse.
Examine the activity status of Rac1 and its downstream effectors. Our current data suggest
that the Rac1 GTPase signaling pathway could play a role in the NMDA receptor-mediated spine
loss. We know that the total protein levels are altered in an age-dependent matter, but it is
unclear what affect these changes have on the activity of Rac1 and its downstream effectors.
Activity of Rho GTPases could be measured by pulldown of the activated, GTP-bound form of
the protein from tissue extracts. The activation state of downstream effectors could be studied by
examining their phosphorylation state. Furthermore, the levels and activation status of these
signaling molecules could be measured in other brain regions to determine whether or not the
changes that we have detected in the striatum are specific to that region or are seen in other brain
regions as well.
Determine whether behavioral or cellular phenotypes of NR1-KD mice can be reversed by
additional expression of Wave1 or other Rac1 effectors. We have observed a general
reduction in the levels of Rac1 and its effector Wave1. Although, as mentioned above, we do not
know the activation status of these proteins, it is likely that their function is affected. However,
we do not know the extent to which these changes cause the phenotypes that are observed in
NR1-KD mice, or whether these are bystander effects. To determine the causal role that these
effectors play in altered behaviors and spine number, it will be necessary to attempt to rescue the
phenotypes of NR1-KD mice by increasing the levels or activity of these Rac1 signaling
molecules. To achieve this, Wave1 or Rac1 could be delivered by adeno- or lentivirus into
specific brain regions by cannulation, and then animals could be tested behaviorally. After
sacrifice of the animals, spine density of infected neurons could also be determined.
Examine spine density of cortical pyramidal neurons. In addition to the striatum, the
prefrontal cortex plays an important role in regulating the behaviors we have examined. It is
very likely that the behavioral deficits detected in the NR1-KD mice are due to synaptic changes
in both striatum and prefrontal cortex. To study this, spine density of cortical pyramidal neurons
(layer 5) could be examined in a similar manner to what was performed for striatal neurons.
64
Developmental trajectory of NMDA receptor-mediated synaptic transmission (EPSCs). In
adult NR1-KD mice, we know from published data that the NMDA receptor-mediated EPSCs
are significantly reduced in the striatum following cortical stimulation. Whether the onset of
synaptic dysfunction correlates with the onset of the behavioral impairments still needs to be
investigated. It would be of interest to determine whether changes in the electrophysiology of
striatal neurons precedes spine loss, coincides with spine loss, or is only detected after a certain
degree of spine loss has occurred (for example in adult brain).
Determine if the dopamine tone is changed in the highly hyperactive juvenile NR1-KD
mice. Although all of the mice were hyperactive, the most pronounced locomotor activity was
detected in the three week NR1-KD mice. The three-week hyperactivity pattern was similar to
those seen in mice with elevated dopamine levels. Therefore, it would be of interest to determine
whether dopamine neurotransmission is particularly elevated in juvenile mice, and whether the
“normal” parameters of dopamine transmission seen in adults are the result of adaptive
mechanisms to downregulate dopamine signaling. To examine dopamine tone in juvenile mice,
microdialysis could be performed to measure extracellular levels of dopamine, tissue content of
dopamine could be studied by high performance liquid chromatography (HPLC) to measure
stored dopamine levels, and cyclic voltammetry could be performed with slices or in vivo to
measure dopamine release.
65
References
Adriani, W., and Laviola, G. (2004). Windows of vulnerability to psychopathology and
therapeutic strategy in the adolescent rodent model. Behav Pharmacol 15, 341-352.
Aitken, A. (2006). 14-3-3 proteins: a historic overview. Semin Cancer Biol 16, 162-172.
Alaghband-Rad, J., McKenna, K., Gordon, C. T., Albus, K. E., Hamburger, S. D., Rumsey, J.
M., Frazier, J. A., Lenane, M. C., and Rapoport, J. L. (1995). Childhood-onset schizophrenia: the
severity of premorbid course. J Am Acad Child Adolesc Psychiatry 34, 1273-1283.
Amann, L. C., Gandal, M. J., Halene, T. B., Ehrlichman, R. S., White, S. L., McCarren, H. S.,
and Siegel, S. J. (2010). Mouse behavioral endophenotypes for schizophrenia. Brain Res Bull 83,
147-161.
Association, A. P. (2000). Diagnostic and Statistical Manual of Mental Disorders, Vol 4th
edition (Washington, DC).
Barria, A., and Malinow, R. (2005). NMDA receptor subunit composition controls synaptic
plasticity by regulating binding to CaMKII. Neuron 48, 289-301.
Barros, C. S., Calabrese, B., Chamero, P., Roberts, A. J., Korzus, E., Lloyd, K., Stowers, L.,
Mayford, M., Halpain, S., and Muller, U. (2009). Impaired maturation of dendritic spines
without disorganization of cortical cell layers in mice lacking NRG1/ErbB signaling in the
central nervous system. Proc Natl Acad Sci U S A 106, 4507-4512.
Battig, K., Rosvold, H. E., and Mishkin, M. (1960). Comparison of the effects of frontal and
caudate lesions on delayed response and alternation in monkeys. J Comp Physiol Psychol 53,
400-404.
Blanpied, T. A., Scott, D. B., and Ehlers, M. D. (2002). Dynamics and regulation of clathrin
coats at specialized endocytic zones of dendrites and spines. Neuron 36, 435-449.
Bliss, T. V., and Collingridge, G. L. (1993). A synaptic model of memory: long-term
potentiation in the hippocampus. Nature 361, 31-39.
Bourne, J. N., and Harris, K. M. (2008). Balancing structure and function at hippocampal
dendritic spines. Annu Rev Neurosci 31, 47-67.
Brennand, K. J., Simone, A., Jou, J., Gelboin-Burkhart, C., Tran, N., Sangar, S., Li, Y., Mu, Y.,
Chen, G., Yu, D., et al. (2011). Modelling schizophrenia using human induced pluripotent stem
cells. Nature 473, 221-225.
Brigman, J. L., Wright, T., Talani, G., Prasad-Mulcare, S., Jinde, S., Seabold, G. K., Mathur, P.,
Davis, M. I., Bock, R., Gustin, R. M., et al. (2010). Loss of GluN2B-containing NMDA
66
receptors in CA1 hippocampus and cortex impairs long-term depression, reduces dendritic spine
density, and disrupts learning. J Neurosci 30, 4590-4600.
Cahill, M. E., Xie, Z., Day, M., Photowala, H., Barbolina, M. V., Miller, C. A., Weiss, C.,
Radulovic, J., Sweatt, J. D., Disterhoft, J. F., et al. (2009). Kalirin regulates cortical spine
morphogenesis and disease-related behavioral phenotypes. Proc Natl Acad Sci U S A 106,
13058-13063.
Chatterton, J. E., Awobuluyi, M., Premkumar, L. S., Takahashi, H., Talantova, M., Shin, Y., Cui,
J., Tu, S., Sevarino, K. A., Nakanishi, N., et al. (2002). Excitatory glycine receptors containing
the NR3 family of NMDA receptor subunits. Nature 415, 793-798.
Chen, L., and Huang, L. Y. (1992). Protein kinase C reduces Mg2+ block of NMDA-receptor
channels as a mechanism of modulation. Nature 356, 521-523.
Chen, L. Y., Rex, C. S., Casale, M. S., Gall, C. M., and Lynch, G. (2007). Changes in synaptic
morphology accompany actin signaling during LTP. J Neurosci 27, 5363-5372.
Chen, Y. J., Johnson, M. A., Lieberman, M. D., Goodchild, R. E., Schobel, S., Lewandowski, N.,
Rosoklija, G., Liu, R. C., Gingrich, J. A., Small, S., et al. (2008). Type III neuregulin-1 is
required for normal sensorimotor gating, memory-related behaviors, and corticostriatal circuit
components. J Neurosci 28, 6872-6883.
Chumakov, I., Blumenfeld, M., Guerassimenko, O., Cavarec, L., Palicio, M., Abderrahim, H.,
Bougueleret, L., Barry, C., Tanaka, H., La Rosa, P., et al. (2002). Genetic and physiological data
implicating the new human gene G72 and the gene for D-amino acid oxidase in schizophrenia.
Proc Natl Acad Sci U S A 99, 13675-13680.
Ciabarra, A. M., Sullivan, J. M., Gahn, L. G., Pecht, G., Heinemann, S., and Sevarino, K. A.
(1995). Cloning and characterization of chi-1: a developmentally regulated member of a novel
class of the ionotropic glutamate receptor family. J Neurosci 15, 6498-6508.
Cingolani, L. A., and Goda, Y. (2008). Actin in action: the interplay between the actin
cytoskeleton and synaptic efficacy. Nat Rev Neurosci 9, 344-356.
Coulter, C. L., Happe, H. K., and Murrin, L. C. (1997). Dopamine transporter development in
postnatal rat striatum: an autoradiographic study with [3H]WIN 35,428. Brain Res Dev Brain
Res 104, 55-62.
Cull-Candy, S., Brickley, S., and Farrant, M. (2001). NMDA receptor subunits: diversity,
development and disease. Curr Opin Neurobiol 11, 327-335.
Das, S., Sasaki, Y. F., Rothe, T., Premkumar, L. S., Takasu, M., Crandall, J. E., Dikkes, P.,
Conner, D. A., Rayudu, P. V., Cheung, W., et al. (1998). Increased NMDA current and spine
density in mice lacking the NMDA receptor subunit NR3A. Nature 393, 377-381.
Davies, C. A., Mann, D. M., Sumpter, P. Q., and Yates, P. O. (1987). A quantitative
morphometric analysis of the neuronal and synaptic content of the frontal and temporal cortex in
patients with Alzheimer's disease. J Neurol Sci 78, 151-164.
67
Debanne, D., Daoudal, G., Sourdet, V., and Russier, M. (2003). Brain plasticity and ion
channels. J Physiol Paris 97, 403-414.
DeKosky, S. T., and Scheff, S. W. (1990). Synapse loss in frontal cortex biopsies in Alzheimer's
disease: correlation with cognitive severity. Ann Neurol 27, 457-464.
Divac, I., Rosvold, H. E., and Szwarcbart, M. K. (1967). Behavioral effects of selective ablation
of the caudate nucleus. J Comp Physiol Psychol 63, 184-190.
Dunah, A. W., and Standaert, D. G. (2001). Dopamine D1 receptor-dependent trafficking of
striatal NMDA glutamate receptors to the postsynaptic membrane. J Neurosci 21, 5546-5558.
Dunah, A. W., Wang, Y., Yasuda, R. P., Kameyama, K., Huganir, R. L., Wolfe, B. B., and
Standaert, D. G. (2000). Alterations in subunit expression, composition, and phosphorylation of
striatal N-methyl-D-aspartate glutamate receptors in a rat 6-hydroxydopamine model of
Parkinson's disease. Mol Pharmacol 57, 342-352.
Duncan, G. E., Moy, S. S., Lieberman, J. A., and Koller, B. H. (2006). Typical and atypical
antipsychotic drug effects on locomotor hyperactivity and deficits in sensorimotor gating in a
genetic model of NMDA receptor hypofunction. Pharmacol Biochem Behav 85, 481-491.
Duncan, G. E., Moy, S. S., Perez, A., Eddy, D. M., Zinzow, W. M., Lieberman, J. A., Snouwaert,
J. N., and Koller, B. H. (2004). Deficits in sensorimotor gating and tests of social behavior in a
genetic model of reduced NMDA receptor function. Behav Brain Res 153, 507-519.
Dzirasa, K., Ramsey, A. J., Takahashi, D. Y., Stapleton, J., Potes, J. M., Williams, J. K.,
Gainetdinov, R. R., Sameshima, K., Caron, M. G., and Nicolelis, M. A. (2009).
Hyperdopaminergia and NMDA receptor hypofunction disrupt neural phase signaling. J
Neurosci 29, 8215-8224.
Eichenbaum, H. (2004). Hippocampus: cognitive processes and neural representations that
underlie declarative memory. Neuron 44, 109-120.
El-Husseini, A. E., Schnell, E., Chetkovich, D. M., Nicoll, R. A., and Bredt, D. S. (2000). PSD-
95 involvement in maturation of excitatory synapses. Science 290, 1364-1368.
Elman, I., Sigler, M., Kronenberg, J., Lindenmayer, J. P., Doron, A., Mendlovic, S., and Gaoni,
B. (1998). Characteristics of patients with schizophrenia successive to childhood attention deficit
hyperactivity disorder (ADHD). Isr J Psychiatry Relat Sci 35, 280-286.
Etienne-Manneville, S., and Hall, A. (2002). Rho GTPases in cell biology. Nature 420, 629-635.
Fallin, M. D., Lasseter, V. K., Avramopoulos, D., Nicodemus, K. K., Wolyniec, P. S., McGrath,
J. A., Steel, G., Nestadt, G., Liang, K. Y., Huganir, R. L., et al. (2005). Bipolar I disorder and
schizophrenia: a 440-single-nucleotide polymorphism screen of 64 candidate genes among
Ashkenazi Jewish case-parent trios. Am J Hum Genet 77, 918-936.
Fan, M. M., and Raymond, L. A. (2007). N-methyl-D-aspartate (NMDA) receptor function and
excitotoxicity in Huntington's disease. Prog Neurobiol 81, 272-293.
68
Fischer, M., Kaech, S., Wagner, U., Brinkhaus, H., and Matus, A. (2000). Glutamate receptors
regulate actin-based plasticity in dendritic spines. Nat Neurosci 3, 887-894.
Fong, D. K., Rao, A., Crump, F. T., and Craig, A. M. (2002). Rapid synaptic remodeling by
protein kinase C: reciprocal translocation of NMDA receptors and calcium/calmodulin-
dependent kinase II. J Neurosci 22, 2153-2164.
Forrest, D., Yuzaki, M., Soares, H. D., Ng, L., Luk, D. C., Sheng, M., Stewart, C. L., Morgan, J.
I., Connor, J. A., and Curran, T. (1994). Targeted disruption of NMDA receptor 1 gene abolishes
NMDA response and results in neonatal death. Neuron 13, 325-338.
Fujii, Y., Shibata, H., Kikuta, R., Makino, C., Tani, A., Hirata, N., Shibata, A., Ninomiya, H.,
Tashiro, N., and Fukumaki, Y. (2003). Positive associations of polymorphisms in the
metabotropic glutamate receptor type 3 gene (GRM3) with schizophrenia. Psychiatr Genet 13,
71-76.
Fukazawa, Y., Saitoh, Y., Ozawa, F., Ohta, Y., Mizuno, K., and Inokuchi, K. (2003).
Hippocampal LTP is accompanied by enhanced F-actin content within the dendritic spine that is
essential for late LTP maintenance in vivo. Neuron 38, 447-460.
Gainetdinov, R. R., Jones, S. R., and Caron, M. G. (1999). Functional hyperdopaminergia in
dopamine transporter knock-out mice. Biol Psychiatry 46, 303-311.
Gao, X. M., Sakai, K., Roberts, R. C., Conley, R. R., Dean, B., and Tamminga, C. A. (2000).
Ionotropic glutamate receptors and expression of N-methyl-D-aspartate receptor subunits in
subregions of human hippocampus: effects of schizophrenia. Am J Psychiatry 157, 1141-1149.
Garey, L. J., Ong, W. Y., Patel, T. S., Kanani, M., Davis, A., Mortimer, A. M., Barnes, T. R.,
and Hirsch, S. R. (1998). Reduced dendritic spine density on cerebral cortical pyramidal neurons
in schizophrenia. J Neurol Neurosurg Psychiatry 65, 446-453.
Gerber, D. J., Hall, D., Miyakawa, T., Demars, S., Gogos, J. A., Karayiorgou, M., and
Tonegawa, S. (2003). Evidence for association of schizophrenia with genetic variation in the
8p21.3 gene, PPP3CC, encoding the calcineurin gamma subunit. Proc Natl Acad Sci U S A 100,
8993-8998.
Geschwind, D. H., and Levitt, P. (2007). Autism spectrum disorders: developmental
disconnection syndromes. Curr Opin Neurobiol 17, 103-111.
Glantz, L. A., and Lewis, D. A. (2000). Decreased dendritic spine density on prefrontal cortical
pyramidal neurons in schizophrenia. Arch Gen Psychiatry 57, 65-73.
Goley, E. D., and Welch, M. D. (2006). The ARP2/3 complex: an actin nucleator comes of age.
Nat Rev Mol Cell Biol 7, 713-726.
Grosshans, D. R., Clayton, D. A., Coultrap, S. J., and Browning, M. D. (2002). LTP leads to
rapid surface expression of NMDA but not AMPA receptors in adult rat CA1. Nat Neurosci 5,
27-33.
69
Gu, Z., Jiang, Q., Fu, A. K., Ip, N. Y., and Yan, Z. (2005). Regulation of NMDA receptors by
neuregulin signaling in prefrontal cortex. J Neurosci 25, 4974-4984.
Hahn, C. G., Wang, H. Y., Cho, D. S., Talbot, K., Gur, R. E., Berrettini, W. H., Bakshi, K.,
Kamins, J., Borgmann-Winter, K. E., Siegel, S. J., et al. (2006). Altered neuregulin 1-erbB4
signaling contributes to NMDA receptor hypofunction in schizophrenia. Nat Med 12, 824-828.
Halene, T. B., Ehrlichman, R. S., Liang, Y., Christian, E. P., Jonak, G. J., Gur, T. L., Blendy, J.
A., Dow, H. C., Brodkin, E. S., Schneider, F., et al. (2009). Assessment of NMDA receptor NR1
subunit hypofunction in mice as a model for schizophrenia. Genes Brain Behav 8, 661-675.
Hallett, P. J., Dunah, A. W., Ravenscroft, P., Zhou, S., Bezard, E., Crossman, A. R., Brotchie, J.
M., and Standaert, D. G. (2005). Alterations of striatal NMDA receptor subunits associated with
the development of dyskinesia in the MPTP-lesioned primate model of Parkinson's disease.
Neuropharmacology 48, 503-516.
Hallett, P. J., Spoelgen, R., Hyman, B. T., Standaert, D. G., and Dunah, A. W. (2006). Dopamine
D1 activation potentiates striatal NMDA receptors by tyrosine phosphorylation-dependent
subunit trafficking. J Neurosci 26, 4690-4700.
Hantraye, P., Riche, D., Maziere, M., and Isacson, O. (1990). A primate model of Huntington's
disease: behavioral and anatomical studies of unilateral excitotoxic lesions of the caudate-
putamen in the baboon. Exp Neurol 108, 91-104.
Hayashi-Takagi, A., Takaki, M., Graziane, N., Seshadri, S., Murdoch, H., Dunlop, A. J., Makino,
Y., Seshadri, A. J., Ishizuka, K., Srivastava, D. P., et al. (2010). Disrupted-in-Schizophrenia 1
(DISC1) regulates spines of the glutamate synapse via Rac1. Nat Neurosci 13, 327-332.
Hering, H., and Sheng, M. (2003). Activity-dependent redistribution and essential role of
cortactin in dendritic spine morphogenesis. J Neurosci 23, 11759-11769.
Hikosaka, O., Sakamoto, M., and Usui, S. (1989). Functional properties of monkey caudate
neurons. III. Activities related to expectation of target and reward. J Neurophysiol 61, 814-832.
Hill, J. J., Hashimoto, T., and Lewis, D. A. (2006). Molecular mechanisms contributing to
dendritic spine alterations in the prefrontal cortex of subjects with schizophrenia. Mol Psychiatry
11, 557-566.
Hokyo, A., Kanazawa, T., Uenishi, H., Tsutsumi, A., Kawashige, S., Kikuyama, H., Glatt, S. J.,
Koh, J., Nishimoto, Y., Matsumura, H., et al. (2010). Habituation in prepulse inhibition is
affected by a polymorphism on the NMDA receptor 2B subunit gene (GRIN2B). Psychiatr Genet
20, 191-198.
Hotulainen, P., and Hoogenraad, C. C. (2010). Actin in dendritic spines: connecting dynamics to
function. J Cell Biol 189, 619-629.
Hotulainen, P., Llano, O., Smirnov, S., Tanhuanpaa, K., Faix, J., Rivera, C., and Lappalainen, P.
(2009). Defining mechanisms of actin polymerization and depolymerization during dendritic
spine morphogenesis. J Cell Biol 185, 323-339.
70
Hutsler, J. J., and Zhang, H. (2010). Increased dendritic spine densities on cortical projection
neurons in autism spectrum disorders. Brain Res 1309, 83-94.
Hynd, M. R., Scott, H. L., and Dodd, P. R. (2001). Glutamate(NMDA) receptor NR1 subunit
mRNA expression in Alzheimer's disease. J Neurochem 78, 175-182.
Ide, M., and Lewis, D. A. (2010). Altered cortical CDC42 signaling pathways in schizophrenia:
implications for dendritic spine deficits. Biol Psychiatry 68, 25-32.
Ikeda, M., Hikita, T., Taya, S., Uraguchi-Asaki, J., Toyo-oka, K., Wynshaw-Boris, A., Ujike, H.,
Inada, T., Takao, K., Miyakawa, T., et al. (2008). Identification of YWHAE, a gene encoding
14-3-3epsilon, as a possible susceptibility gene for schizophrenia. Hum Mol Genet 17, 3212-
3222.
Javitt, D. C. (2007). Glutamate and schizophrenia: phencyclidine, N-methyl-D-aspartate
receptors, and dopamine-glutamate interactions. Int Rev Neurobiol 78, 69-108.
Javitt, D. C., and Zukin, S. R. (1991). Recent advances in the phencyclidine model of
schizophrenia. Am J Psychiatry 148, 1301-1308.
Jentsch, J. D., and Roth, R. H. (1999). The neuropsychopharmacology of phencyclidine: from
NMDA receptor hypofunction to the dopamine hypothesis of schizophrenia.
Neuropsychopharmacology 20, 201-225.
Jocoy, E. L., Andre, V. M., Cummings, D. M., Rao, S. P., Wu, N., Ramsey, A. J., Caron, M. G.,
Cepeda, C., and Levine, M. S. (2011). Dissecting the contribution of individual receptor subunits
to the enhancement of N-methyl-d-aspartate currents by dopamine D1 receptor activation in
striatum. Front Syst Neurosci 5, 28.
Kantrowitz, J. T., and Javitt, D. C. (2010). N-methyl-d-aspartate (NMDA) receptor dysfunction
or dysregulation: the final common pathway on the road to schizophrenia? Brain Res Bull 83,
108-121.
Karlsgodt, K. H., Sun, D., Jimenez, A. M., Lutkenhoff, E. S., Willhite, R., van Erp, T. G., and
Cannon, T. D. (2008). Developmental disruptions in neural connectivity in the pathophysiology
of schizophrenia. Dev Psychopathol 20, 1297-1327.
Kellendonk, C., Simpson, E. H., and Kandel, E. R. (2009). Modeling cognitive endophenotypes
of schizophrenia in mice. Trends Neurosci 32, 347-358.
Kim, Y., Sung, J. Y., Ceglia, I., Lee, K. W., Ahn, J. H., Halford, J. M., Kim, A. M., Kwak, S. P.,
Park, J. B., Ho Ryu, S., et al. (2006). Phosphorylation of WAVE1 regulates actin polymerization
and dendritic spine morphology. Nature 442, 814-817.
Krystal, J. H., Karper, L. P., Seibyl, J. P., Freeman, G. K., Delaney, R., Bremner, J. D.,
Heninger, G. R., Bowers, M. B., Jr., and Charney, D. S. (1994). Subanesthetic effects of the
noncompetitive NMDA antagonist, ketamine, in humans. Psychotomimetic, perceptual,
cognitive, and neuroendocrine responses. Arch Gen Psychiatry 51, 199-214.
71
Labrie, V., Fukumura, R., Rastogi, A., Fick, L. J., Wang, W., Boutros, P. C., Kennedy, J. L.,
Semeralul, M. O., Lee, F. H., Baker, G. B., et al. (2009). Serine racemase is associated with
schizophrenia susceptibility in humans and in a mouse model. Hum Mol Genet 18, 3227-3243.
Lahti, A. C., Koffel, B., LaPorte, D., and Tamminga, C. A. (1995). Subanesthetic doses of
ketamine stimulate psychosis in schizophrenia. Neuropsychopharmacology 13, 9-19.
Lahti, A. C., Weiler, M. A., Tamara Michaelidis, B. A., Parwani, A., and Tamminga, C. A.
(2001). Effects of ketamine in normal and schizophrenic volunteers. Neuropsychopharmacology
25, 455-467.
Lan, J. Y., Skeberdis, V. A., Jover, T., Grooms, S. Y., Lin, Y., Araneda, R. C., Zheng, X.,
Bennett, M. V., and Zukin, R. S. (2001). Protein kinase C modulates NMDA receptor trafficking
and gating. Nat Neurosci 4, 382-390.
Landis, D. M., and Reese, T. S. (1983). Cytoplasmic organization in cerebellar dendritic spines. J
Cell Biol 97, 1169-1178.
Landles, C., and Bates, G. P. (2004). Huntingtin and the molecular pathogenesis of Huntington's
disease. Fourth in molecular medicine review series. EMBO Rep 5, 958-963.
Lau, C. G., and Zukin, R. S. (2007). NMDA receptor trafficking in synaptic plasticity and
neuropsychiatric disorders. Nat Rev Neurosci 8, 413-426.
Laurie, D. J., and Seeburg, P. H. (1994). Regional and developmental heterogeneity in splicing
of the rat brain NMDAR1 mRNA. J Neurosci 14, 3180-3194.
Lavezzari, G., McCallum, J., Dewey, C. M., and Roche, K. W. (2004). Subunit-specific
regulation of NMDA receptor endocytosis. J Neurosci 24, 6383-6391.
Lee, F. H., Fadel, M. P., Preston-Maher, K., Cordes, S. P., Clapcote, S. J., Price, D. J., Roder, J.
C., and Wong, A. H. (2011). Disc1 point mutations in mice affect development of the cerebral
cortex. J Neurosci 31, 3197-3206.
Leuner, B., Falduto, J., and Shors, T. J. (2003). Associative memory formation increases the
observation of dendritic spines in the hippocampus. J Neurosci 23, 659-665.
Li, B., Otsu, Y., Murphy, T. H., and Raymond, L. A. (2003). Developmental decrease in NMDA
receptor desensitization associated with shift to synapse and interaction with postsynaptic
density-95. J Neurosci 23, 11244-11254.
Li, Y., Erzurumlu, R. S., Chen, C., Jhaveri, S., and Tonegawa, S. (1994). Whisker-related
neuronal patterns fail to develop in the trigeminal brainstem nuclei of NMDAR1 knockout mice.
Cell 76, 427-437.
Li, Z., Aizenman, C. D., and Cline, H. T. (2002). Regulation of rho GTPases by crosstalk and
neuronal activity in vivo. Neuron 33, 741-750.
72
Lin, Y., Skeberdis, V. A., Francesconi, A., Bennett, M. V., and Zukin, R. S. (2004). Postsynaptic
density protein-95 regulates NMDA channel gating and surface expression. J Neurosci 24,
10138-10148.
Lipton, S. A. (2005). The molecular basis of memantine action in Alzheimer's disease and other
neurologic disorders: low-affinity, uncompetitive antagonism. Curr Alzheimer Res 2, 155-165.
Liu, H., Heath, S. C., Sobin, C., Roos, J. L., Galke, B. L., Blundell, M. L., Lenane, M.,
Robertson, B., Wijsman, E. M., Rapoport, J. L., et al. (2002). Genetic variation at the 22q11
PRODH2/DGCR6 locus presents an unusual pattern and increases susceptibility to
schizophrenia. Proc Natl Acad Sci U S A 99, 3717-3722.
Loschmann, P. A., De Groote, C., Smith, L., Wullner, U., Fischer, G., Kemp, J. A., Jenner, P.,
and Klockgether, T. (2004). Antiparkinsonian activity of Ro 25-6981, a NR2B subunit specific
NMDA receptor antagonist, in animal models of Parkinson's disease. Exp Neurol 187, 86-93.
Losi, G., Prybylowski, K., Fu, Z., Luo, J., Wenthold, R. J., and Vicini, S. (2003). PSD-95
regulates NMDA receptors in developing cerebellar granule neurons of the rat. J Physiol 548, 21-
29.
Luo, L., Hensch, T. K., Ackerman, L., Barbel, S., Jan, L. Y., and Jan, Y. N. (1996). Differential
effects of the Rac GTPase on Purkinje cell axons and dendritic trunks and spines. Nature 379,
837-840.
Ma, X. M., Kiraly, D. D., Gaier, E. D., Wang, Y., Kim, E. J., Levine, E. S., Eipper, B. A., and
Mains, R. E. (2008). Kalirin-7 is required for synaptic structure and function. J Neurosci 28,
12368-12382.
Malhotra, A. K., Pinals, D. A., Adler, C. M., Elman, I., Clifton, A., Pickar, D., and Breier, A.
(1997). Ketamine-induced exacerbation of psychotic symptoms and cognitive impairment in
neuroleptic-free schizophrenics. Neuropsychopharmacology 17, 141-150.
Mandillo, S., Tucci, V., Holter, S. M., Meziane, H., Banchaabouchi, M. A., Kallnik, M., Lad, H.
V., Nolan, P. M., Ouagazzal, A. M., Coghill, E. L., et al. (2008). Reliability, robustness, and
reproducibility in mouse behavioral phenotyping: a cross-laboratory study. Physiol Genomics 34,
243-255.
Marcotte, E. R., Pearson, D. M., and Srivastava, L. K. (2001). Animal models of schizophrenia:
a critical review. J Psychiatry Neurosci 26, 395-410.
Marsh, P. J., and Williams, L. M. (2006). ADHD and schizophrenia phenomenology: visual
scanpaths to emotional faces as a potential psychophysiological marker? Neurosci Biobehav Rev
30, 651-665.
Martucci, L., Wong, A. H., De Luca, V., Likhodi, O., Wong, G. W., King, N., and Kennedy, J.
L. (2006). N-methyl-D-aspartate receptor NR2B subunit gene GRIN2B in schizophrenia and
bipolar disorder: Polymorphisms and mRNA levels. Schizophr Res 84, 214-221.
73
Massey, P. V., and Bashir, Z. I. (2007). Long-term depression: multiple forms and implications
for brain function. Trends Neurosci 30, 176-184.
Mattson, M. P. (2008). Glutamate and neurotrophic factors in neuronal plasticity and disease.
Ann N Y Acad Sci 1144, 97-112.
Meng, Y., Zhang, Y., Tregoubov, V., Falls, D. L., and Jia, Z. (2003). Regulation of spine
morphology and synaptic function by LIMK and the actin cytoskeleton. Rev Neurosci 14, 233-
240.
Meng, Y., Zhang, Y., Tregoubov, V., Janus, C., Cruz, L., Jackson, M., Lu, W. Y., MacDonald, J.
F., Wang, J. Y., Falls, D. L., and Jia, Z. (2002). Abnormal spine morphology and enhanced LTP
in LIMK-1 knockout mice. Neuron 35, 121-133.
Messias, E. L., Chen, C. Y., and Eaton, W. W. (2007). Epidemiology of schizophrenia: review of
findings and myths. Psychiatr Clin North Am 30, 323-338.
Middleton, F. A., Peng, L., Lewis, D. A., Levitt, P., and Mirnics, K. (2005). Altered expression
of 14-3-3 genes in the prefrontal cortex of subjects with schizophrenia.
Neuropsychopharmacology 30, 974-983.
Mirnics, K., Middleton, F. A., Lewis, D. A., and Levitt, P. (2001). Analysis of complex brain
disorders with gene expression microarrays: schizophrenia as a disease of the synapse. Trends
Neurosci 24, 479-486.
Mohn, A. R., Gainetdinov, R. R., Caron, M. G., and Koller, B. H. (1999). Mice with reduced
NMDA receptor expression display behaviors related to schizophrenia. Cell 98, 427-436.
Monyer, H., Burnashev, N., Laurie, D. J., Sakmann, B., and Seeburg, P. H. (1994).
Developmental and regional expression in the rat brain and functional properties of four NMDA
receptors. Neuron 12, 529-540.
Morishita, W., Marie, H., and Malenka, R. C. (2005). Distinct triggering and expression
mechanisms underlie LTD of AMPA and NMDA synaptic responses. Nat Neurosci 8, 1043-
1050.
Moy, S. S., Nadler, J. J., Perez, A., Barbaro, R. P., Johns, J. M., Magnuson, T. R., Piven, J., and
Crawley, J. N. (2004). Sociability and preference for social novelty in five inbred strains: an
approach to assess autistic-like behavior in mice. Genes Brain Behav 3, 287-302.
Nakazawa, T., Kuriu, T., Tezuka, T., Umemori, H., Okabe, S., and Yamamoto, T. (2008).
Regulation of dendritic spine morphology by an NMDA receptor-associated Rho GTPase-
activating protein, p250GAP. J Neurochem 105, 1384-1393.
Nakazawa, T., Watabe, A. M., Tezuka, T., Yoshida, Y., Yokoyama, K., Umemori, H., Inoue, A.,
Okabe, S., Manabe, T., and Yamamoto, T. (2003). p250GAP, a novel brain-enriched GTPase-
activating protein for Rho family GTPases, is involved in the N-methyl-d-aspartate receptor
signaling. Mol Biol Cell 14, 2921-2934.
74
Nash, J. E., Fox, S. H., Henry, B., Hill, M. P., Peggs, D., McGuire, S., Maneuf, Y., Hille, C.,
Brotchie, J. M., and Crossman, A. R. (2000). Antiparkinsonian actions of ifenprodil in the
MPTP-lesioned marmoset model of Parkinson's disease. Exp Neurol 165, 136-142.
Nimchinsky, E. A., Sabatini, B. L., and Svoboda, K. (2002). Structure and function of dendritic
spines. Annu Rev Physiol 64, 313-353.
O'Brien, J. A., and Lummis, S. C. (2006). Diolistic labeling of neuronal cultures and intact tissue
using a hand-held gene gun. Nat Protoc 1, 1517-1521.
O'Connor, J. J., Rowan, M. J., and Anwyl, R. (1994). Long-lasting enhancement of NMDA
receptor-mediated synaptic transmission by metabotropic glutamate receptor activation. Nature
367, 557-559.
Ohtsuki, T., Sakurai, K., Dou, H., Toru, M., Yamakawa-Kobayashi, K., and Arinami, T. (2001).
Mutation analysis of the NMDAR2B (GRIN2B) gene in schizophrenia. Mol Psychiatry 6, 211-
216.
Okamoto, K., Nagai, T., Miyawaki, A., and Hayashi, Y. (2004). Rapid and persistent modulation
of actin dynamics regulates postsynaptic reorganization underlying bidirectional plasticity. Nat
Neurosci 7, 1104-1112.
Oray, S., Majewska, A., and Sur, M. (2006). Effects of synaptic activity on dendritic spine
motility of developing cortical layer v pyramidal neurons. Cereb Cortex 16, 730-741.
Penzes, P., Cahill, M. E., Jones, K. A., VanLeeuwen, J. E., and Woolfrey, K. M. (2011).
Dendritic spine pathology in neuropsychiatric disorders. Nat Neurosci 14, 285-293.
Pilowsky, L. S., Bressan, R. A., Stone, J. M., Erlandsson, K., Mulligan, R. S., Krystal, J. H., and
Ell, P. J. (2006). First in vivo evidence of an NMDA receptor deficit in medication-free
schizophrenic patients. Mol Psychiatry 11, 118-119.
Rampon, C., Tang, Y. P., Goodhouse, J., Shimizu, E., Kyin, M., and Tsien, J. Z. (2000).
Enrichment induces structural changes and recovery from nonspatial memory deficits in CA1
NMDAR1-knockout mice. Nat Neurosci 3, 238-244.
Ramsey, A. J. (2009). NR1 knockdown mice as a representative model of the glutamate
hypothesis of schizophrenia. Prog Brain Res 179, 51-58.
Ramsey, A. J., Laakso, A., Cyr, M., Sotnikova, T. D., Salahpour, A., Medvedev, I. O., Dykstra,
L. A., Gainetdinov, R. R., and Caron, M. G. (2008). Genetic NMDA receptor deficiency disrupts
acute and chronic effects of cocaine but not amphetamine. Neuropsychopharmacology 33, 2701-
2714.
Ramsey, A. J., Milenkovic, M., Oliveira, A. F., Escobedo-Lozoya, Y., Seshadri, S., Salahpour,
A., Sawa, A., Yasuda, R., and Caron, M. G. (2011). Impaired NMDA receptor transmission
alters striatal synapses and DISC1 protein in an age-dependent manner. Proc Natl Acad Sci U S
A 108, 5795-5800.
75
Rapoport, J. L., Addington, A. M., Frangou, S., and Psych, M. R. (2005). The
neurodevelopmental model of schizophrenia: update 2005. Mol Psychiatry 10, 434-449.
Roche, K. W., Standley, S., McCallum, J., Dune Ly, C., Ehlers, M. D., and Wenthold, R. J.
(2001). Molecular determinants of NMDA receptor internalization. Nat Neurosci 4, 794-802.
Rodal, A. A., Sokolova, O., Robins, D. B., Daugherty, K. M., Hippenmeyer, S., Riezman, H.,
Grigorieff, N., and Goode, B. L. (2005). Conformational changes in the Arp2/3 complex leading
to actin nucleation. Nat Struct Mol Biol 12, 26-31.
Rogers, R. D., Andrews, T. C., Grasby, P. M., Brooks, D. J., and Robbins, T. W. (2000).
Contrasting cortical and subcortical activations produced by attentional-set shifting and reversal
learning in humans. J Cogn Neurosci 12, 142-162.
Ryu, J., Liu, L., Wong, T. P., Wu, D. C., Burette, A., Weinberg, R., Wang, Y. T., and Sheng, M.
(2006). A critical role for myosin IIb in dendritic spine morphology and synaptic function.
Neuron 49, 175-182.
Samii, A., Nutt, J. G., and Ransom, B. R. (2004). Parkinson's disease. Lancet 363, 1783-1793.
Saneyoshi, T., Fortin, D. A., and Soderling, T. R. (2010). Regulation of spine and synapse
formation by activity-dependent intracellular signaling pathways. Curr Opin Neurobiol 20, 108-
115.
Scott, D. B., Blanpied, T. A., and Ehlers, M. D. (2003). Coordinated PKA and PKC
phosphorylation suppresses RXR-mediated ER retention and regulates the surface delivery of
NMDA receptors. Neuropharmacology 45, 755-767.
Segal, D. S., and Mandell, A. J. (1974). Long-term administration of d-amphetamine:
progressive augmentation of motor activity and stereotypy. Pharmacol Biochem Behav 2, 249-
255.
Sekino, Y., Kojima, N., and Shirao, T. (2007). Role of actin cytoskeleton in dendritic spine
morphogenesis. Neurochem Int 51, 92-104.
Selkoe, D. J. (2002). Alzheimer's disease is a synaptic failure. Science 298, 789-791.
Shankar, G. M., Bloodgood, B. L., Townsend, M., Walsh, D. M., Selkoe, D. J., and Sabatini, B.
L. (2007). Natural oligomers of the Alzheimer amyloid-beta protein induce reversible synapse
loss by modulating an NMDA-type glutamate receptor-dependent signaling pathway. J Neurosci
27, 2866-2875.
Shipp, S. (2007). Structure and function of the cerebral cortex. Curr Biol 17, R443-449.
Simpson, E. H., Kellendonk, C., and Kandel, E. (2010). A possible role for the striatum in the
pathogenesis of the cognitive symptoms of schizophrenia. Neuron 65, 585-596.
76
Snyder, E. M., Nong, Y., Almeida, C. G., Paul, S., Moran, T., Choi, E. Y., Nairn, A. C., Salter,
M. W., Lombroso, P. J., Gouras, G. K., and Greengard, P. (2005). Regulation of NMDA receptor
trafficking by amyloid-beta. Nat Neurosci 8, 1051-1058.
Snyder, E. M., Philpot, B. D., Huber, K. M., Dong, X., Fallon, J. R., and Bear, M. F. (2001).
Internalization of ionotropic glutamate receptors in response to mGluR activation. Nat Neurosci
4, 1079-1085.
Soderling, S. H., Guire, E. S., Kaech, S., White, J., Zhang, F., Schutz, K., Langeberg, L. K.,
Banker, G., Raber, J., and Scott, J. D. (2007). A WAVE-1 and WRP signaling complex regulates
spine density, synaptic plasticity, and memory. J Neurosci 27, 355-365.
Soderling, S. H., Langeberg, L. K., Soderling, J. A., Davee, S. M., Simerly, R., Raber, J., and
Scott, J. D. (2003). Loss of WAVE-1 causes sensorimotor retardation and reduced learning and
memory in mice. Proc Natl Acad Sci U S A 100, 1723-1728.
Sorra, K. E., and Harris, K. M. (2000). Overview on the structure, composition, function,
development, and plasticity of hippocampal dendritic spines. Hippocampus 10, 501-511.
Spencer, E. K., and Campbell, M. (1994). Children with schizophrenia: diagnosis,
phenomenology, and pharmacotherapy. Schizophr Bull 20, 713-725.
Stahl, S. M. (2007). Beyond the dopamine hypothesis to the NMDA glutamate receptor
hypofunction hypothesis of schizophrenia. CNS Spectr 12, 265-268.
Starling, A. J., Andre, V. M., Cepeda, C., de Lima, M., Chandler, S. H., and Levine, M. S.
(2005). Alterations in N-methyl-D-aspartate receptor sensitivity and magnesium blockade occur
early in development in the R6/2 mouse model of Huntington's disease. J Neurosci Res 82, 377-
386.
Stefansson, H., Sigurdsson, E., Steinthorsdottir, V., Bjornsdottir, S., Sigmundsson, T., Ghosh, S.,
Brynjolfsson, J., Gunnarsdottir, S., Ivarsson, O., Chou, T. T., et al. (2002). Neuregulin 1 and
susceptibility to schizophrenia. Am J Hum Genet 71, 877-892.
Sucher, N. J., Akbarian, S., Chi, C. L., Leclerc, C. L., Awobuluyi, M., Deitcher, D. L., Wu, M.
K., Yuan, J. P., Jones, E. G., and Lipton, S. A. (1995). Developmental and regional expression
pattern of a novel NMDA receptor-like subunit (NMDAR-L) in the rodent brain. J Neurosci 15,
6509-6520.
Talbot, K., Eidem, W. L., Tinsley, C. L., Benson, M. A., Thompson, E. W., Smith, R. J., Hahn,
C. G., Siegel, S. J., Trojanowski, J. Q., Gur, R. E., et al. (2004). Dysbindin-1 is reduced in
intrinsic, glutamatergic terminals of the hippocampal formation in schizophrenia. J Clin Invest
113, 1353-1363.
Tashiro, A., Minden, A., and Yuste, R. (2000). Regulation of dendritic spine morphology by the
rho family of small GTPases: antagonistic roles of Rac and Rho. Cereb Cortex 10, 927-938.
77
Taya, S., Shinoda, T., Tsuboi, D., Asaki, J., Nagai, K., Hikita, T., Kuroda, S., Kuroda, K.,
Shimizu, M., Hirotsune, S., et al. (2007). DISC1 regulates the transport of the NUDEL/LIS1/14-
3-3epsilon complex through kinesin-1. J Neurosci 27, 15-26.
Tepper, J. M., and Bolam, J. P. (2004). Functional diversity and specificity of neostriatal
interneurons. Curr Opin Neurobiol 14, 685-692.
Thompson, P. M., Vidal, C., Giedd, J. N., Gochman, P., Blumenthal, J., Nicolson, R., Toga, A.
W., and Rapoport, J. L. (2001). Mapping adolescent brain change reveals dynamic wave of
accelerated gray matter loss in very early-onset schizophrenia. Proc Natl Acad Sci U S A 98,
11650-11655.
Tingley, W. G., Ehlers, M. D., Kameyama, K., Doherty, C., Ptak, J. B., Riley, C. T., and
Huganir, R. L. (1997). Characterization of protein kinase A and protein kinase C
phosphorylation of the N-methyl-D-aspartate receptor NR1 subunit using phosphorylation site-
specific antibodies. J Biol Chem 272, 5157-5166.
Tolias, K. F., Bikoff, J. B., Burette, A., Paradis, S., Harrar, D., Tavazoie, S., Weinberg, R. J., and
Greenberg, M. E. (2005). The Rac1-GEF Tiam1 couples the NMDA receptor to the activity-
dependent development of dendritic arbors and spines. Neuron 45, 525-538.
Torres, G. E., Gainetdinov, R. R., and Caron, M. G. (2003). Plasma membrane monoamine
transporters: structure, regulation and function. Nat Rev Neurosci 4, 13-25.
Uitti, R. J., Rajput, A. H., Ahlskog, J. E., Offord, K. P., Schroeder, D. R., Ho, M. M., Prasad, M.,
Rajput, A., and Basran, P. (1996). Amantadine treatment is an independent predictor of
improved survival in Parkinson's disease. Neurology 46, 1551-1556.
Ultanir, S. K., Kim, J. E., Hall, B. J., Deerinck, T., Ellisman, M., and Ghosh, A. (2007).
Regulation of spine morphology and spine density by NMDA receptor signaling in vivo. Proc
Natl Acad Sci U S A 104, 19553-19558.
van Galen, E. J., and Ramakers, G. J. (2005). Rho proteins, mental retardation and the
neurobiological basis of intelligence. Prog Brain Res 147, 295-317.
Vonsattel, J. P., and DiFiglia, M. (1998). Huntington disease. J Neuropathol Exp Neurol 57, 369-
384.
Walsh, D. M., and Selkoe, D. J. (2004). Deciphering the molecular basis of memory failure in
Alzheimer's disease. Neuron 44, 181-193.
Waltereit, R., and Weller, M. (2003). Signaling from cAMP/PKA to MAPK and synaptic
plasticity. Mol Neurobiol 27, 99-106.
Wayman, G. A., Davare, M., Ando, H., Fortin, D., Varlamova, O., Cheng, H. Y., Marks, D.,
Obrietan, K., Soderling, T. R., Goodman, R. H., and Impey, S. (2008). An activity-regulated
microRNA controls dendritic plasticity by down-regulating p250GAP. Proc Natl Acad Sci U S A
105, 9093-9098.
78
Wegner, A. M., Nebhan, C. A., Hu, L., Majumdar, D., Meier, K. M., Weaver, A. M., and Webb,
D. J. (2008). N-wasp and the arp2/3 complex are critical regulators of actin in the development
of dendritic spines and synapses. J Biol Chem 283, 15912-15920.
Weickert, C. S., Straub, R. E., McClintock, B. W., Matsumoto, M., Hashimoto, R., Hyde, T. M.,
Herman, M. M., Weinberger, D. R., and Kleinman, J. E. (2004). Human dysbindin (DTNBP1)
gene expression in normal brain and in schizophrenic prefrontal cortex and midbrain. Arch Gen
Psychiatry 61, 544-555.
Wong, A. H., Macciardi, F., Klempan, T., Kawczynski, W., Barr, C. L., Lakatoo, S., Wong, M.,
Buckle, C., Trakalo, J., Boffa, E., et al. (2003). Identification of candidate genes for psychosis in
rat models, and possible association between schizophrenia and the 14-3-3eta gene. Mol
Psychiatry 8, 156-166.
Xie, Z., Srivastava, D. P., Photowala, H., Kai, L., Cahill, M. E., Woolfrey, K. M., Shum, C. Y.,
Surmeier, D. J., and Penzes, P. (2007). Kalirin-7 controls activity-dependent structural and
functional plasticity of dendritic spines. Neuron 56, 640-656.
Xiong, Z. G., Raouf, R., Lu, W. Y., Wang, L. Y., Orser, B. A., Dudek, E. M., Browning, M. D.,
and MacDonald, J. F. (1998). Regulation of N-methyl-D-aspartate receptor function by
constitutively active protein kinase C. Mol Pharmacol 54, 1055-1063.
Yoshihara, Y., De Roo, M., and Muller, D. (2009). Dendritic spine formation and stabilization.
Curr Opin Neurobiol 19, 146-153.
Young, A. B., Greenamyre, J. T., Hollingsworth, Z., Albin, R., D'Amato, C., Shoulson, I., and
Penney, J. B. (1988). NMDA receptor losses in putamen from patients with Huntington's disease.
Science 241, 981-983.
79
List of Publications and Abstracts
Publication
Ramsey, A. J., M. Milenkovic, et al. (2011). "Impaired NMDA receptor transmission alters
striatal synapses and DISC1 protein in an age-dependent manner." Proc Natl Acad Sci U S A
108(14): 5795-800.
Abstract
Marija Milenkovic, Ana Oliveira, Yasmin Escobedo-Loyoza, Ryohei Yasuda and Amy J.
Ramsey (2010). “Developmental Trajectory of Synaptic Changes in a Mouse Model of
Schizophrenia”. Canadian Neuroscience Meeting, Ottawa, Canada.
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