The committee for Noelle Catherine Anastasio certifies that this is the approved
version of the following dissertation:
Effects of Perinatal Phencyclidine Treatment on Neuronal
Viability, N-Methyl-D-Aspartate Receptor Regulation and
Schizophrenic-like Behaviors
Committee:
Kenneth M. Johnson, Ph.D. ,Supervisor
Kathryn A. Cunningham, Ph.D.
Kelly T. Dineley, Ph.D.
Joel P. Gallagher, Ph.D.
Geoffrey T. Swanson, Ph.D.
______________________________
Dean, Graduate School
Effects of Perinatal Phencyclidine Treatment on Neuronal
Viability, N-Methyl-D-Aspartate Receptor Regulation and
Schizophrenic-like Behaviors
By
Noelle Catherine Anastasio, M.S.
Dissertation
Presented to the Faculty of the Graduate School of Biomedical Sciences of
The University of Texas Medical Branch at Galveston
in Partial Fulfillment of the Requirements
for the Degree of
Doctor of Philosophy
The University of Texas Medical Branch
March, 2009
for my grandfather
Acknowledgements
I would like to express my gratitude to my advisor, Dr. Ken Johnson, for
his support and encouragement. He pushed and challenged me to always do my
best and his trust in me allowed me to develop in to the independent scientist
that I am today. I wish to also thank the members of my committee, Drs. Kathryn
Cunningham, Kelly Dineley, Joel Gallagher and Geoffrey Swanson for their
expertise and advice, both in and out of the laboratory. This work was supported
by a Ruth L. Kirschstein NRSA F31 Fellowship from the National Institute on
Drug Abuse.
I would also like to thank Dr. Cheng Z. Wang and Dr. Gang Lei for their
technical expertise and countless scientific discussions concerning not only my
studies, but the field of schizophrenia animal models as well. I must of course
mention Yan Xia, aka “Sunshine”; with her there was never a dull moment in the
lab. Her ability to make me laugh and her constant support and encouragement
made coming to work a joy. I must also thank Dr. Marcy Jordan for her
friendship, ability to answer any question I had about the inner workings of
UTMB, the endless science discussions, and most importantly, for always being
there when I needed her most.
On a personal note, I must thank Tad Conaty for his love, encouragement
and patience throughout this process. To my parents, thank you for nurturing the
inner scientist in me as a child by encouraging me to always ask questions and
when the answer was unknown to discover a way to answer it myself. I must
also thank you for being my biggest fans and for supporting me every step of the
way as I pursued a higher education. A special thanks to my sister, Bethany,
who has taught me to not take it all so seriously.
iv
Effects of Perinatal Phencyclidine Treatment on Neuronal
Viability, N-Methyl-D-Aspartate Receptor Regulation and
Schizophrenic-like Behaviors
Publication No._____________
Noelle Catherine Anastasio, Ph.D.
The University of Texas Medical Branch, 2009
Supervisor: Kenneth M. Johnson
Clinical use of phencyclidine (PCP), a potent dissociative anesthetic, was
abandoned as a result of reports of post-operative hallucinations and disoriented
behavior; further, illicit use has substantially diminished because of its
psychotomimetic properties. PCP intoxication in humans has also been shown
to mimic both the positive and negative symptoms of schizophrenia as well as
exacerbate psychoses in schizophrenics. PCP elicits its major actions as a
noncompetitive NMDA receptor (NMDAR) antagonist.
Administration to
immature rats has been shown to cause neurotoxicity in a regional and treatment
dependent manner and development of schizophrenic-like behaviors later in life.
The purpose of this study is to determine the mechanism and functional
consequences of PCP-induced regulation of the NMDAR in association with
neurotoxicity in the frontal cortex and if there is a causal relationship between cell
death and the development of aberrant behaviors in perinatal rats. The first
specific aim of this project will focus on delineating the mechanisms of regulation
of the NMDAR, specifically the role of synthesis and trafficking of the receptor
following acute and sub-chronic PCP administration. Specific aim 2 is designed
to determine the functionality of the cortical NMDAR following both acute and
sub-chronic PCP treatment by assessing NMDA- and glycine-dependent
activation of 3H-MK-801 binding. The subunit composition of the NMDAR that
mediates PCP-induced neuronal neurotoxicity is currently unknown; therefore,
we designed specific aim 3 to show that the synaptic or extrasynaptic localization
of NMDA NR2A and 2B receptors may dictate function and neuronal
susceptibility to cell death. Finally, the purpose of specific aim 4 is to
demonstrate that neurotoxicity underlies the development of behaviors which
model several aspects of schizophrenia, including the positive symptoms
(locomotor sensitization), the negative symptoms (social interaction and social
discrimination) and deficits in sensorimotor gating (PPI of acoustic startle). Work
v
within this dissertation discovered that PCP induces neurotoxicity in developing
pups in a manner that is highly regulated affecting the distribution, composition,
number, and function of NMDAR and that this loss of cortical neurons and altered
cortical landscape is associated with behavioral deficits that are similar to both
the positive and negative symptoms of schizophrenia.
vi
Table of Contents
List of Tables ..................................................................................................... xi
List of Figures ................................................................................................... xii
List of Figures ................................................................................................... xii
List of Abbreviations ........................................................................................ xv
Chapter 1: Introduction ...................................................................................... 1
Phencyclidine (PCP) and noncompetitive NMDA receptor antagonists ......... 1
The NMDA Receptor ..................................................................................... 3
Schizophrenia................................................................................................ 4
Pathophysiology of schizophrenia ................................................................. 5
NMDAR-mediated neurotoxicity and aberrant behaviors in rodent models of
schizophrenia ................................................................................................ 7
Hypothesis 1 .............................................................................................. 12
Specific Aim 1a ............................................................................................ 13
Specific Aim 1b ............................................................................................ 14
Hypothesis 2. ............................................................................................. 14
Specific Aim 2 .............................................................................................. 15
Hypothesis 3 .............................................................................................. 16
Specific Aim 3a ............................................................................................ 16
Specific Aim 3b ............................................................................................ 17
Specific Aim 3c ............................................................................................ 17
Hypothesis 4 .............................................................................................. 18
Specific Aim 4a ............................................................................................ 18
Specific Aim 4b ............................................................................................ 19
vii
Chapter 2: NR2A-Containing Synaptic NMDARs Mediate PCP-Induced
Cortical Neurotoxicity and the Subsequent Development of Schizophrenialike Behaviors ................................................................................................... 20
Introduction ............................................................................................... 20
Materials and Methods .............................................................................. 23
Animals .................................................................................................... 23
Drugs. ...................................................................................................... 23
Organotypic slice culture.......................................................................... 24
In vivo experimental design. .................................................................... 24
Terminal dUTP Nick-End Labeling (TUNEL)............................................ 25
Caspase-3 Activity. .................................................................................. 25
Co-Immunoprecipitation and Western Blotting. ........................................ 26
Antibodies. ............................................................................................... 27
Locomotor Activity. .................................................................................. 28
Statistical Analysis ................................................................................... 28
Results ....................................................................................................... 28
Discussion ................................................................................................. 41
Chapter 3: Differential Regulation of the NMDAR by Acute and Sub-chronic
Phencyclidine Administration ......................................................................... 48
Introduction ............................................................................................... 49
Materials and methods.............................................................................. 51
Animals. ................................................................................................... 51
Drugs. ...................................................................................................... 51
Experimental design. ............................................................................... 52
Sub-cellular fractionation. ........................................................................ 52
Western blot analysis. .............................................................................. 53
Antibodies. ............................................................................................... 53
Results ....................................................................................................... 54
Discussion ................................................................................................. 63
Chapter 4: Alterations in NMDAR Binding of 3H-MK-801 in Response to
Postnatal PCP Administration ......................................................................... 67
Introduction ............................................................................................... 68
viii
Materials and methods.............................................................................. 71
Animals. ................................................................................................... 71
Drugs. ...................................................................................................... 71
Experimental design. ............................................................................... 72
3H-MK-801 binding. .................................................................................. 72
Results ....................................................................................................... 74
Discussion ................................................................................................. 82
Chapter 5: Atypical Antischizophrenic Drugs Prevent Changes in Cortical
NMDARs and Behavior Following Sub-chronic PCP Administration .......... 86
Introduction ............................................................................................... 86
Materials and Methods .............................................................................. 89
Animals. ................................................................................................... 89
Drugs. ...................................................................................................... 89
Experimental design. ............................................................................... 90
Sub-cellular fractionation. ........................................................................ 90
Western blot analysis. .............................................................................. 91
Antibodies. ............................................................................................... 92
Pre-pulse inhibition (PPI) of acoustic startle. ........................................... 92
Locomotor Activity. .................................................................................. 92
Statistical analysis. .................................................................................. 93
Results ....................................................................................................... 93
Discussion ............................................................................................... 106
Chapter 6: Modeling the Negative Symptoms of Schizophrenia ............... 113
Introduction ............................................................................................. 113
Materials and Methods ............................................................................ 116
Animals. ................................................................................................. 116
Drugs. .................................................................................................... 116
Experimental design. ............................................................................. 116
Social Interaction Test. .......................................................................... 117
Social Discrimination Task. .................................................................... 117
Statistical Analysis. ................................................................................ 118
ix
Results ..................................................................................................... 118
Discussion ............................................................................................... 123
Chapter 7: Conclusions and Future Directions .......................................... 127
General Conclusions............................................................................... 127
Specific Aim 1 ............................................................................................ 130
Specific Aim 2 ............................................................................................ 134
Specific Aim 3 ............................................................................................ 135
Specific Aim 4 ............................................................................................ 138
Future Directions ..................................................................................... 139
References ...................................................................................................... 144
x
List of Tables
Table 5.1 Effects of PCP (10 mg/kg) on weight (g) gain in developing rats ..... 94
xi
List of Figures
Figure 2.1. Representative photomicrographs showing TUNEL-positive staining
in corticostriatal organotypic slice cultures. ........................................................ 30
Figure 2.2. Effects of PCP administration on neurotoxicity in corticostriatal slice
cultures. .............................................................................................................. 31
Figure 2.3. Effects of neonatal PCP and selective NR2A and NR2B antagonists
on frontal cortex neurotoxicity in PN7 rats. ......................................................... 32
Figure 2.4. Effects of neonatal PCP and selective NR2A and NR2B antagonists
on striatal neurotoxicity in PN7 rats. ................................................................... 33
Figure 2.5. Effects of neonatal PCP and selective NR2A and NR2B antagonists
on frontal cortex neurotoxicity in young rats. ...................................................... 34
Figure 2.6. Effects of neonatal PCP and selective NR2A and NR2B antagonists
on striatal neurotoxicity in young rats. ................................................................ 35
Figure 2.7. Effects of PCP administration on the localization of NR2A and NR2B
in the synaptic or extrasynaptic milieu. ............................................................... 37
Figure 2.8. Locomotor sensitization induced by a 4 mg/kg PCP challenge to
either sub-chronic PCP or PEAQX treated animals. ........................................... 39
Figure 2.9. Effect of selective blockade of NR2A and NR2B subunits on
locomotor sensitization. ...................................................................................... 40
Figure 3.1. Acute PCP administration selectively up-regulates NR1/NR2B in the
frontal cortex of perinatal rats. ............................................................................ 56
Figure 3.2. Sub-chronic PCP treatment up-regulates NR1/NR2A in the frontal
cortex of perinatal rats. ....................................................................................... 57
Figure 3.3. Effects of acute and sub-chronic PCP administration on the NMDAR
subunits in the cortical ER fraction. .................................................................... 58
Figure 3.4. Acute PCP treatment selectively alters levels of PSD-95 in the
frontal cortex of perinatal rats. ............................................................................ 59
Figure 3.5. Effects of acute and sub-chronic PCP administration on the
activation of calpain in the frontal cortex. ............................................................ 61
xii
Figure 3.6. Effects of MDL28170 (calpain inhibitor) on the cortical membrane
expression of NMDAR subunits following acute PCP treatment. ........................ 62
Figure 4.1. Effects of agonist stimulation (100 µM NMDA + 30 µM glycine) on
3H-MK-801 binding in the frontal cortex of saline and PN7 PCP treated rat pups.
........................................................................................................................... 75
Figure 4.2. Effects of agonist (100 µM NMDA + 30 µM glycine) stimulation on
3H-MK-801 binding in the frontal cortex of PN7, 9, & 11 PCP treated rat pups. . 76
Figure 4.3. 3H-MK-801 displacement by the NR2B selective antagonist ifenprodil
in acute PCP treated animals. ............................................................................ 78
Figure 4.4. 3H-MK-801 displacement by the NR2B selective antagonist ifenprodil
in sub-chronic PCP treated animals. .................................................................. 79
Figure 4.5. 3H-MK-801 displacement by the NR2A selective antagonist NVPAAM007 .............................................................................................................. 80
Figure 4.6. 3H-MK-801 displacement by the NR2A preferring antagonist PEAQX
........................................................................................................................... 81
Figure 5.1. Effects of PCP administration (PN7 only) on PPI of acoustic startle.
........................................................................................................................... 96
Figure 5.2. Effects of PCP administration (PN7 only) the development of
locomotor sensitization. ...................................................................................... 97
Figure 5.3. Effects of subchronic PCP administration (PN7, 9 & 11) on PPI of
acoustic startle.................................................................................................... 98
Figure 5.4. Effects of subchronic PCP administration (PN7, 9 & 11) on the
development of locomotor sensitization. ............................................................. 99
Figure 5.5. Pharmacological antagonism of PCP treatment on PN7 effects on
NR1 and NR2B in the frontal cortex. ................................................................ 102
Figure 5.6. Pharmacological antagonism of PCP treatment on PN7, 9, & 11induced up-regulation on NR1 and NR2A in the frontal cortex. ........................ 103
Figure 5.7. Effects of a single injection of PCP on PN7 on the membrane protein
expression of NR1 and NR2B in the striatum. .................................................. 104
Figure 5.8. Antagonism of PCP treatment on PN7, 9, & 11-induced downregulation on NR1 in the striatum. .................................................................... 105
xiii
Figure 6.1. Effects of neonatal PCP administration on social interaction and
locomotor activity in pre-and post-pubescent male pups. ................................. 120
Figure 6.2. Effects of neonatal PCP administration on social interaction and
locomotor activity in pre- and post-pubescent females. .................................... 121
Figure 6.3. Effects of perinatal PCP treatment on PN7, 9, & 11 on social
discrimination in adult (PN56-58) animals. ....................................................... 122
xiv
List of Abbreviations
5-HT
Serotonin
7-Cl KYNA
7-chloro Kynurenic Acid
ACh
Acetylcholine
AChE
Acetyl cholinesterase
AMPA
α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate
ANOVA
Analysis of Variance
APV
(2R)-Amino-5-phosphonovaleric Acid
BDNF
Brain Derived Neurotrophic Factor
Bmax
Maximum bound
CB
Calbindin
CHX
Cycloheximide
CR
Calretinin
CRE
cAMP Response Element
CREB
cAMP Response Element Binding
DA
Dopamine
DAG
Diacylglycerol
DAOA
D-amino Acid Oxidase Activator
DIV
Day in vitro
DTNBP1
Dystrobrevin Binding Protein 1
ER
Endoplasmic Reticulum
ERK
Extracellular Signal-Regulated Kinase
GABA
Gamma-Aminobutyric Acid
xv
GK
Guanylate Kinase
GRM3
Metabotropic Glutamate Receptor 3 Gene
GSK
Glycogen Synthase Kinase
i.p.
Intraperitoneal
IC50
Concentration of inhibitor that inhibits 50% response
KA
Kainate
LTD
Long-term depression
LTP
Long-term potentiation
MAGUK
Membrane-Associated Guanylate Kinase
MDL28170
Calpain Inhibitor III
MK-801
Dizocilpine
NE
Norepinephrine
NF-κB
Nuclear Factor kappa B
NMDAR
N-methyl-D-aspartate receptor
NR1
NMDA receptor subunit 1
NR2
NMDA receptor subunit 2
NRG1
Neuregulin-1
NVP-AAM007
[(R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4tetrahydro-quinoxalin-5-yl)-methyl]-phosphonic acid
PCP
Phencyclidine OR 1-(1-phenylcyclohexyl) piperidine
hydrochloride
PDI
Protein Disulfide Isomerase
PEAQX
[[(1S)-1-(4-Bromophenyl) ethyl] amino] (1, 2, 3, 4-tetrahydro-2, 3dioxo-5-quinoxalinyl) methyl
PI3K
Phosphotidylinositide-3 Kinase
xvi
PKC
Protein Kinase C
PLC
Phospholipase C
PN
Postnatal Day
PPI
Pre-pulse Inhibition
PSD-95
Post-Synaptic Density 95
PV
Parvalbumin
RGS4
Regulator of G-protein Signaling 4
s.c.
Subcutaneous
SAP102
Synapse Associated Protein 102
SAP97
Synapse Associated Protein 97
SBP
α-II spectrin specific breakdown products
SH3
Src Homology Domain 3
TUNEL
Terminal d-UTP Nick End Labeling
xvii
Chapter 1: Introduction
Phencyclidine (PCP) and noncompetitive NMDA receptor antagonists
Phencyclidine [1-(1-phenylcyclohexyl) piperidine hydrochloride or PCP]
was originally developed in the 1950s for use as a surgical anesthetic (Johnson
& Jones 1990). Despite its effectiveness at producing a state of dissociative
anesthesia with little cardio-respiratory depression, clinical use was abandoned
because patients reported post-surgical hallucinations, delirium and disoriented
behavior (Morris et al. 2005). Due to its hallucinogenic effects and availability,
PCP appeared on the drug of abuse scene in the mid-1960’s and was known as
“angel dust”, “hog” or the PeaCe Pill (Morris et al. 2005, Johnson & Jones 1990).
However, illicit use of PCP fell out of favor because of its psychotomimetic
properties (Johnson & Jones 1990).
PCP intoxication in humans has been
shown to induce a psychotic state in healthy individuals which mimics both the
positive and negative symptoms of schizophrenia as well as exacerbates
psychoses in schizophrenics (Luby et al. 1962, Javitt & Zukin 1991, Steinpreis
1996). Ketamine, a structurally related arylcycloalkylamine, is a shorter acting
dissociative anesthetic that is still used clinically in veterinary medicine and as a
pediatric anesthetic in the treatment of burn victims during wound dressing
changes (Hirsch et al. 1997). Like PCP, ketamine is also abused illegally and is
known on the street as “special K” or “vitamin K” (Morris et al. 2005). Users
report dream-like states and hallucinations; however, high doses can result in
respiratory depression, delirium and amnesia and schizomimetic liked effects
(Krystal et al. 1994).
Dizocilpine (MK-801) is a noncompetitive N-methyl-D-
aspartate receptor (NMDAR) antagonist that possesses a higher affinity and
specificity for the NMDAR compared to PCP and was originally developed to
serve as a neuroprotectant following stroke and can also serve as an
anticonvulsant (Kochhar et al. 1991, Vanderschuren et al. 1998, Olney et al.
1
1989b).
Neither the abuse potential of MK-801 nor its intoxication effect in
humans is known, but it is self-administered by animals (Beardsley et al. 1990).
PCP possesses a complex and broad pharmacology, affecting various
neurotransmitter systems and ion channels (Lodge & Johnson 1990, Johnson &
Jones 1990, Morris et al. 2005), including voltage-dependent sodium and
potassium channels (French-Mullen & Rogawski 1992, Vincent et al. 1983). PCP
appears to largely act as a noncompetitive NMDAR antagonist (Ki of 50-100 nM)
(Anis et al. 1983) as an open channel blocker, binding within the channel pore in
a voltage and use-dependent manner (Honey et al. 1985, MacDonald et al. 1987,
Johnson & Jones 1990). PCP also has been shown to act behaviorally as an
anticholinergic in locomotor activity and stereotypy (Johnson & Jones 1990,
Finnegan et al. 1976, Murray & Horita 1979). Interestingly, it has also been
shown dose-dependently to act as a cholinomimetic and a weak inhibitor of
acetyl cholinesterase (AChE) and butyryl cholinesterase (Maayani et al. 1974,
Snell et al. 1988); however, this was not well correlated with rotorod activity or
the discriminative stimulus properties of PCP (Johnson & Jones 1990, Johnson
1982).
PCP also inhibits the σ binding site (Largent et al. 1984), norepinephrine
(NE) reuptake (Taube et al. 1975, Garey & Heath 1976), and serotonin (5-HT)
reuptake as well as reduces 5-HT turnover (Smith et al. 1977). PCP blockade of
the NMDAR can also lead to the release of 5-HT in the prefrontal cortex and
hippocampus. The subsequent activation of 5-HT receptors, specifically 5-HT2A,
results in the increased release of glutamate (Adams & Moghaddam 2001) and
may also underlie the hallucinatory aspects of PCP intoxication (Martin et al.
1998). In addition, PCP blocks dopamine (DA) reuptake (Ki = 0.1µM) (Steinpreis
& Salamone 1993, Garey & Heath 1976, Johnson & Jones 1990) and facilitates
its release [albeit ten times less potent than amphetamine] (Johnson & Jones
1990, Verma & Moghaddam 1996).
DA antagonists are able to antagonize
behavioral effects produced by PCP such as cognitive tasks involving working
memory in addition to hyperlocomotor activity (Castellani & Adams 1981, Verma
2
& Moghaddam 1996), suggesting a role for DA in the expression of these
behaviors.
However, these varying actions of PCP are less potent than its
actions on the NMDAR; therefore, it is likely that the psychotomimetic actions of
PCP are mostly due to its blockade of the NMDAR (Johnson & Jones 1990,
Morris et al. 2005), but inhibition of monoamine transport may also play a role.
The NMDA Receptor
The NMDAR is a member of the ionotropic glutamate family of receptors,
which also includes α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)
and kainate (KA) receptors. These receptors act in concert to mediate excitatory
neurotransmission in the mammalian brain. The NMDAR is unique among the
glutamate receptors in that it requires binding of glutamate to the NR2 subunit
and binding of the co-agonist glycine to the NR1 subunit as well as alleviation of
the voltage-dependent Mg2+ block in order for the receptor to be open and
functionally active.
Glutamate receptors promote various phases of neuronal
development, including neuronal migration, differentiation, and synaptogenesis
(Komuro & Rakic 1993).
Further, in the developing brain, specifically during a
stage of rapid neurite growth and synapse formation known as the brain growth
spurt (Olney et al. 2002), the NMDAR is one of the primary mediators of
glutamatergic excitatory neurotransmission and synaptic plasticity and is
intricately linked to the development of long-term depression (LTD) and long-term
potentiation (LTP) in learning and memory models (Lynch & Guttmann 2002).
Nevertheless, in a process known as excitotoxicity, overactivation of NMDA
receptors triggers a rapid rise of internal calcium resulting in the activation of prodeath signaling cascades as well as phospholipases, endonucleases, and
proteases which can lead to cytoskeletal, membrane and DNA damage and
ultimately apoptosis and/or necrosis (Rothman & Olney 1995).
3
Schizophrenia
Schizophrenia is a complex neuropsychiatric disorder, characterized by
the presence of both positive (paranoia, hallucinations, delusions) and negative
symptoms (emotional and social withdrawal, anhedonia, depression) as well as
cognitive impairments, such as working memory and attention deficits
(McGlashan 1996b).
2000).
The etiology currently remains unknown (Weinberger
It afflicts around 1% of the population worldwide and shows strong
genetic tendencies (Lewis & Lieberman 2000, Bromet & Fennig 1999). It has
been described as a neurodevelopmental disorder since symptoms of the first
psychotic break do not appear until early adulthood and may result from a
combination of susceptible genes as well as unknown environmental factors
(Weinberger 1987, Weinberger 1996, Pilowsky et al. 1993, Harrison & Owen
2003). The primary pathological insult, in at least some cases of schizophrenia,
is thought to originate in the late second to early third trimester in utero; a stage
of brain development in the human that corresponds to the first three weeks of
postnatal life in the rat (Benes 1991, Murray et al. 1992, Pilowsky et al. 1993).
The functional consequences of this insult do not become evident until after
puberty, a time at which many of the affected neural networks reach maturity
(Weinberger 1987). That is, damage to the brain remains relatively clinically
silent until normal development and neuronal maturity (e.g. dendritic pruning and
alterations in cytoarchitecture) unmasks behavioral abnormalities caused by the
affected structures (Marenco & Weinberger 2000).
Schizophrenia occurs equally in males and females, although onset
typically appears earlier in men than women (peak ages of onset are 20–28
years and 26–32, respectively) (Castle et al. 1991).
Interestingly, those
diagnosed with schizophrenia are more likely to have been born in winter or
spring (Davies et al. 2003), delivered during obstetric complications (Geddes &
Lawrie 1995), or had a prenatal exposure to infections (e.g. influenza), all of
4
which provide additional support for a link between in utero pathology and risk of
developing the disease (Brown 2006).
Pathophysiology of schizophrenia
A hyper-dopaminergic state is thought to underlie the pathophysiology of
schizophrenia and is based on evidence that indirect-acting DA agonists, such as
amphetamine, are capable of mimicking the positive symptoms of schizophrenia
(Carlsson 1988). In further support of this DA hypothesis of the disease, the
clinical potency of all of the early “typical” antipsychotics (e.g. haloperidol and the
phenothiazines) to effectively alleviate the hallucinations, paranoia, and
delusions associated with schizophrenia is strongly correlated with their affinity
for DA D2 receptors (Bunney et al. 2000, Moghaddam 1994). However, typical
antipsychotics are not effective at alleviating or controlling the negative
symptoms or cognitive deficits of the disease.
antipsychotics,
termed
“atypical”
Thus, introduction of newer
antipsychotics
(clozapine,
olanzapine,
risperidone), which are effective at treating both positive and negative symptoms,
suggested that DA may not be the only player in schizophrenia, since atypical
antipsychotics possess relatively high affinity for DA, 5-HT, histamine, α1
noradrenergic, and muscarinic ACh receptors (Bondolfi et al. 1998, Large 2007).
The
NMDA
hypofunction
(reduced
channel
function)
theory
of
schizophrenia is based on the major finding that noncompetitive NMDAR
antagonists, including PCP, ketamine, and MK-801 mimic multiple aspects of
schizophrenia. Transient inactivation of NMDA receptors by administration of
PCP or MK-801 to rodents results in neurotoxicity and structural damage in brain
regions somewhat similar to those in which damage is present in schizophrenics
(Olney et al. 1989b, Olney & Farber 1995b). Structural damage to the cingulate
cortex, hippocampus, parahippocampal gyrus and entorhinal cortex has been
observed in postmortem analysis of schizophrenic brains (Kovelman & Scheibel
1984, Bogerts 1993). Further, it was reported that high doses of PCP or MK-801
5
cause neuronal vacuolization in the adult rat retrosplenial and posterior cingulate
cortices (Olney et al. 1989b). Also, repeated administration of either MK-801 or
PCP in adult rats has been reported to cause neurodegeneration (silver-positive)
in the anterior cingulate, parietal, temporal, piriform, and entorhinal cortices, as
well as the hippocampus and amygdala (Olney & Farber 1995b).
In
schizophrenics, there is also evidence for a reduction in mismatch negativity
responses following a sensory cue [ketamine has also been reported to reduce
mismatch negativity (Umbricht et al. 2000)]; this response has been shown to be
mediated by the NMDAR and is an additional indication of NMDAR hypofunction
(Javitt et al. 1996, Shelley et al. 1991).
Normally, activation of NMDARs on GABAergic interneurons (primarily
parvalbumin (PV)-containing basket cells) in the basal ganglia results in an
inhibition of excitatory neurotransmission to cortical pyramidal neurons; however,
PCP-induced inhibition of the NMDAR on these interneurons results in a
disinhibition of neurotransmission, producing an increase in excitation of
pyramidal cells in the cortex [confirmed in the rat by electrophysiology (Jackson
et al. 2004), metabolic imaging (Sharp et al. 2001, Vaisanen et al. 2004) as well
as elevated glutamate release (Moghaddam et al. 1997)]. The subsequent and
prolonged overactivation of glutamate receptors on the pyramidal neurons leads
to cellular stress and ultimately excitotoxic cell death, (Olney & Farber 1995a,
Large 2007). Cortical disinhibition has been reported in schizophrenia as evident
by an increase in brain metabolic activity (Friston 1992, Friston et al. 1992,
Lisman et al. 2008).
Further, examination of post-mortem brain tissue from
schizophrenics shows a loss of PV-containing interneurons, with no effect on
calbindin- or calretinin-containing interneurons in the frontal cortex (and
hippocampus) (Beasley & Reynolds 1997, Beasley et al. 2002, Danos et al.
1998, Reynolds & Beasley 2001).
Malfunctioning communication between
pyramidal neurons and PV-containing interneurons has been proposed to also
affect DA neurotransmission, linking the hyper-dopaminergic theory and the
6
NMDA hypofunction theory (Lisman et al. 2008). In fact, NMDAR antagonists
have been shown to stimulate dopamine release in animal models (Pawlowski et
al. 1990, Verma & Moghaddam 1996) suggesting that the hyper-dopaminergic
state in schizophrenia is indeed a consequence of NMDA hypofunction (Grace
1991, Carlsson & Carlsson 1990), most likely originating in the hippocampus
(Floresco et al. 2001, Legault et al. 2000), a brain region implicated in the
manifestation of psychosis and deficits in working and short-term memory
characteristic of schizophrenia (Lisman et al. 2008).
NMDAR-mediated neurotoxicity and aberrant behaviors in rodent models of
schizophrenia
Based on Olney’s hypothesis and clinical evidence linking deficits in
NMDAR synaptic transmission and disinhibition or neurotransmission to the
pathophysiology of schizophrenia (Mouri et al. 2007), the effects of acute and
chronic PCP or MK-801 treatment has been extensively studied in the adult rat
as an animal model of the disease (Andine et al. 1999, Hanania et al. 1999,
Hargreaves & Cain 1995, Jentsch et al. 1997, Johnson et al. 1998, Moghaddam
et al. 1997, Ogren & Goldstein 1994, Phillips et al. 2001).
However, since
symptoms of the disease do not typically manifest in the human until late
adolescence or early adulthood, many researchers, including those in this
laboratory, investigate the effects of NMDAR blockade in developing rodents
(Weinberger 1987, Ikonomidou et al. 1999, Wang et al. 2001, Wang & Johnson
2005). Neurodegeneration during early stages of brain development has been
shown following PCP or MK-801 treatment in the cortex, hippocampus, and
striatum, all of which are regions of the brain implicated in schizophrenia
(Ikonomidou et al. 1999). In addition, acute PCP treatment on postnatal day
(PN) 7, results in neurodegeneration (positive silver staining) in the frontal cortex,
striatum, and hippocampus within 9 hours of treatment (Wang & Johnson 2005).
This laboratory has also reported the presence of both non-specific
7
neurodegeneration (positive silver staining) and apoptotic neurons (terminal dUTP nick end labeling (TUNEL) positive) in the cortex, but not the in the striatum
or hippocampus, following sub-chronic PCP administration on PN7, 9, & 11
(Wang et al. 2001, Wang & Johnson 2005), thereby suggesting either a
developmental or pharmacological tolerance in these regions. Pretreatment with
olanzapine was able to prevent the appearance of neurotoxic indices following
sub-chronic postnatal PCP administration, suggesting that this treatment
paradigm may model certain aspects of schizophrenia in rats (Wang et al. 2001)
Schizophrenics have an impaired ability to filter external sensory
information, a process known as sensorimotor gating (Bunney et al. 2000).
Deficits in sensorimotor gating are thought to contribute to thought disorder and
cognitive fragmentation characteristic of the disease (Braff & Geyer 1990). Prepulse inhibition (PPI) of acoustic startle response in animals is used as an animal
model of sensorimotor gating.
PPI of acoustic startle is a measure of the
reduction of the startle response when a smaller non-startling acoustic stimulus
(pre-pulse) is presented 80-120 ms prior to the startling stimulus (pulse)
(Swerdlow et al. 1994). Acute PCP treatment produces an inhibition in PPI in
adult rats, similar to that seen in schizophrenic patients (Martinez et al. 2000a).
Typical antipsychotics, such as haloperidol, are not able to reverse deficits in PPI
caused by acute PCP treatment in adult rats (Geyer et al. 2001) or in prepubertal rats (Martinez et al. 2002), but can reverse the effects of DA agonists,
e.g. apomorphine.
However, atypical antipsychotics, including clozapine,
olanzapine, and quetiapine, are effective at alleviating acute PCP inhibition of
PPI in adult rats (Geyer et al. 2001, Ballmaier et al. 2001, Martinez et al. 2002)
as well as pre-pubescent rats (PN24-28, 10 mg/kg PCP on PN PN7, 9, & 11
(Wang et al. 2001), but not following acute PCP treatment to pups (PN16-19) or
pre-pubertal (PN45) rats (Martinez et al. 2002).
Additional behavioral consequences of acute PCP administration to adult
rats include increases in locomotor activity, stereotypy, ataxia, circling, head8
weaving and walking backwards (Steinpreis 1996, Castellani & Adams 1981,
Geyer & Ellenbroek 2003). In general, alterations in motoric behaviors such as
these are thought to be mediated by DA and model the positive symptoms of
schizophrenia, since they can be blocked by typical antipsychotics such as
haloperidol (Geyer & Ellenbroek 2003, Steinpreis 1996). DA antagonists have
been shown to inhibit PCP-induced locomotor activity, turning, and ataxia
(Tsutsumi et al. 1995); more specifically, D1 and D2 receptors may play a role in
acute PCP-induced hyperlocomotion, while only D2 receptors appear to regulate
chronic PCP-induced hyperlocomotor activity (Phillips et al. 2001).
Chronic
administration of PCP to adult rats results in locomotor sensitization (Hanania et
al. 1999, Johnson et al. 1998). Locomotor sensitization induced by chronic PCP
can be inhibited by pretreatment with the atypical antipsychotic clozapine
(Johnson et al. 1998), which possesses affinity for D2 and D4 receptors, 5-HT2,
5-HT3, 5-HT6 and 5-HT7 receptors, α1 noradrenergic receptors, muscarinic ACh
receptors and histamine receptors (Lieberman 1993).
Atropine, a selective
muscarinic antagonist, was able to partially inhibit chronic PCP-induced
locomotor sensitization; however, ketanserin, a 5-HT2A/2C receptor antagonist,
had no effect (Phillips et al. 2001). These results suggest a regulatory role for
ACh and DA in the manifestations of PCP-induced hyperlocomotor activity in
adult rats. We have previously reported that sub-chronic PCP administration
(PN7, 9, & 11) to developing rat pups can enhance locomotor activity and induce
the development of locomotor sensitization following PCP challenge as well as
deficits in PPI of acoustic startle (Anastasio & Johnson 2008a, Wang et al. 2001),
both of which can be alleviated by treatment with atypical antipsychotics. Acute
and repeated PCP treatment, but not amphetamine, of rats also results in deficits
in social interaction (a hallmark of the negative symptoms of schizophrenia) and
cognitive deficits as measured by the social interaction test and delayed spatial
alternation task, respectively (Sams-Dodd 1999, Koros et al. 2007, Sams-Dodd
1998, Wang et al. 2001).
Chronic treatment (21 days) with the atypical
9
antipsychotic, clozapine, but not the typical antipsychotic haloperidol reversed
the PCP-induced deficits in social behavior (Sams-Dodd 1996).
Deficits in
selective attention and information processing, i.e. the inability to differentiate
relevant from irrelevant information is an additional debilitating negative symptom
of schizophrenia (Goldberg & Gold 1995). Acute PCP administration to adult rats
has been reported to produce a reduced novelty discrimination index [time spent
investigating the familiar juvenile compared to time spent investigating novel
juvenile] (Terranova et al. 2005). Further, adult rats which received PCP during
the perinatal stage (PN7, 9, & 11) also present with a deficit in the novelty
discrimination index (Depoortere et al. 2005, Terranova et al. 2005, Harich et al.
2007a).
These deficits could be reversed with the atypical antipsychotic
clozapine (Terranova et al. 2005), supporting this behavior model as a suitable
model of the attention processing deficits present in schizophrenia.
Several studies from this laboratory have investigated the possible
mechanisms by which PCP may elicit its neurotoxic effects and produce
alterations in behavior in rats, specifically by measuring the effects of PCP on the
expression of the NMDAR.
The NMDAR is composed of multiple subunits
including NR1, NR2A-D and NR3A/B; a functional receptor is typically composed
of a NR1 subunit with one of the four NR2 subunits, forming a heterotetrameric
complex.
The NR1/NR2 complex differs in regard to its regional pattern of
expression in the brain, its regulation by phosphorylation, proteases, polyamines
or protons, its electrophysiological and functional properties, and its affinity for
cytoskeletal post-synaptic density proteins (Paoletti & Neyton 2007, Kohr 2006,
Waxman & Lynch 2005, Westbrook et al. 1997, Cull-Candy et al. 2001). Each
subunit of the NMDAR contains an extracellular N-terminal region, three
membrane spanning domains, one intramembrane loop and an intracellular Cterminal region. The C-terminal region of the NR2 subunit serves as a binding
site for intracellular anchoring and trafficking proteins such as post-synaptic
density 95 (PSD-95), synapse associated protein 102 (SAP102), and synapse
10
associated protein 97 (SAP97) and also links second messenger cascades to the
NMDAR, thereby modulating the interaction of the receptor with various
intracellular signaling cascades (Niethammer et al. 1996, Bi et al. 1998b,
Wechsler & Teichberg 1998, Wu & Lynch 2006, Bi et al. 1998a, Guttmann et al.
2001, Guttmann et al. 2002, Zheng et al. 1999). Chronic PCP treatment of adult
rats (20 mg/kg x 5 days) resulted in increases in the NMDAR subunit NR1 mRNA
in the olfactory tubercle, piriform cortex, frontal cortex and anterior striatum
(Wang et al. 1999).
In addition, this treatment of adult rats also produced
locomotor sensitization that was accompanied by an increase in NMDA-induced
release of striatal
14C-GABA
and 3H-ACh, which was correlated with an increase
in NR1 immunoreactivity, suggesting that alterations in NMDAR function in the
striatum may underlie PCP-induced locomotor sensitization (Hanania & Johnson
1999). In developing rats, sub-chronic PCP treatment of pups on PN7, 9, & 11
resulted in increased expression of NR1 mRNA in the frontal cortex, striatum,
nucleus accumbens and olfactory cortex that was inhibited by pretreatment with
the atypical antipsychotic olanzapine (Wang et al. 2001). This confirmed that
increases in NR1 protein levels in the frontal cortex of PCP-treated pups
coincided with increases in NR1 protein that was observed in PCP-treated adult
rats (Hanania et al. 1999). Up-regulation of NR1 protein in the frontal cortex
caused by sub-chronic PCP administration was associated with increases in
TUNEL positive cells as well as an increase in the Bax (pro-apoptotic protein) to
Bcl-XL (anti-apoptotic protein) ratio, both of which were inhibited by olanzapine
pretreatment (Wang et al. 2001). These results parallel studies using 3H-MK-801
binding to show increases in NMDAR density (McDonald et al. 1990b, Gao &
Tamminga 1994) as well as studies showing the delayed occurrence of cortical
and striatal neurotoxicity induced by MK-801 or PCP (Ikonomidou et al. 1999,
Wang et al. 2001, Wang & Johnson 2005). The exact mechanisms underlying
the regulation of the NMDAR following acute or sub-chronic PCP administration
11
and the relationship between neurotoxicity have yet to be established and merit
further investigation.
The overall purpose of the present study was to characterize the
regulation, function, and localization of NMDAR subunits (NR1, NR2A, and
NR2B) following both acute (PN7) and sub-chronic (PN7, 9, & 11) PCP treatment
in order to gain insight into the mechanism of neurotoxicity and the behavioral
alterations caused by perinatal PCP administration in a developmental animal
model of schizophrenia. The following hypotheses were addressed:
Hypothesis 1:
Differential regulation of NR1 and NR2 subunits in the
frontal cortex following acute or sub-chronic PCP administration results
from differences in synthesis and/or intracellular trafficking of the
subunits.
NMDA antagonists including PCP, MK-801 and ethanol have been shown
to regulate the membrane expression levels of the NMDAR through changes in
protein synthesis or trafficking of the NMDAR complex from intracellular
compartments to the membrane (Dong et al. 2004, Sircar et al. 1996, Suvarna et
al. 2005, Wenthold et al. 2003). Alterations in the trafficking of the NMDAR have
been hypothesized to be dependent on its interactions with members of the post
synaptic complex, specifically PSD-95 (Yamada et al. 1999b, Suvarna et al.
2005, Lu et al. 2000, Dong et al. 2004). PSD-95 plays a major role in the cell
surface expression of assembled NR1/NR2 receptor complexes by anchoring the
functional NR1/NR2 complex to the membrane through its interactions with the
terminal four amino acid residues of the NR2 subunit (Wenthold et al. 2003,
Yamada et al. 1999b). It has also been reported that MK-801 treatment to
neonatal rats increases mRNA levels of NR1, NR2A, and NR2D mRNA in the
frontal cortex and striatum (Sircar et al. 1996, Wilson et al. 1998). Sub-chronic
PCP administration has been shown to result in the nuclear translocation of the
transcription factor NF-κB (Wang et al. 2001), which has been shown to lead to
12
the up-regulation of the NR1 promotor and subsequent transcription of the gene
(Liu et al. 2004a).
My previous studies reported that acute PCP treatment increased
membrane levels of NR1 and NR2B protein in the frontal cortex, while no effect
on NR2A levels was detected. Furthermore, NR1 and NR2B protein levels in the
endoplasmic reticulum (ER) fraction were decreased by acute PCP, suggesting
an increased trafficking of NR1/NR2B from the ER to the membrane.
Pretreatment with various antipsychotics had no effect on changes in protein
expression induced by acute PCP.
On the other hand, sub-chronic PCP
treatment caused an increase in NR1 and NR2A expression in both the ER and
membrane fractions of the frontal cortex, while no effect on NR2B expression
was measured in either fraction. The increase in NR1/NR2A in the membrane
fraction was blocked by pretreatment with antipsychotics. Therefore, I proposed
to determine the mechanisms underlying the differences between acute and subchronic PCP-induced regulation of the NMDAR by assessing the synthesis and
trafficking of NR1, NR2A, and NR2B subunits in the frontal cortex and striatum in
vivo as well as the role of DA and 5-HT systems in regulation of the NMDAR.
Specific Aim 1a
Preliminary data shows that acute and sub-chronic PCP administration
results in modest increases in the mRNA of NR1, NR2A, or NR2B in the frontal
cortex 24 hrs later; however, these changes are not well correlated with the
protein expression levels measured at this time point. It has been suggested that
mRNA may be a poor indicator of protein levels, since expression of protein is
not only dependent on transcription of the gene, but also nuclear export of
mRNA, mRNA stability, translational regulation and protein degradation/turnover
(Pradet-Balade et al. 2001). Thus, to address this concern we will use a protein
synthesis inhibitor, cycloheximide (CHX), in order to clarify the role of protein
synthesis in PCP-mediated regulation of the NMDAR subunits. CHX is a broad
13
protein synthesis inhibitor whose mechanism of action involves the reduction of
the elongation rate via an eEF2-independent mechanism (Liu et al. 1997). To
determine the role of protein synthesis in acute and sub-chronic PCP-induced
regulation of the NMDAR, we will measure the protein expression levels of
membrane bound NMDAR subunits in the presence and absence of the protein
synthesis inhibitor, CHX following PCP administration.
Specific Aim 1b
Alterations
in
the
trafficking
of
the
NMDAR
from
intracellular
compartments to the membrane are suggested to be mediated through activation
of the calcium-dependent neutral cysteine protease, calpain (Dong et al. 2004,
Yamashima 2004).To establish the role of calpain-mediated cleavage and
trafficking of the NMDAR from intracellular compartments to the membrane,
tissue from the frontal cortex of acute and sub-chronic PCP treated animals will
be collected and the expression of α-II spectrin (a member of the post-synaptic
density that is enzymatically cleaved directly by calpain) and its calpain specific
breakdown products (SBP) which migrates at 145 and 150 kDa. In addition, to
further clarify the role of calpain in the trafficking of the NMDAR, we will utilize
MDL28170, a selective calpain inhibitor, following acute or sub-chronic PCP
administration, and measure the membrane expression levels of NR1, NR2A,
and NR2B in the frontal cortex and striatum. MDL28170 is a potent calpain
inhibitor (Ki=10 nM in vitro) that has been shown to cross the blood brain barrier
following systemic administration (Li et al. 1998).
Hypothesis 2: Up-regulation of the NMDAR in the frontal cortex by either
acute or sub-chronic PCP treatment of perinatal rats results in a hyperfunctional receptor and is dependent on the subunit composition of the
receptor.
14
The consequences of PCP-induced neurotoxicity are multifaceted and
may involve various compensatory mechanisms, including but not limited to
altering the synaptic expression levels and functional properties of the NMDAR.
It has been reported that postnatal PCP administration (1 and 5 mg/kg on PN515) increased membrane expression levels of the NMDAR as well as maximal
3H-MK-801
binding in the frontal cortex (Sircar 2003). The exact mechanisms
underlying the differential regulation of the NMDAR following acute or subchronic PCP administration and the relationship between NMDAR density and
function are unknown in this animal model of schizophrenia; therefore, the
present study seeks to determine whether the apparent treatment dependent upregulation of the NMDAR subunits corresponds to an increase in its functionality
using a 3H-MK-801 binding assay.
Glutamate and glycine, which promote
opening and activation of the NMDA channel, stimulate radioligand binding in a
well washed membrane preparation, and inhibition by noncompetitive NMDAR
antagonists is dependent on the presence of both agonists (Johnson et al. 1989,
MacDonald et al. 1987). Further, MK-801 binds deep within the NMDAR channel
and binding is influenced by the open state of the channel; therefore, it is
possible to study the regulation and function of the NMDAR by measuring the
binding of radiolabeled MK-801(Johnson et al. 1989).
Specific Aim 2
The functionality of the NMDAR following both acute and sub-chronic PCP
treatment will be determined in the frontal cortex by assessing NMDA- and
glycine-dependent activation of 3H-MK-801 binding. Preliminary data suggest
that in the frontal cortex acute PCP treatment selectively affects the NR1/NR2B
complex, while the NR1/NR2A complex appears to be sensitive to sub-chronic
PCP treatment (Anastasio & Johnson 2008b). Thus, it is anticipated that the
sensitivity of 3H-MK-801 binding to NR2A and NR2B selective antagonists such
15
as NVP-AAM077 and ifenprodil, respectively, will be altered following either
acute or sub-chronic PCP treatment.
Hypothesis 3:
Neuroprotection from PCP administration is mediated
through activation of NR1/NR2A receptors (synaptic) while PCP-induced
neurotoxicity
is
dependent
on
blockade
of
NR1/NR2B
receptors
(extrasynaptic), suggesting that localization of NMDA NR2A and NR2B
receptors may dictate different neurotoxic functions in response to PCP.
The subunit composition of the NMDAR that mediates PCP-induced
neuronal neurotoxicity is currently unknown. Work from the Bading laboratory
has proposed that activation of synaptic NMDARs lead to neuronal survival while
activation of extrasynaptic receptors may lead to neuronal death (Hardingham et
al. 2002). The subunit composition of synaptic and extrasynaptic NMDARs is not
entirely clear and may depend on the stage of development. Early in
development, NR2B receptors dominate in expression compared to NR2A, but a
“switch” occurs later in development and NR2A are then expressed at a higher
level. However, this switch does not rule out the role of NR2B receptors in either
synaptic or extrasynaptic transmission. Recent work from our laboratory
suggests that activation of synaptic receptors, which are thought to be
NR1/NR2A, protects neurons from PCP-induced neurotoxicity (Lei et al. 2008).
Further, we have hypothesized that aberrant behaviors induced by PCP are due
to its neurotoxic effects; therefore, the purpose of this study is to show a
relationship between NR2-blockade mediated neurotoxicity and development of
locomotor sensitization.
Specific Aim 3a
We plan to investigate the role of synaptic and extrasynaptic NMDARs in
PCP-induced neurotoxicity in vitro and in vivo. Selective antagonists at various
doses for NR2A (NVP-AAM007 and PEAQX) and NR2B (ifenprodil) will be
16
administered to organotypic corticostriatal slice cultures as well as developing rat
pups and apoptosis estimated by measurement of caspase-3 enzymatic activity
and histochemical assessment of terminal dUTP nick-end labeling (TUNEL) of
apoptotic neurons.
Specific Aim 3b
The membrane associated guanylate kinase (MAGUK) family of proteins
includes PSD-95 (post-synaptic density 95), SAP102 (synapse associated
protein 102), and SAP97 (synapse associated protein 97). PSD-95 is thought to
bind to the PDZ domain in the C-terminal region of synaptic NR2A or NR2B
subunits (Lu et al. 2000, Wu et al. 2007), while SAP102 is linked to NR2B
subunits either in the synaptic or extrasynaptic space (Wu et al. 2007, Sans et al.
2003) To determine the differential “synaptic” localization of NR2A and NR2B
following either acute or sub-chronic PCP administration in vivo, we will perform
immunoprecipitation studies in washed cortical synaptic membranes and crude
membrane fractions from animals treated with PCP on PN7 (acute studies) or on
PN7, 9, & 11 (sub-chronic studies). First, we will determine the composition of
the NMDAR (NR1+NR2A and/or NR2B) via pull down assays.
determine
the
synaptic
localization
of
the
NR2
In order to
subunits
we
will
immunoprecipitate PSD-95 or SAP102 and then immunoblot with anti-NR1, antiNR2A, anti-NR2B, or anti-actin. To confirm our results, we will pull-down with
NR2A or NR2B and immunoblot for actin, NR1, PSD-95 or SAP102.
Specific Aim 3c
So as to determine the effects of blockade of NR2 subunits on the
development of locomotor sensitization and establish a relationship between
neurotoxicity and development of this behavioral phenomenon, animals will be
treated either acutely or sub-chronically with saline, PCP, PEAQX (NR2Apreferring antagonist) or ifenprodil (NR2B-selective antagonist) on PN7, 9, & 11,
17
allowed to develop and locomotor activity measured following challenge with
either PCP, PEAQX, or ifenprodil on PN28-35.
Hypothesis 4:
Sub-chronic PCP administration to developing rats will
induce locomotor sensitization in response to PCP challenge, deficits in
PPI of acoustic startle as well as deficits in social interaction and social
discrimination, thereby mimicking multiple symptoms of schizophrenia.
PCP intoxication in humans has been shown to mimic both the positive
and negative symptoms of schizophrenia as well as exacerbate psychosis in
schizophrenics. In general, alterations in motoric behaviors are thought to model
the positive symptoms of schizophrenia. Further, PCP disrupts PPI of acoustic
startle in animals and is routinely used to model the sensorimotor gating deficits
of schizophrenia (Geyer et al. 2001, Rasmussen et al. 2007).
Hypofunction in
the prefrontal cortex is thought to underlie the origins of the negative symptoms
of the disease (Goff & Evins 1998). Clinical observations have shown that PCP
intoxicated humans repeatedly present with symptoms which mimic several
negative symptoms of schizophrenia, including social withdrawal. There is a
paucity of studies investigating the PCP perinatal model and its ability to mimic
the negative symptoms of schizophrenia. Therefore, in this study we sought to
determine the effect of PN7 and PN7, 9, 11 PCP administration on multiple
behaviors thought to model different aspects of schizophrenia; specifically
locomotor sensitization, PPI of acoustic startle, social interaction as well as its
effect on social discrimination.
Specific Aim 4a
Measurement of PPI of acoustic startle will performed according to
previously published procedures with minor modifications (Wang et al. 2001,
Wang et al. 2003). Animals will be treated on PN7 or PN7, 9, & 11 with PCP,
allowed to age and PPI measured on PN24-26.
18
The ability of atypical
antipsychotics to prevent the PCP-induced deficits in PPI will also be determined.
Repeated PCP administration causes a progressive augmentation of locomotor
activity (Xu & Domino 1994), referred to as sensitization. Sensitization is also
thought to be an important index related to psychosis as well as movement and
thought disorders in schizophrenia (Robbins 1990); therefore, we will determine if
acute or sub-chronic PCP administration results in locomotor sensitization as well
as the effects of atypical antipsychotics (e.g. olanzapine and risperidone) to
prevent it.
Specific Aim 4b
Social interaction is best defined as the time a pair of animals spend in an
active social interaction (sniffing and grooming its partner, close following,
mounting and crawling over or under its partner) with or without direct physical
contact (Mouri et al. 2007). Male and female Sprague-Dawley rat pups will be
treated on PN 7, 9, and 11 with PCP and then tested for deficits in social
interaction on PN28-29 (pre-pubescent) and PN54 (post-pubescent). Deficits in
social novelty discrimination, i.e. the inability of an adult rat to discriminate
between a familiar and a novel juvenile, is thought to serve as a suitable animal
model
of
the
selective
attention
processing
deficits
characteristic
of
schizophrenia (Engelmann et al. 1995). Animals will be treated on PN7, 9, & 11
with saline or PCP allowed to age and tested as adults on PN58-60. The time of
investigative behavior of or by the adult (anogenital exploration, sniffing with
direct contact, crawling over each other, grooming, play behaviors) towards each
of the two juveniles will be recorded. The time spent investigating the familiar
juvenile divided by the time spent investigating the novel juvenile is defined as
the novelty discrimination index.
19
Chapter 2: NR2A-Containing Synaptic NMDARs Mediate
PCP-Induced Cortical Neurotoxicity and the Subsequent
Development of Schizophrenia-like Behaviors
The relationship between PCP-induced neurotoxicity in perinatal rats and
the development of schizophrenic-like behaviors is uncertain, as is the role of
NMDAR subunits in both. We used antagonists selective for NR2A- and NR2Bcontaining NMDARs to test the hypothesis that the behavioral and neurotoxic
effects of PCP are mediated by blockade of synaptic NMDARs which are thought
to predominantly consist of NR1/NR2A subunits, rather than NR1/NR2B
subunits. Sprague-Dawley rats were treated on PN7 (acute) or on PN7, 9, & 11
(subchronic) with PCP (10 mg/kg), PEAQX (selective NR2A antagonist, 10, 20,
or 40 mg/kg, s.c.), or ifenprodil (selective NR2B antagonists, 1, 5, or 10 mg/kg,
s.c.) and sacrificed to measure caspase-3 activity as an index of apoptosis or
allowed to age and tested for locomotor sensitization to PCP challenge on PN2835. PCP or PEAQX on both PN7 and PN7, 9, & 11 markedly elevated caspase-3
activity in the cortex while ifenprodil showed no effect. Striatal neurotoxicity was
evident after treatment with PCP or PEAQX on PN7 or only after sub-chronic
treatment with a high dose of PEAQX (20 mg/kg). Animals treated with PCP or
PEAQX on PN7, 9 and 11 showed a sensitized locomotor response to PCP
challenge on PN28-35. Ifenprodil treatment had no effect on either measure.
Therefore, PCP blockade of cortical synaptic NR1/NR2A, rather than NR1/NR2B,
appears to be responsible for PCP-induced neurotoxicity and the development of
long-lasting behavioral deficits.
Introduction
NMDAR antagonists including PCP and ketamine are drugs of abuse that
induce amnesia, delirium, and schizophrenia-like symptoms (Luby et al. 1962,
Javitt & Zukin 1991). PCP is thought to be one of the most dangerous drugs of
20
abuse; depending on the dose administered it can act as a hallucinogen,
stimulant, depressant and/or anesthetic.
In addition to the psychotomimetic
symptoms produced by PCP, several parallels exist between the effects of PCP
administration and schizophrenia, specifically altered cerebral blood flow,
glucose utilization and neurotoxicity in brain regions including, but not limited to
the frontal cortex, striatum, and hippocampus all of which are thought to be
afflicted in schizophrenics (Olney et al. 1989a, Olney & Farber 1995b, Strous &
Javitt 1996). The similarities between PCP-induced neurotoxicity in animals and
its ability to mimic schizophrenia led to the hypo-glutamatergic hypothesis of
schizophrenia (Olney & Farber 1995a).
PCP is an open channel blocker of NMDA receptors, binding within the
channel pore in a voltage and use dependent manner (Honey et al. 1985,
MacDonald et al. 1987, Johnson & Jones 1990). The NMDAR is a voltage-gated
channel and member of the glutamate family of excitatory neurotransmitters,
which is activated following binding of glutamate and the co-agonist glycine and
is permeable to Ca2+ and is chiefly localized to the postsynaptic neuronal
membrane. The NMDAR is primarily a heterodimeric complex formed by the
combination of two of the obligatory NR1 subunits and two of the four NR2
subunits (A-D) and less commonly, it is also known to form a heterotrimeric
complex including NR1, NR2A and NR2B (Kutsuwada et al. 1992, Kohr 2006,
Cull-Candy et al. 2001).
The composition of the NMDAR complex varies in
distribution and possesses unique pharmacological and physiological properties
in the brain (Lynch & Guttmann 2001, Cull-Candy et al. 2001).
During early postnatal development of the rat, NMDARs are the principal
mediator of glutamatergic neurotransmission and are also highly responsive to
over-stimulation resulting in excitotoxic neuronal death (Ben-Ari et al. 1997).
Extensive evidence indicates that activation of synaptic NMDARs [predominantly
NR1/NR2A (Tovar & Westbrook 1999, Cull-Candy et al. 2001)] promote neuronal
survival through increased synaptic function and Ca2+ entry leading to activation
21
of the CREB pathway. However, activation of extrasynaptic receptors
[predominantly NR1/NR2B (Cull-Candy et al. 2001, Tovar & Westbrook 1999)]
leads to neuronal death by shutoff of the pro-survival CREB pathway as well as
inactivation of activated ERK and its downstream signaling partners (Soriano &
Hardingham 2007, Hardingham et al. 2002, Hardingham & Bading 2002, Ivanov
et al. 2006).
Although acute blockade of NMDA receptors by MK-801 or PCP results in
wide-spread neurotoxicity (Ikonomidou et al. 1999), this laboratory has reported
that subchronic PCP treatment of perinatal rats on PN7, 9 and 11 results in a
cumulative apoptosis in the frontal cortex (Wang & Johnson 2007, Wang &
Johnson 2005, Wang et al. 2001) that is associated with long-lasting behavioral
deficits that resemble certain aspects of schizophrenic-like behaviors (Anastasio
& Johnson 2008a, Wang et al. 2001). For example, this treatment caused an
olanzapine-sensitive deficit in pre-pulse inhibition (PPI) of acoustic startle as
measured on PN24-26, as well as a sensitized locomotor response to low-dose
PCP (4 mg/kg) measured on PN28-35 (Anastasio & Johnson 2008a). These
studies help to establish the validity of perinatal neurodegeneration induced by
PCP along with its behavioral sequelae as a potential animal model of
schizophrenia. However, the means by which PCP causes neurodegeneration is
not completely understood, nor is the relationship between neurotoxicity and the
development of abnormal behaviors later in life.
The primary goal of this study was to determine whether PCP-induced
neurotoxicity and its detrimental behavioral effects could be attributed to
blockade of either synaptic or extrasynaptic NMDA receptors. Since NR2A
receptors are thought to be localized predominantly in the synapse and NR2B
receptors are believed to be mostly extrasynaptic (Cull-Candy et al. 2001), we
proposed that if PCP-induced neurodegeneration and behavioral deficits were
the consequence of blockade of synaptic, rather than extrasynaptic NMDA
receptors, then the effects of PCP would be mimicked by the NR2A antagonists
22
NVP-AAM007 and PEAQX (Feng et al. 2004), but not by the NR2B antagonist,
ifenprodil (Williams 2001). To this end, we compared the effects of PEAQX, or its
stereomer NVP-AAM007 (Auberson et al. 2002) and ifenprodil to PCP treatment
in measures of apoptosis (both in organotypic corticostriatal slices and in vivo)
and the development of locomotor sensitization to PCP challenge on PN28-35.
The results of these experiments allow us to propose that neurotoxicity and the
subsequent alterations in early adolescence behavior that results from PCP
administration during a critical stage of brain development in the rat is
predominantly due to blockade of synaptic NR2A-containing, rather than NR2Bcontaining receptors.
Materials and Methods
Animals.
Timed, day 14 pregnant female Sprague-Dawley rats were obtained from
Charles River Laboratories (Wilmington, MA).
The dams were housed
individually with a regular 12h light-dark cycle (lights on 0700, off at 1900) with
food and water ad libitum. Following parturition, male and female pups from four
dams were combined and randomly cross-fostered to one of the four lactating
dams. Each litter consisted of ten to twelve pups. On postnatal (PN) day 2.5,
some animals were sacrificed by decapitation and their brains used for
organotypic brain slice culture. Other pups were treated on PN7 or on PN7, 9
and 11 and killed by decapitation in experiments described below or allowed to
age and used for behavioral studies.
All experiments were conducted in
accordance with NIH regulations and with approval of the University of Texas
Medical Branch at Galveston Institutional Animal Care and Use Committee.
Drugs.
PCP was acquired from the National Institute on Drug Abuse (NIDA,
Rockville, MD) and dissolved in 0.9% NaCl. NVP-AAM007 was a generous gift
23
provided to us by Dr. Yves Auberson (Novartis Institute for Biomedical Research,
Basel, Switzerland).
[[[(1S)-1-(4-Bromophenyl) ethyl] amino] (1, 2, 3, 4-
tetrahydro-2, 3-dioxo-5-quinoxalinyl) methyl] phosphonic acid tetrasodium
hydrate (PEAQX) and ifenprodil (+)-tartrate salt were purchased from SigmaAldrich (St. Louis, MO) and dissolved in 0.9% NaCl. Doses were chosen based
on our own work with these compounds as well as the published ED50 values.
Organotypic slice culture.
Corticostriatal slice cultures were prepared as previously prescribed (Xia
et al. 2008) In brief, 2.5 day-old rat pups were sacrificed by decapitation. The
brains were removed quickly and cut into 400-μm-thick coronal sections by a
McIlwain tissue chopper under sterile conditions.
Three adjacent frontal
corticostriatal slices with morphology comparable to levels between A5.3 and
A6.8 mm in P10 rats (Sherwood & Timiras 1970) were placed and cultured in
inserts with a porous and translucent membrane (Culture Plate Insert,
MILLIPORE Co, Bedford, MA) at the interface between medium and CO 2enriched atmosphere.
The initial culture medium was a mixture of 25%
inactivated horse serum, 25% Hank's balanced salt solution, and 50% OPTIMEM culture medium, supplemented with 25 mM D-glucose and 1%
penicillin/streptomycin. On DIV 3, the medium was switched to serum-free
Neurobasal medium supplemented with 25 mM D-glucose, 1 mM glutamine, 2%
B-27, and 1% penicillin/streptomycin. The medium was changed twice a week
thereafter. Slices were used in experiments on DIV9.
In vivo experimental design.
Male and female rat pups were treated on PN 7 (acute) or on PN 7, 9, and
11 (sub-chronic) with 10 mg/kg PCP (s.c.), 10, 20 or 40 mg/kg PEAQX (s.c.), 10
mg/kg NVP-AAM007 (s.c.), or 1, 5, or 10 mg/kg ifenprodil (i.p.).
For the
neurotoxicity studies, animals were sacrificed and the brains were processed for
24
measurement of caspase-3 activity as described below.
For the behavioral
studies, animals were assessed for locomotor sensitization in response to PCP
challenge (4 mg/kg, i.p.) on PN28-35.
Terminal dUTP Nick-End Labeling (TUNEL).
Slices used for TUNEL labeling were collected 12 hours [(time of peak cell
death in the slice (Wang & Johnson 2007)] after PCP treatment. Slices were first
rinsed with 0.01 M PBS (pH 7.2) and then fixed with ice-cold 2% paraformaldehyde
in 0.1 M PBS (pH 7.2) at room temperature for 1 hour. After washing with 0.01M
PBS (pH 7.2), slices were dehydrated and rehydrated in ethanol (70%, 90%, 100%,
90%, 70%, PBS), incubated with pepsin (0.04% in 10 mM HCl) for 15 min followed
by quenching of endogenous peroxidase with 3.0% hydrogen peroxide in methanol
for 10 min. After washing with PBS and pre-incubation with TdT (terminal
deoxynucleotidyl transferase) reaction buffer (30 mM Tris-HCl, pH 7.2, 140 mM Na
cacodylate and 1 mM CoCl2) for 15 min, slices were incubated with biotin-16-dUTP
(10 nmoles/ml) and TdT (200 U/ml) in the TdT buffer in a humidified chamber for 2
hours at 37°C. Slices were then washed in PBS, incubated with the Vectastain ABC
reagents for 60 min and stained with a filtered mixture of Vector SG peroxidase
substrate.
Caspase-3 Activity.
Organotypic slice samples used for caspase-3 activity measurement were
always collected 12 hours after PCP treatment; in vivo samples were collected 8 hrs
[peak time point for PCP-induced caspase-3 activation in the frontal cortex and
striatum (Wang & Johnson 2007, Wang & Johnson 2005)] after the injection on PN7
or 8 hrs after the last of the three injections on PN7, 9, & 11. Measurement of
caspase-3 activity was carried out as previously described (Wang & Johnson 2007).
Briefly, samples were sonicated in ice-cold lysis buffer containing 25 mM HEPES
(pH 7.4), 5 mM MgCl2, 1.5 mM EDTA, 1.0 mM EGTA, 1 mM DTT, 0.1% Triton X-100
25
and 1% protease inhibitor cocktail. After cooling on ice for 15 min, the sonicates
were centrifuged at 13,000 x g for 5 min at 4°C. The supernatants were then
collected for measurement of caspase-3 activity. Protein concentration was
measured by using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). To
measure the enzyme activity, each sample was prepared in two parallel sets. One
set consisted of an equal volume of supernatant and assay buffer (100 mM HEPES,
pH 7.4, containing 2 mM dithiothreitol, 0.1% CHAPS, and 1% sucrose). The other
set was a mix of equal volume of the same supernatant and assay buffer containing
the selective caspase-3 inhibitor, Z-DEVD-FMK (0.5 µM). After incubation at room
temperature for 15 min, the caspase-3 substrate, Ac-DEVD-AFC (25 µM), was
added and the samples were then incubated at 37°C for 60 min. Fluorescence
resulting from cleavage of the substrate was measured using a microplate
fluorometer (Fluoroskan Ascent, Labsystems, Helsinki, Finland) at excitation and
emission wavelengths of 405 and 510 nm, respectively. 7-Amino-4-trifluoromethylcumarin (AFC) was used as a fluorescent standard. Caspase-3 activity was
calculated as the difference of enzyme activities in samples incubated without and
with the caspase-3 inhibitor and then normalized to the total protein concentration.
Co-Immunoprecipitation and Western Blotting.
Crude synaptosomal protein extracts were prepared from 2 mm sections
corresponding to 4.7 to 2.7 mm anterior to Bregma for the frontal cortex and 0.7
mm to -1.3 mm for the striatum as previously described (Anastasio & Johnson
2008b) with minor modifications
Brain sections were homogenized (10 times
w/v) in ice cold Krebs-sucrose solution (125 mM NaCl, 1.2 mM KCl, 1.2 mM
MgSO4, 1.2 mM CaCl2, 22 mM Na2CO3, 1 mM NaH2PO4, 10 mM glucose, 0.32
M sucrose). Just prior to use, a protease inhibitor cocktail, consisting of 4-(2aminoethyl) benzenesulfonyl fluoride (AEBSF), pepstatin A, E-64, bestatin,
leupeptin, and aprotinin without metal chelators (Sigma-Aldrich, St Louis, MO) at
a concentration of 10 μL/mL was added to the lysis buffer. The homogenate was
26
then centrifuged at 1000 x g at 4oC. The supernatant (S1) was collected and
centrifuged at 16,000 x g at 4oC for 20 minutes to pellet the crude synaptosomes;
the resultant pellet was re-suspended in buffer and stored at -80oC. Total protein
concentrations were determined using the BCA (bicinchoninic acid) protein
assay© (Pierce Chemical, Rockford, IL).
In immunoprecipitation experiments,
100 µg membrane protein was incubated with antibodies (2 µg) against PSD-95,
SAP102, NR2A, or NR2B and the immune complexes were collected with 50 µl
of
protein
A/G–Sepharose
beads
(gently
shaken
overnight
at
4°C).
Immunoprecipitates were then washed 3 times with ice-cold PBS, and
resuspended in 2x Laemmli sample buffer, incubated in 90 0C water for 5 min,
and centrifuged at 15,000 x g for 1 min. The supernatant was subjected to SDSPAGE and transferred to polyvinylidene difluoride (PVDF) membranes (0.2 μm)
in a Mini Electrotransfer Unit (Bio-Rad, Hercules, CA) overnight. The blotting
was performed by repeated stripping and reprobing with anti-NR1, anti-NR2A,
anti-NR2B, anti-PSD-95, and anti-SAP102.
Analysis was carried out using
enhanced chemiluminescence (ECL) plus Western blotting detection reagents
(Amersham Biosciences, Piscataway, NJ).
The bands corresponding to the
various proteins of interest were scanned and densitometrically analyzed by
using an automatic imaging analysis system (Alpha Innotech Corporation, San
Leandro, CA).
All quantitative analyses were normalized to β-actin (after
stripping [Reblot mild, Chemicon International, Temecula, CA]).
Antibodies.
Monoclonal anti-NR1, anti-NR2A, and anti-NR2B antibodies were
purchased from BD Biosciences (San Jose, CA). Monoclonal anti-PSD-95 and
anti-SAP102 were purchased from Cell Signaling (Danvers, MA).
Primary
antibody dilution was 1:500-1:1000. Secondary antibodies were purchased from
Zymed (Invitrogen Corporation, Carlsbad, CA) and used at a concentration of
1:5000.
27
Locomotor Activity.
On the day of testing, animals were placed in locomotor activity chambers
and allowed to habituate for 30 minutes prior to a challenge dose of PCP (4
mg/kg, i.p.), PEAQX (4 mg/kg, i.p.) or ifenprodil (5 mg/kg, i.p.). Locomotor activity
was measured for an additional 90 minutes in an open-field activity system (San
Diego Instruments, San Diego, CA) which consisted of a square enclosure with
Plexiglas walls (40 x 40 x 40 cm). Horizontal activity was measured with a 4 x 4
photobeam matrix which recorded both central and peripheral activity in 5 min
bins as previously described (Anastasio & Johnson 2008a).
Statistical Analysis.
Group comparisons were specifically defined before the beginning of each
experiment; therefore, planned comparisons were performed instead of an
overall F test in a multifactorial ANOVA (Keppel 1982). Statistical comparisons
for each biochemical and for locomotor sensitization experiments were
conducted using a one-way ANOVA. All values are presented as mean ± SEM.
The null hypothesis was rejected at p<0.05.
Results
Inasmuch as this laboratory has previously characterized PCP-induced
neurotoxicity in the corticostriatal slice culture (Wang & Johnson 2005, Xia et al.
2008) we decided to build on these studies and to investigate which NMDAR
subunit may mediate cell death. Organotypic brain slices can serve as an in vitro
model that conserves the biologically relevant structural and functional features
of in vivo tissues (Vickers & Fisher 2004), while also allowing easier manipulation
of drugs than possible in vivo. PCP (3 µM) and the NR2A antagonist NVPAAM007 (100 nM, 1µM, and 10 µM) caused robust TUNEL-positive staining
compared to control (Figure 2.1). No apoptosis was evident in slices treated
28
with 100 nM, 1 µM, or 10 µM ifenprodil (Figure 2.1). Measurement of caspase-3
activity also showed that 3 µM PCP and 3 µM PEAQX produced similar caspase3 activation, while NVP-AAM007 was approximately three-fold more potent
(Figure 2.2). Ifenprodil had no effect on caspase-3 activity in the slice culture
(Figure 2.2).
As previously reported, PCP (10 mg/kg) treatment on either PN7 or on
PN7, 9, & 11 results in robust caspase-3 activation in the frontal cortex (Figures
2.3 and 2.5, respectively) (Wang & Johnson 2007). NVP-AAM007 (10 mg/kg)
also significantly increased cleaved caspase-3 compared to control (data not
shown) and blockade of NR2A subunits with PEAQX dose dependently
increased cortical caspase-3 activity following both acute (Figure 2.3) and subchronic administration (Figure 2.5). In contrast, as observed in vitro, blockade of
NR2B subunits with ifenprodil on PN7 (Figure 2.3) or on PN7, 9, & 11 had no
significant effect on cleavage of caspase-3 at 1, 5 or 10 mg/kg in the frontal
cortex (Figure 2.5). PCP administration or PEAQX (20 mg/kg and 40 mg/kg) on
PN7 resulted in a 6-8 fold increase in caspase-3 activity in the striatum, but no
effect of ifenprodil was observed (Figure 2.4).
Similarly, subchronic
administration of 20 mg/kg PEAQX treatment on PN7, 9, & 11 caused an 8-fold
increase in caspase-3 activity in the striatum, but no effect of PCP or ifenprodil
was observed in this brain region (Figure 2.6). The highest dose of PEAQX (40
mg/kg) was toxic to the animals when administered on PN7, 9, & 11, i.e. 8 hrs
after the third injection all of the pups had lost ~33% of their starting body weight
suggesting a failure to thrive.
29
Figure 2.1.
Representative photomicrographs showing TUNEL-positive
staining in corticostriatal organotypic slice cultures. Cultures were treated
on DIV 9 for 12 hrs with 3 μM PCP, various doses of NVP-AAM007, or various
doses of ifenprodil. PCP and NVP-AAM007 dose dependently caused robust
apoptosis compared to control; no cell death was evident in ifenprodil treated
cultures. N=3/treatment
30
Figure 2.2. Effects of PCP administration on neurotoxicity in corticostriatal
slice cultures. 3 μM PCP (DIV 9 for 12 hrs) to corticostriatal organotypic slice
cultures activates caspase-3.
NVP-AAM007 and PEAQX dose-dependently
activate caspase-3 while ifenprodil does not.
control (one-way ANOVA with Dunnett’s method)
31
N=5-7/treatment, *p<0.05 vs.
PN7-Frontal Cortex
Figure 2.3. Effects of neonatal PCP and selective NR2A and NR2B
antagonists on frontal cortex neurotoxicity in PN7 rats. Sprague-Dawley rat
pups were treated on PN7 with saline, 10 mg/kg PCP (s.c.), various doses of
PEAQX (s.c.), or various doses of ifenprodil (i.p.). PCP and PEAQX resulted in
caspase-3 activation in the frontal cortex 8 hrs after treatment. N=5-7/treatment,
*p<0.05 vs. saline (one-way ANOVA with Holm-Sidak method)
32
PN7-Striatum
Figure 2.4. Effects of neonatal PCP and selective NR2A and NR2B
antagonists on striatal neurotoxicity in PN7 rats. Sprague-Dawley rat pups
were treated on PN7 with saline, 10 mg/kg PCP (s.c.), various doses of PEAQX
(s.c.), or various doses of ifenprodil (i.p.). PCP and PEAQX resulted in caspase-3
activation in the striatum 8 hrs after treatment. N=5-7/treatment, *p<0.05 vs.
saline (one-way ANOVA with Holm-Sidak method)
33
PN7, 9, &11-Frontal Cortex
Figure 2.5.
Effects of neonatal PCP and selective NR2A and NR2B
antagonists on frontal cortex neurotoxicity in young rats. Sprague-Dawley
rat pups were treated on PN7, 9, & 11 with saline, 10 mg/kg PCP (s.c.), various
doses of PEAQX (s.c.), or various doses of ifenprodil (i.p.). PCP and PEAQX
resulted in caspase-3 activation in the frontal cortex 8 hrs after the last of 3
injections.
N=5-7/treatment,
*p<0.05
Bonferroni's post hoc test)
34
vs.
saline
(one-way
ANOVA
with
PN7, 9, & 11-Striatum
Figure 2.6.
Effects of neonatal PCP and selective NR2A and NR2B
antagonists on striatal neurotoxicity in young rats. Sprague-Dawley rat pups
were treated on PN7, 9, & 11 with saline, 10 mg/kg PCP (s.c.), various doses of
PEAQX (s.c.), or various doses of ifenprodil (i.p.). PCP and PEAQX resulted in
caspase-3 activation in the striatum 8 hrs after the last of three injections. N=57/treatment,*p<0.05 vs. saline (one-way ANOVA with Holm-Sidak method)
35
Since, the sub-chronic paradigm more closely models the schizophrenic
disease state (Anastasio & Johnson 2008a), this treatment paradigm was carried
through for the remaining experiments in this study. In order to determine the
subunit composition of synaptic and extrasynaptic NMDAR receptors following
sub-chronic PCP administration, we took advantage of the predominately
synaptic localization of PSD-95 and the extrasynaptic localization of SAP102 and
performed co-immunoprecipitation (Sans et al. 2000, van Zundert et al. 2004).
Quantification of these results indicate that PCP treatment on PN7, 9, & 11
increases the association of PSD-95 with NR2A, but not NR2B (Figure 2.7A).
Sub-chronic PCP administration results in a significant increase in the
association of both NR2A and NR2B with SAP102 in the cortical synaptosomal
fraction (Figure 2.7B). To verify these results, reverse pull-down assays were
performed by precipitating with the NR2 subunit antibodies and subsequent
probing for PSD-95 and SAP102 with specific antibodies for these proteins.
These experiments yielded results similar to those obtained above (data not
shown). Further, to authenticate the presence of fully assembled and functional
NMDARs, all membranes were probed for the obligatory NR1 subunit. The NR1
subunit was associated with NR2A and NR2B in both synaptic and extrasynaptic
fractions (data not shown).
36
Figure 2.7. Effects of PCP administration on the localization of NR2A and
NR2B in the synaptic or extrasynaptic milieu. Cortical crude synaptosomal
extract (100 µg) from rat pups 8 hrs after treatment were incubated with 2 µg
antibody against PSD-95 or SAP102. The blots were probed sequentially, after
stripping, with antibodies against NR2A or NR2B. Quantified results indicate in
A) PCP treatment on PN7, 9, & 11 increases the association of NR2A with PSD95. B) Sub-chronic PCP treatment results in an increase in the association of
NR2A and NR2B with SAP102.
Values shown are mean ± SEM n=4/treatment
group, * p < 0.05 (Student’s t- test)
37
To better understand the relationship between PCP-induced neurotoxicity
and the development of adverse behaviors in the rat, we extended our previous
study (Anastasio & Johnson 2008a) and investigated the effects of selective
NR2A and NR2B antagonists on the development of locomotor sensitization.
Sub-chronic PCP and PEAQX (10 and 20 mg/kg) showed enhanced locomotor
activity in response to PCP challenge (4 mg/kg) compared to saline pretreatment
(F3, 73=4.99) (Figure 2.8A); however, pretreatment with either dose of ifenprodil
(1 or 5 mg/kg) did not enhance the locomotor response to PCP challenge. The
time courses of the locomotor responses to PCP challenge for all treatment
groups are shown in Figure 2.8B. We did not determine the effects of 10 mg/kg
ifenprodil in the behavior studies because the lower doses had no effect on
cortical neurotoxicity or locomotor sensitization.
The reciprocal locomotor
sensitization experiments were also performed in order to determine whether
blockade of either the NR2A or NR2B subunits alone can activate a sensitized
locomotor response similar to PCP challenge.
The effects of PEAQX or
ifenprodil challenge on locomotor activity following treatment during development
with either saline, PCP, PEAQX, or ifenprodil are shown in Figures 2.9A and
2.9C.
Acute challenge with either PEAQX (4 mg/kg) (F2,23=2.098) or ifenprodil
(5 mg/kg) (F2,32=1.342) on PN28-35 did not significantly increase locomotor
activity in rats treated sub-chronically with either PCP, PEAQX or ifenprodil on
PN7, 9 and 11. Similarly treated rats were also co-challenged with both PEAQX
and ifenprodil on PN28-35 and no alterations in locomotor activity were observed
(data not shown).
38
Figure 2.8. Locomotor sensitization induced by a 4 mg/kg PCP challenge to
either sub-chronic PCP or PEAQX treated animals.
Animals treated sub-
chronically with ifenprodil do not show a sensitized response to PCP challenge.
(left) Time course of locomotor activity (right) N=8-26/treatment group *p<0.05
vs. saline (one-way ANOVA with Holm-Sidak method)
39
Figure 2.9. Effect of selective blockade of NR2A and NR2B subunits on
locomotor sensitization.
Neither PEAQX nor ifenprodil challenge results in
locomotor sensitization as measured on PN28-35 (A and C). The time course of
locomotor activity is shown in panels B and D. N=8/treatment group.
40
Discussion
Neither the mechanism of PCP-induced neurotoxicity in perinatal rats, nor
the association between neurotoxicity and the development of behavioral deficits
as the animal matures, is completely understood. Further, the role played by
specific NMDAR subunits in either is undefined. Therefore, the focus of this
study was to investigate the role of NR2A and NR2B receptors in PCP-induced
neurotoxicity and the development of aberrant behaviors in the immature rat.
Through a pharmacological approach, we discovered that administration of
NR2A-preferring antagonists mimicked PCP-induced neurotoxicity in the frontal
cortex, while administration of a selective NR2B antagonist had no effect. This
strongly suggests that cortical neurotoxicity following PCP administration is due
to blockade of NMDA receptors that contain NR2A subunits, rather than those
that contain NR2B subunits. Moreover, blockade of NR1/NR2A receptors during
the early postnatal period results in locomotor sensitization in response to PCP
challenge.
During the embryonic stage and shortly after birth, neurons primarily
express NR1/NR2B-containing NMDARs (Monyer et al. 1994, Akazawa et al.
1994).
Within the first postnatal week, NR2A subunit mRNA expression is up-
regulated during a period of rapid synapse formation (Akazawa et al. 1994,
Monyer et al. 1994).
The increase in synaptic NR2A-containing NMDARs is
thought to correlate with a gradual decrease of NR2B-containing synaptic
NMDARs. Generally, NR2B-containing receptors translocate during early
development to the extrasynaptic space; however, depending on the brain
region, some may remain in the synaptic space (Cull-Candy et al. 2001, Thomas
et al. 2006).
Excessive over-activation of synaptic NMDARs results in Ca2+
overload and neuronal excitotoxicity. Blockade of synaptic NMDAR activation
has been shown to be neuroprotective in animal models of stroke and seizure
(Lei et al. 2008, Lee et al. 1999, Hardingham & Bading 2002, Hardingham et al.
2002). Generally, it appears that synaptic NMDARs induce a coordinate up41
regulation of newly identified pro-survival genes and down-regulation of prodeath genes while extrasynaptic NMDARs appear not to activate pro-survival
genes, but instead induce a putative Ca2+ activated Cl- channel that kills neurons
(Hardingham & Bading 2003, Hardingham & Bading 2002, Hardingham et al.
2002).
Further, it has been suggested that it is the spatial distribution and not
the subunit composition per se that determines the NMDAR-dependent signaling
(Ivanov et al. 2006) and its downstream consequences. Conversely, the division
of function between synaptic and extrasynaptic NMDARs has been challenged
by data showing that excitotoxic insults are preferentially mediated by NR2B
containing receptors, regardless of localization, while NR2A receptors exerted a
neuroprotective effect against both NMDAR-mediated and non-NMDARmediated toxic insults (Liu et al. 2007, Zhang et al. 2007).
Here, we hypothesized that PCP-induced neurotoxicity is in part mediated
through blockade of synaptic NR1/NR2A receptors. We compared two NR2A
preferential antagonists, NVP-AAM007 and PEAQX, to ifenprodil, a selective
NR2B subunit antagonist. NVP-AAM007 and PEAQX have greater than 10-fold
selectivity for NR2A (Ki=5.4 nM) compared to NR2B (Ki=67 nM), though only a 2fold selectivity over the NR2C subunit (Ki=11.6 nM), which is sparingly found in
the frontal cortex (Feng et al. 2004). Ifenprodil is a relatively selective inhibitor of
the polyamine binding site on the NR2B subunit and has a 400-fold lower IC50
for NR2B-containing receptors compared to NR2A or NR2C-containing receptors
(Williams 2001). We first compared these antagonists in cultured organotypic
slices and observed robust neurotoxicity following incubation with either PCP or
the NR2A-preferring antagonists, NVP-AAM007 and PEAQX. However, no effect
of the NR2B-selective antagonist, ifenprodil, was observed. Although PEAQX
and NVP-AAM007 are not truly specific for the NR2A subunit over the NR2B
subunit, the lack of effect of ifenprodil strongly support the hypothesis that PCPinduced neurotoxicity is mediated largely through its blockade of NR2Acontaining receptors.
At this time in our model system it is not possible to
42
definitively state that the NR1/NR2A receptors that are blocked and mediate
PCP-induced neurotoxicity are located only in the synapse. Nevertheless,
pharmacological manipulation with preferential NR2A and NR2B antagonists
suggests that the subunit composition does play a prominent role in neurotoxicity
in the frontal cortex of the developing rat, though PCP-induced neurotoxicity may
be dependent on both the spatial distribution and subunit composition.
This laboratory has previously reported that acute PCP administration on
PN7 causes apoptosis in the cortex and striatum, but evidence of toxicity
following PCP administration on PN7, 9 and 11 is found only in the cortex (Wang
& Johnson, 2005; Wang & Johnson 2007). In the current experiments, we again
observed
a
developmental and
pharmacological
specific
PCP-mediated
neurotoxicity; such that caspase-3 activation was observed in the striatum
following a single injection on PN7, yet no PCP-mediated neurotoxicity was
observed in the striatum following treatment on PN7, 9, & 11. We have previously
attributed this to “tolerance” or an undefined, developmentally regulated,
susceptibility mechanism. Interestingly, in the current study, unlike PCP, the
NR2A-preferring antagonist, PEAQX, induced neurotoxicity following sub-chronic
treatment on PN7, 9, & 11.
At this time, the reason for this discrepancy between PEAQX and PCPmediated neurotoxicity is unknown; however, we speculate that the difference
between PCP and PEAQX may be related to the neuronal type, specifically the
striatal interneurons, affected by these compounds.
This laboratory has
reported that a single injection of PCP on PN7 does not cause a loss of
GABAergic interneurons containing either parvalbumin (PV), calretinin (CR) or
calbindin (CB) in the adult striatum (PN56), but does cause a loss of PVcontaining interneurons in the frontal cortex (Wang et al. 2008).
Chen and
Reiner (1996) demonstrated that there is a differential distribution of NR2A/NR2B
subunits on striatal neuron types, such that somatostatin- and about half of the
PV-containing interneurons do not possess either NR2A or NR2B subunits
43
(somatostatin and PV-containing interneurons account for approximately onepercent of all striatal neurons). Cholinergic interneurons and the majority of the
identified striatal projection, striatonigral, enkephalin-containing, and striatal
matrix neurons do express both NR2A or NR2B (Chen & Reiner 1996);
therefore, PEAQX-induced neurotoxicity may be due to its blockade of these
striatal neurons which express the NR2A subunit, e.g. cholinergic interneurons
and striatal projection neurons.
Further, these cholinergic interneurons have
been reported to possess a greater resistance to NMDA-mediated toxicity
compared to the striatal projection neurons (Araki et al. 1994, FigueredoCardenas et al. 1994). It has been postulated that these cholinergic interneurons
do not express AMPA receptors (Cheng et al. 1996); therefore, without AMPA
receptor activation and depolarization, it would not be possible to alleviate the
voltage-dependent Mg2+ block of NMDARs and the open channel blocker, PCP,
would not be able to gain entry to the NMDA channel or inhibit its function,
thereby sparing these neurons. Thus, this may explain the temporal neurotoxic
effects of PCP, i.e. a single injection of PCP results in the death and clearance of
the projection neurons leaving behind the heartier cholinergic interneurons which
are resistant to PCP-induced neurotoxicity, but are sensitive to PEAQX blockade.
While division of NR2A and NR2B receptors into synaptic and
extrasynaptic pools, respectively, is not completely true in all instances, it
provides a framework for this study.
Furthermore, although the biological
relevance of the segregation of NMDARs into synaptic and extrasynaptic locales
is incompletely understood, the mechanism of targeting the NMDAR to these
different sites is thought to involve the C-terminal tail of the NR2 subunit and its
interactions with PDZ domain proteins in the postsynaptic density (Prybylowski et
al. 2005).
The PDZ proteins play a fundamental role in linking the NMDAR to
intracellular signaling cascades (Prybylowski et al. 2005). Further, the differential
association with the MAGUK (membrane-associated guanylate kinase) family
members may be vital not only for the regulation and expression of the NMDAR,
44
but also for the localization of NMDAR subtypes during development and
subsequently, the response to noxious agents, such as PCP (Kim & Sheng 2004,
Cousins et al. 2008). PSD-95 is the most highly enriched PDZ protein in the
postsynaptic density and is thought to predominantly associate in vivo with
NR2A, while SAP102 is commonly expressed in dendrites, axons and cytoplasm
as well as the synaptic and extrasynaptic spaces and associates with primarily
with NR2B (Kim & Sheng 2004, Sans et al. 2000). There is also a developmental
expression pattern for SAP102 and PSD-95, whereby SAP102 predominates in
the synapse early in postnatal life and then PSD-95 gradually increases in the
synaptic space as the animal matures (Kim & Sheng 2004). Interestingly, this
developmental pattern mirrors the developmental “switch” in expression pattern
of NR2B to NR2A, supporting earlier reports that in immature synapses NR2BSAP102 complexes are predominant, but as the animals age, NR2B subunits
may be translocated to the extrasynaptic space as NR2A-PSD-95 expression
increases in the mature synaptic milieu (Sans et al. 2000, Kim & Sheng 2004,
Cull-Candy et al. 2001) .
We
performed
co-immunoprecipitation
studies
to
determine
the
localization of cortical NMDARs in a crude synaptoneurosomal preparation from
postnatal cortex.
Several studies have shown the number of NMDARs, their
subunit composition, as well as their postsynaptic linkers can be altered following
administration of NMDA antagonists including MK-801, ethanol and PCP (Dong
et al. 2004, Anastasio & Johnson 2008b, Sircar et al. 1996, Suvarna et al. 2005,
du Bois et al. 2009). These alterations in expression of the NMDAR have been
suggested to be due to changes in synthesis, trafficking of the receptor from
intracellular compartments to the membrane, or lateral diffusion within the
membrane (Anastasio & Johnson 2008b, Tovar & Westbrook 2002, Choquet &
Triller 2003).
We demonstrated that following PCP treatment on PN7, 9, & 11,
there is an apparent compensatory up-regulation in the surviving neurons of
NR1/NR2A/PSD-95, while no statistically significant effect on NR1/NR2B/PSD-95
45
was measured. This response to PCP-induced neurotoxicity by neighboring
neurons may represent a protective mechanism to prevent further damage to the
brain and/or restore cortical NMDA-mediated neurotransmission and synaptic
communication. An increase in the association of both NR2A and NR2B with
SAP102 following PCP administration was also observed. While it is reasonable
to postulate that these receptors exists as functional heterodimers, the
segregation of these subunits into a heterotrimeric receptors in either the
synaptic or extrasynaptic space cannot be ruled out. Interestingly, we observed
no effect of PCP treatment on the relationship between NR1 and SAP102 (data
not shown). An alternative explanation of this is that in the extrasynaptic space
there is an increase in NR2A or NR2B subunits that is not associated with NR1,
suggesting the possibility of an effect on the composition of the NMDAR resulting
in the insertion and expression of a nonfunctional NMDAR linked to SAP102
most likely in the extrasynaptic milieu, but may also be in the synaptic locale.
This would also represent an alternative, pro-survival compensatory mechanism
rendering the neuron less susceptible to glutamate toxicity (Vanhoutte & Bading
2003).
The neurotoxicity evident in the frontal cortex following either a single
injection or multiple injections suggests a role for altered cortical function in either
the poor performance of certain behavioral tasks or the development of aberrant
behaviors (Wang & Johnson 2007, Wang & Johnson 2005, Wang et al. 2001).
The neurodevelopmental theory of schizophrenia states that damage to the brain
during a critical stage of brain maturity (PN7-11 in the rat) may account for the
development of mental illness later in life due to impaired synapse formation and
neurite outgrowth (Olney et al. 2002, du Bois & Huang 2007).
Therefore, we
tested the hypothesis that PCP-induced cortical neurotoxicity plays a role in the
development of locomotor sensitization and that both involve blockade of
synaptic NR1/NR2A subunits during a critical stage in brain development. This
study established that PCP administration on PN7, 9, & 11 resulted in an
46
enhanced locomotor response to PCP challenge on PN28-35, and a similar
result was observed following treatment with the NR2A antagonist, PEAQX. On
the other hand, animals treated with ifenprodil on PN7, 9, & 11 exhibited no
change in locomotor activity in response to PCP challenge. Thus, these data
strongly support the hypothesis that blockade of NR2A receptors by PCP
treatment on PN7, 9 and 11 and the subsequent cell death alters the architecture
of the cortex as well as glutamatergic and GABAergic synaptic transmission (du
Bois et al. 2009, Olney & Farber 1995a) resulting in
the development of a
sensitized locomotor response to PCP challenge.
Interestingly, these experiments demonstrate that the effect of the PCP
challenge on test day (PN28-35) to either developmentally treated PCP or
PEAQX animals is not due to the immediate blockade of either NR2A or NR2B
by PCP. That is, since challenge with either PEAQX or ifenprodil, or a
combination of the two, did not result in an enhanced locomotor response, then it
follows that blockade of either NR2A or NR2B alone or in combination is
insufficient to produce an increased behavioral response and suggests that the
expression of PCP-induced sensitization requires activation of mechanisms apart
from blocking either NR2A or NR2B independently or simultaneously. It is quite
possible that the actual expression of sensitization requires NMDA receptorindependent effects of PCP such as blockade of DA or 5-HT uptake or inhibition
of cholinergic receptors or K+ channels (Verma & Moghaddam 1996, Johnson &
Jones 1990, Lodge & Johnson 1990).
In summary, PCP induces neurotoxicity in developing pups in a manner
that is highly regulated and is dependent on the distribution as well as the
composition of the NMDAR. The consequences of this loss of cortical neurons
and altered cortical landscape are associated with behavioral deficits that are
similar to the positive symptoms of schizophrenia.
Further, future work
delineating the role of NR2 subunits in the neurotoxic process may lead to the
development of future pharmacotherapeutics for schizophrenia.
47
Chapter 3: Differential Regulation of the NMDAR by
Acute and Sub-chronic Phencyclidine Administration
Neurodegeneration induced by the NMDAR antagonist phencyclidine
(PCP) has been used to model the pathogenesis of schizophrenia in the
developing rat. Acute and sub-chronic administration of PCP in perinatal rats
results in different patterns of neurodegeneration. The potential role of an
alteration in the membrane expression of NMDARs in PCP-induced degeneration
is unknown. Acute PCP treatment on PN7 increased membrane levels of both
NR1 and NR2B proteins in the frontal cortex; conversely, NR1 and NR2B protein
levels in the endoplasmic reticulum (ER) fraction were decreased. Acute PCP
administration also resulted in increased membrane cortical protein levels of
postsynaptic density-95 (PSD-95), as well as the activation of calpain, which
paralleled the observed increase in membrane expression of NR1 and NR2B.
Further, administration of the calpain inhibitor, MDL28170, prevented PCPinduced up-regulation of NR1 and NR2B. On the other hand, sub-chronic PCP
treatment on PN 7, 9 and 11 caused an increase in NR1 and NR2A expression,
which was accompanied by an increase in both NR1 and NR2A in the ER
fraction. Sub-chronic PCP administration did not alter levels of PSD-95 and had
no effect on activation of calpain. These data suggest that increased trafficking
accounts for up-regulation of cortical NR1/NR2B subunits following acute PCP
administration, while increased protein synthesis likely accounts for the increased
expression of NR1/NR2A following sub-chronic PCP treatment of the developing
rat.
These
results
are
discussed
in
the
context
of
the
differential
neurodegeneration caused by acute and subchronic PCP administration in the
developing rat brain.
48
Introduction
PCP is a potent noncompetitive NMDAR antagonist [Ki~100 nM (Anis et
al. 1983)] that has been shown to mimic both the positive and negative
symptoms of schizophrenia in humans and to exacerbate psychosis in
schizophrenics (Luby et al. 1962, Javitt & Zukin 1991).
Since the symptoms of
schizophrenia do not appear until early adulthood, Weinberger (1987) postulated
that the etiology of schizophrenia may be developmental in nature and that the
primary pathological insult may occur in utero or early in postnatal development
(Benes 1991, Murray et al. 1992, Pilowsky et al. 1993). The nature of this insult
is unknown, but it could involve glutamatergic hypofunction and may involve a
loss of certain cortical interneurons and/or an altered organization of cortical
connectivity (Benes 1991, Benes 1995, Lewis 1997). This led to investigation of
the neurotoxic effects of acute PCP or MK-801 during development in the rat (Lei
et al. 2008, Ikonomidou et al. 1999, Wang & Johnson 2005, Wang & Johnson
2007). While acute PCP administration on PN7 causes wide-spread apoptosis
(Ikonomidou et al. 1999, Wang et al. 2005, Wang & Johnson 2007) evidence of
apoptosis including positive caspase-3 immunoreactivity and TUNEL staining is
restricted to the cortex following sub-chronic PCP administration on postnatal day
(PN) 7, 9, and 11 (Wang et al. 2001, Wang & Johnson 2007, Wang & Johnson
2005). These data suggest the presence of a developmentally regulated
tolerance in these regions (Wang et al. 2001, Wang & Johnson 2007, Wang &
Johnson 2005).
Over activation of NMDARs has been shown to cause damage that will
eventually kill neurons in a process known as excitotoxicity (Lynch & Guttmann
2002). It is generally agreed that activation of NMDARs causes an increase in
Ca2+ influx leading to intracellular Ca2+ overload (Abdel-Hamid & Tymianski 1997,
Garthwaite & Garthwaite 1986, Lei et al. 1992), activation of pro-death proteins
including caspase-3 and ultimately neurotoxicity. Other studies have shown that
NMDAR-mediated neurotoxicity involves increased superoxide formation and the
49
activation of the proteolytic enzyme caspase-3 resulting in increased NF-κB
nuclear transport. These mechanisms are activated by excessive intracellular
Ca2+ levels (McInnis et al. 2002, Qin et al. 2000, Takadera et al. 1999), most
likely secondary to increased NMDAR function and support the hypothesis that
PCP-induced neurotoxicity may be mediated by increases in the NMDAR density
(Wang et al. 2005). Thus, further investigation of PCP-induced regulation of the
NMDAR in conjunction with neurotoxicity may shed light on the molecular
mechanisms underlying the psychotomimetic properties of PCP, particularly
those
mediating
the
schizophrenia-like
symptoms
following
perinatal
administration (Wang et al. 2001).
The NMDAR is composed of multiple subunits including NR1, NR2A-D
and NR3A/B. The NR1 subunit forms a heteromeric complex with one of the four
NR2 (A-D) subunits (Kutsuwada et al. 1992, Monyer et al. 1994).
The
composition of the NR1/NR2 complex differs in regard to their regional pattern of
expression, their regulation by phosphorylation, polyamines or protons, their
electrophysiological properties, and their affinity for cytoskeletal proteins (Paoletti
& Neyton 2007, Kohr 2006, Waxman & Lynch 2005, Westbrook et al. 1997).
Each subunit of the NMDAR contains an extracellular N-terminal region, three
membrane spanning domains, one intramembrane loop and an intracellular Cterminal region. The C-terminal region of the NR2 subunit is thought to be a
substrate for the calcium-activated neutral protease calpain (Wu & Lynch 2006,
Wechsler & Teichberg 1998, Guttmann et al. 2001, Guttmann et al. 2002, Bi et
al. 1998a, Bi et al. 1998b). Further, this region serves as a binding site for
intracellular anchoring proteins such as PSD-95, SAP102, and SAP97 and also
links second messenger cascades to the NMDAR, thereby modulating the
relations of
the
receptor with
various
intracellular signaling
cascades
(Niethammer et al. 1996, Bi et al. 1998b, Wechsler & Teichberg 1998, Wu &
Lynch 2006, Bi et al. 1998a, Guttmann et al. 2001, Guttmann et al. 2002, Zheng
et al. 1999)). Furthermore, the C terminus has been suggested to play a strong
50
role in regulating trafficking of the NR1 subunit (Wenthold et al. 2003) and posttranslational modifications of this region play a role in the activity of the NMDAR
localization and function (Guttmann et al. 2002).
Therefore, the purpose of the present study was to characterize the effect
of both acute (PN7) and sub-chronic (PN7, 9, & 11) PCP treatment on the
regulation of the NMDAR subunits (NR1, NR2A, and NR2B) as well as PSD-95,
a member of the NMDAR postsynaptic density complex, in the frontal cortex in
order to determine the possible role of NMDAR trafficking and expression in
PCP-induced neurodegeneration and later behavioral deficits.
Materials and methods
Animals.
Timed, day 14 pregnant female Sprague-Dawley rats were obtained from
Charles River Laboratories (Wilmington, MA).
The dams were housed
individually with a regular 12h light-dark cycle (lights on 0700, off at 1900) with
food and water ad libitum. Following parturition, male and female pups from four
dams were combined and randomly cross-fostered to one of the four lactating
dams.
Each litter consisted of ten to twelve pups.
All experiments were
conducted in accordance with the NIH and the Institutional Animal Care and Use
Committee.
Drugs.
Phencyclidine was acquired from the National Institute on Drug Abuse
(NIDA, Rockville, MD) and dissolved in 0.9% NaCl.
MDL28170 (10 mg/kg,
Sigma-Aldrich, St. Louis, MO) was dissolved in 1% DMSO. Doses were chosen
based on prior experiments that addressed PCP-induced regulation of the
NMDAR (Wang et al. 2001) and our own work with the compounds. Injections
were administered i.p. or s.c. in 1 mL/kg of vehicle.
51
Experimental design.
Male and female rat pups were treated on either PN7 (acute) or on PN 7,
9, and 11 (sub-chronic) with 10 mg/kg subcutaneous (s.c.) PCP or saline. In the
acute studies, pups were sacrificed by decapitation on PN7 at 0, 4, 8, or 24 hours
following saline/vehicle (control, time=0 hours), PCP/vehicle treatment.
In the
acute MDL28170 studies, pups were treated with PCP on PN7 or post-treatment
with MDL28170 (10 mg/kg, i.p., every 2 hours for 8 hours) and then sacrificed by
decapitation 24 hours following saline/ vehicle, PCP/vehicle, MDL28170/vehicle,
MDL28170+PCP/vehicle treatment.
In the sub-chronic studies, pups were
sacrificed by decapitation 24 hours following the last injection of the
aforementioned drug regimens on PN7, 9 and 11. For both acute and subchronic biochemical studies, the frontal cortex was dissected as described below
and used for Western blot analysis.
Sub-cellular fractionation.
Protein extracts were prepared from 2 mm sections corresponding to 4.7
to 2.7 mm anterior to Bregma for the frontal cortex as previously described with
some modifications (Wang et al. 2001, Paxinos & Watson 1986). Cortical brain
sections were homogenized in 500 μL of lysis buffer with the aid of an automatic
tissue grinder (Kontes Pellet Pestle Motor, Kimble / Kontes, Vineland, New
Jersey). The lysis buffer consisted of 10 mM HEPES (pH 7.4), 1 mM EDTA, 2
mM EGTA, and 500 μM DTT. Just prior to use, protease inhibitor cocktail [4-(2aminoethyl) benzenesulfonyl fluoride (AEBSF), pepstatin A, E-64, bestatin,
leupeptin, and aprotinin without metal chelators (Sigma-Aldrich, St Louis, MO)] at
a concentration of 10 μL/mL was added to the lysis buffer. The homogenate was
then centrifuged at 1000 x g at 4oC for 10 minutes to pellet the nuclear protein
fraction (P1). The supernatant (S1) was collected and centrifuged at 8000 x g at
4oC for 30 minutes to pellet the membrane bound protein fraction (P2). The
cytoplasmic fraction (S2) was collected and stored at -80oC. P2 (membrane
52
fraction) was re-suspended in homogenization buffer and centrifuged at 20,000 x
g at 4oC for 30 minutes; the resultant pellet was re-suspended in lysis buffer +
1% SDS, boiled for 10 minutes and stored at -80oC. To isolate endoplasmic
reticulum (ER) protein, the S2 fraction was centrifuged at 100,000 x g at 4 oC for
60 minutes. P3 (ER fraction) was re-suspended in lysis buffer and stored at 80oC.
Total protein concentrations were determined using the BCA
(bicinchoninic acid) protein assay© (Pierce Chemical, Rockford, IL).
Western blot analysis.
Equal amounts of protein were separated on 10% Bis-Tris gels
(Invitrogen, NY) using SDS-PAGE with a MES-SDS running buffer system, pH
7.4. Following electrophoresis (110 V for 2 hours), proteins were transferred to
polyvinylidene difluoride (PVDF) membranes (0.2 μm) in a Mini Electrotransfer
Unit (Bio-Rad, Hercules, CA) overnight.
The membrane was blocked in 5%
nonfat milk, followed by incubation with the primary antibody in 1% milk for 2
hours at room temperature. Following washes (3 x 10 minutes) in TBS+0.1%
Tween 20 (TBST), the membrane was incubated with horseradish peroxidase
conjugated secondary antibodies for 1 hour at room temperature. Analysis was
carried out using the enhanced chemiluminescence (ECL) plus Western blotting
detection reagents (Amersham Biosciences, Piscataway, NJ).
corresponding to the various proteins of
interest were
The bands
scanned and
densitometrically analyzed by using an automatic imaging analysis system
(Alpha Innotech Corporation, San Leandro, CA). All quantitative analyses were
normalized to β-actin (after stripping [Reblot mild, Chemicon International,
Temecula, CA]).
Antibodies.
The monoclonal anti-NR1, anti-NR2A, anti-NR2B, and anti-protein
disulfide isomerase (PDI) were purchased from BD Biosciences (San Jose, CA).
53
The monoclonal anti-PSD-95 and anti-αII spectrin antibodies were purchased
from Chemicon International (Temecula, CA).
Primary antibody dilution was
1:500-1:1000. Secondary antibodies were purchased from Zymed (Invitrogen
Corporation, Carlsbad, CA) and used at a concentration of 1:5000.
Results
It has been reported that neurotoxicity caused by non-competitive NMDAR
antagonists is dependent on development with the most vulnerable time existing
in the early postnatal period in rats (Ikonomidou et al. 1999, Wang & Johnson
2007, Wang & Johnson 2005). In order to ascertain the effects of acute PCP (10
mg/kg) treatment on the expression of NMDAR subunits, membrane protein from
the frontal cortex was extracted on PN 7 at 0, 4, 8 and 24 hours after saline or
PCP
treatment.
Saline
treated
animals
sacrificed
immediately
after
administration (t=0 hour) on PN7 served as the control in the biochemical studies
of the time course of acute PCP effects.
Figure 3.1A shows representative
Western blots of NR1 and NR2B from the frontal cortex of acute PCP or saline
treated perinatal rats at all time points examined on PN7.
Quantitative analysis
revealed that acute PCP treatment produced a 3-fold increase in membrane
bound NR1 and NR2B subunits in the frontal cortex at 8 hours and this persisted
for 24 hours (Figure 3.1B). The calpain-degraded NR2B breakdown product,
molecular weight 140 kDa, was increased 80% following acute PCP treatment
(saline 0.485 ± 0.161; PCP 0.868 ± 0.216, n=6 for both groups, p<0.05 paired ttest). There was no effect of acute PCP treatment on membrane bound NR2A
protein levels in the frontal cortex (Figure 3.1B).
To investigate the effects of sub-chronic PCP administration on the
concentration of the membrane NR1, NR2A, and NR2B in the frontal cortex, rat
pups were treated with either saline or PCP on PN7, 9, & 11 and were sacrificed
on PN12 (24 hours following the last injection).
Cortical membrane bound
protein extracts were then subjected to Western blot analysis.
54
Figure 3.2A
shows representative Western blots of NR1 and NR2A subunits from the frontal
cortex. Sub-chronic PCP treatment caused a 3-fold increase in membrane NR1
and a 10-fold increase in NR2A protein levels in the frontal cortex with no effect
on NR2B protein expression (Figure 3.2B).
To determine the possible mechanism by which PCP regulates the
expression of cortical NMDAR subunits, protein residing in the endoplasmic
reticulum (ER) fraction was isolated from saline and acute PCP or sub-chronic
PCP treated animals and subjected to Western blot analysis. To verify that the
ER fraction was successfully isolated, we probed for protein disulfide isomerase
(PDI, an ER housekeeping protein); no changes in ER PDI protein levels were
measured following acute or sub-chronic PCP administration and PDI was not
detected in the membrane protein fraction (data not shown).
Acute PCP
treatment produced a decrease in NR1 and NR2B protein levels in the ER
fraction of the frontal cortex on PN7 at 24 hours following administration (Figure
3.3). Conversely, sub-chronic PCP treatment produced an increase in NR1 and
NR2A protein levels in the ER fraction measured 24 hours following the last of
the three injections (Figure 3.3).
These results indicate that acute PCP
administration may cause an increase in trafficking of the NR1 and NR2B
subunits from intracellular compartments to the membrane while sub-chronic
PCP treatment induces new synthesis of the NR1 and NR2A subunits.
In
addition, PSD-95 protein expression levels in the membrane fraction of frontal
cortex were also increased following 8 hours of PCP treatment and remained
elevated 24 hours after treatment (Figure 3.4B). However, following sub-chronic
PCP administration, the β-actin normalized values for cortical membrane PSD-95
protein (0.973 ± 0.214, N=4) were not significantly different from saline treated
animals (0.717 ± 0.0781, N=4, p=0.306, Figure 3.4B).
Figure 3.4A shows
representative Western blots of cortical PSD-95 protein expression from acute
and sub-chronic PCP treated animals.
55
Figure 3.1. Acute PCP administration selectively up-regulates NR1/NR2B in
the frontal cortex of perinatal rats. A) Representative western blots from the
frontal cortex of saline or acute PCP treated animals at all time points examined
on PN7. B) Quantitative analysis of the effects of acute PCP treatment on NR1,
NR2A and NR2B membrane bound protein levels in the frontal cortex
(N=15/group). *p<0.05 vs. SAL NR1 (one-way ANOVA with Bonferroni's post hoc
test) #p<0.05 vs. SAL NR2B (one-way ANOVA with Bonferroni's post hoc test)
56
Figure 3.2.
Sub-chronic PCP treatment up-regulates NR1/NR2A in the
frontal cortex of perinatal rats.
A) Representative western blots from the
frontal cortex of saline or sub-chronic PCP (10 mg/kg, N=15) treated animals. B)
Quantitative analysis of the effect of sub-chronic PCP on NMDAR subunit
proteins in the frontal cortex. (N=15/group)
*p<0.05 vs. SAL NR1 (one-way
ANOVA with Bonferroni’s post hoc test) ^p<0.05 vs. SAL NR2A (one-way
ANOVA with Bonferroni’s post hoc test)
57
Figure 3.3. Effects of acute and sub-chronic PCP administration on the
NMDAR subunits in the cortical ER fraction.
Quantitative analysis of the
effect of acute and sub-chronic PCP (10 mg/kg) on ER protein levels of NMDAR
subunits in the frontal cortex. *p<0.05 vs. acute saline (Student’s t-test) ^p<0.05
vs. sub-chronic saline (Student’s t-test)
58
Figure 3.4. Acute PCP treatment selectively alters levels of PSD-95 in the
frontal cortex of perinatal rats. A) Representative western blot showing acute
PCP treatment (10 mg/kg, n=4) produced a time-dependent increase in
membrane levels of PSD-95 while sub-chronic PCP treatment has no effect on
the levels of membrane PSD-95 protein in the frontal cortex.
B) Quantitative
analysis of protein expression from acute and sub-chronic PCP treatment on
PSD-95 protein levels in the frontal cortex. (N=4/group) *p<0.05 vs. acute SAL
(one-way ANOVA with Bonferroni’s post hoc test)
59
To further define the mechanism of PCP-induced regulation of the
NMDAR, we examined the role of calpain (calcium-dependent neutral cysteine
protease).
Cleavage of the cytoskeletal proteins alpha-II spectrin by calpain
between Tyr1176 and Gly1177 produces two αII spectrin specific breakdown
products (SBP) of molecular weights 150 and 145 kDa, which are calpainspecific spectrin breakdown products and antibodies to these fragments are
available commercially; therefore, one can indirectly measure calpain activation
through Western blot analysis of SBPs (Zhou & Baudry 2006, Nath et al. 1996, Bi
et al. 1997, Wu & Lynch 2006). Acute PCP treatment resulted in an increase in
the αII-spectrin calpain SBP while sub-chronic PCP treatment had no effect on
αII-spectrin calpain SBP expression levels (Figure 3.5B). To further clarify the
role of calpain in PCP-induced trafficking of the NMDAR subunits, we treated
animals following acute PCP administration with the selective calpain inhibitor,
MDL28170 (10 mg/kg, every 2 hrs for 8 hrs, i.p.). MDL28170 is a potent calpain
inhibitor (Ki=10 nM in vitro) that has been shown to cross the blood brain barrier
following systemic administration (Li et al. 1998). Animals were sacrificed 24
hours after the PCP injection and Western blot analysis of cortical membrane
NR1 and NR2B was performed. PCP administration resulted in an up-regulation
of both NR1 and NR2B, which was blocked by post-treatment with MDL28170
(Figure 3.6). These data lend support for the role of calpain activation in acute
PCP-induced changes in the trafficking of NR1/NR2B.
60
Figure 3.5. Effects of acute and sub-chronic PCP administration on the
activation of calpain in the frontal cortex.
A) Representative western blot
showing acute PCP treatment (10 mg/kg, n=4) produced an increase in
membrane levels of calpain SBP (145/150 kDa) while sub-chronic PCP treatment
has no effect on the levels of calpain SBP (145/150 kDa) protein in the frontal
cortex. B) Quantitative analysis shows that acute PCP treatment causes an
increase in the activation of calpain as measured by the cleavage of αII-spectrin
while sub-chronic PCP has no effect. *p<0.05 vs. acute saline (Student’s t-test)
61
Figure 3.6.
Effects of MDL28170 (calpain inhibitor) on the cortical
membrane expression of NMDAR subunits following acute PCP treatment.
Quantitative analysis shows that acute PCP (10 mg/kg, 24 hrs after treatment)
treatment causes an increase in membrane levels of NR1 and NR2B and that
administration of the calpain inhibitor, MDL28170 (10 mg/kg i.p.), blocks upregulation of both NR1 and NR2B. MDL28170 was administered on PN7 every 2
hrs following PCP treatment for 8 hrs. (N=6/group) *p<0.05 vs. NR1 SAL (One
way ANOVA with Tukey’s test) ^p<0.05 vs. NR2B SAL (One way ANOVA with
Student-Newman-Keuls method)
62
Discussion
NMDAR antagonists including PCP, MK-801 and ethanol have been
shown to regulate the membrane expression levels of the NMDAR through
changes in protein synthesis or trafficking of the complex from intracellular
compartments to the membrane (Dong et al. 2004, Sircar et al. 1996, Suvarna et
al. 2005). Previous studies reported an increase in NR1 and NR2A mRNA in the
cortex and striatum of the neonatal rat 4 hours after MK-801 treatment (Wilson et
al. 1998). These results are consistent with an increase in NMDAR membrane
protein (McDonald et al. 1990a, Gao & Tamminga 1994) and the delayed
occurrence of toxicity induced by MK-801 or PCP (Ikonomidou et al. 1999, Wang
et al. 2001, Wang & Johnson 2005).
The present study demonstrated that acute PCP treatment on PN7
increased the levels of membrane NR1 and NR2B protein expression in the
frontal cortex at 8 and 24 hours following PCP administration. Further, analysis
of the cortical ER fraction demonstrated a decrease in NR1 and NR2B protein
following acute PCP administration. To understand the mechanism by which the
up-regulation of NR1 and NR2B occurs, we first examined PCP-induced changes
in the expression of PSD-95. The membrane associated guanylate kinase family
(MAGUK) of proteins, specifically PSD-95, play key roles in NMDAR trafficking to
the membrane (Dong et al. 2004, Lu et al. 2000, Suvarna et al. 2005), clustering
at the membrane, and stabilization of the NMDAR complex and organization of
the post-synaptic architecture (Mauceri et al. 2007, Wu et al. 2007, Lu et al.
2000, Yamada et al. 1999a). Acute PCP administration produced an increase in
membrane cortical protein levels of PSD-95, which paralleled the observed
increase in membrane NR1 and NR2B expression in the frontal cortex.
Therefore, increased trafficking of assembled NMDAR from the ER to the
membrane in conjunction with intact PSD-95 following acute PCP administration
would account for the apparent up-regulation of NR1/NR2B composed receptors
in the frontal cortex.
63
Activation of the NMDA receptor results in the influx of Ca 2+ which in turn
activates the calcium-dependent neutral cysteine protease, calpain, among many
other key calcium-dependent signaling molecules.
Calpain activation has been
suggested to play a role in excitotoxic neuronal death (Saido et al. 1994, Brorson
et al. 1995, Araujo et al. 2004, Zhou & Baudry 2006) as well as
neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases
(Etienne & Baudry 1987, Crocker et al. 2003, Vanderklish & Bahr 2000).
Excessive activation of calpain results in the enzymatic cleavage of cytoskeletal
proteins, loss of structural integrity and disturbances in axonal transport
(Yamashima 2004).
The C-terminal regions of the NR2A (cleavage position
Phe1279; Ser1330) and NR2B subunits have been shown to be substrates of
calpain (Bi et al. 1998a, Bi et al. 1998b, Guttmann et al. 2001), both in vivo and in
vitro (Wu et al. 2007, Wu & Lynch 2006).
Here we report that acute PCP
administration results in a robust activation of calpain 24 hrs following treatment.
Wu and colleagues (2005) suggested that after overactivation of
NMDARs, calpain will cleave and degrade the NMDAR, resulting in downregulation of its function and protection of the neuron from excitotoxic cell death;
on the other hand, it is possible that following acute PCP administration the
protective effects of calpain are abolished; therefore, the cell death cascade is
activated. Based on functional studies using I 125-MK-801 binding to NR1/NR2B,
it has been suggested that in excitotoxic settings, calpain cleavage of NR2B
creates novel, active fragments (140, 130, and 115-120 kDa) which remain on
the extracellular surface and function similar to the native receptor (Liao et al.
2001, Simpkins et al. 2003). Taken together, our results support the possibility
that cortical neurotoxicity measured on PN7 in perinatal rats is due to a hyperfunctional NR1/NR2B complex; however, this interpretation is complicated by
recent time course studies from this laboratory. It has been shown that following
a single injection of PCP on PN7, caspase-3 is activated by 2 hrs in the frontal
cortex; furthermore, TUNEL staining appears 6 hrs after treatment, peaks at 16
64
hrs and is cleared by 24 hrs (Wang & Johnson 2007, Wang & Johnson 2005).
This study reports that the time course of up-regulation of NR1 and NR2B in the
frontal
cortex
following
acute
PCP
treatment
occurs
after
neuronal
neurodegeneration has been initiated (Wang & Johnson 2007, Wang & Johnson
2005), suggesting that the up-regulation of NR1 and NR2B must also arise in
surviving neurons where up-regulation potentially could play a compensatory role
in these neurons.
Nonetheless, it is also possible that NR1 and NR2B up-
regulation plays a significant role in neurotoxicity following acute administration of
PCP.
Sub-chronic PCP treatment of pups resulted in increased expression of
NR1 mRNA and polypeptide in the frontal cortex (Wang et al. 2001). In addition
to up-regulation of NR1, the current study found that sub-chronic PCP
administration resulted in increased expression of membrane NR2A in the frontal
cortex, with no change in NR2B levels. Furthermore, analysis of the cortical ER
protein fraction revealed increased NR1 and NR2A protein levels following subchronic PCP treatment, suggesting that up-regulation may be due to increased
synthesis of these subunits. While the mechanism underlying this up-regulation
is unknown, it is known that the NR1 gene contains a binding site for the
transcription factor NF-κB (Liu et al. 2004a). Further, it has been reported that
binding to the NR1 NF-κB site up-regulates the NR1 promotor and subsequent
transcription of the gene through interactions with Sp1/Sp3 factors.
This is
relevant in that this laboratory previously reported that sub-chronic PCP
treatment increased nuclear translocation of NF-κB (Wang et al. 2001).
Therefore, PCP-induced activation of NF-κB may signal increased synthesis of
the NR1 subunit, resulting in the observed up-regulation of the protein. These
data along with the neurotoxic effects of sub-chronic PCP treatment in vivo
support the possibility that up-regulation of NR1 and NR2A in the frontal cortex
may be due to an increase in new protein synthesis.
65
We suggest that the mechanism of regulation of the NMDAR is treatmentdependent. This is supported by our observation of the lack of effect of subchronic PCP administration on the levels of PSD-95 and calpain activation.
Disruption of the NR2A-PSD-95 interface by either palmitoylation of PSD-95 or
elimination of the PSD-95 binding site on NR2A leaves NR2A susceptible to
cleavage by calpain (Wu & Lynch 2006, Dong et al. 2004). Alternatively, it has
been suggested that PSD-95 may also be a substrate for calpain and it has been
shown that NMDA treatment of organotypic hippocampal cultures produced
activation of calpain and the subsequent cleavage of PSD-95 (Lu et al. 2000).
Truncation of PSD-95 would then lead to an unstable NR1/NR2A/PSD-95
complex in the membrane and cause the internalization and down-regulation of
the assembled NMDAR (Dong et al., 2004).
It is reasonable then to predict
that the up-regulation of NR1/NR2B following acute PCP administration is due to
activation of calpain-mediated trafficking while up-regulation of NR1/NR2A
following sub-chronic PCP administration is due to an increase in protein
synthesis as well as a stable NR1/NR2A/PSD-95 complex and the subsequent
forward trafficking of this complex into the synapse.
These conclusions are
consistent with recent studies suggesting that calpain mediated regulation of the
NMDAR is dependent on the NR2 subunit present (2A resistant, 2B sensitive) as
well as the developmental age and brain region examined (Dong et al. 2006).
Furthermore, it has been recently reported that NMDA-induced protein synthesis
is dependent on the presence of the NR2A, but not the NR2B subunit (Tran et al.
2007).
In summary, this study provides evidence that two distinct mechanisms
are likely involved in the differential regulation of NMDAR subunits following
perinatal acute and sub-chronic PCP administration. These data are consistent
with the hypothesis that changes in NMDAR density may play a role in PCPinduced neurotoxicity in the frontal cortex in the developing rat.
66
Chapter 4: Alterations in NMDAR Binding of 3H-MK-801
in Response to Postnatal PCP Administration
PCP is known to block the function of N-methyl-D-aspartate receptors
(NMDAR) through binding to the interior of the open ion channel. We have
previously reported that the NR1/NR2B receptor is up-regulated following a
single injection of PCP while the NR1/NR2A receptor is up-regulated in response
to sub-chronic PCP treatment (see chapter 3).
It is hypothesized that up-
regulation of the NMDAR protein is associated with increased receptor function.
To test this hypothesis, NMDAR function was estimated by measuring the
binding of 3H-MK-801, a high-affinity open channel blocker, that binds to the
NMDAR channel interior in a manner that reflects the relative degree of channel
opening (Jones et al. 1989) Rat pups were treated on PN7 (acute) or on PN 7, 9,
11 (sub-chronic) with 10 mg/kg PCP or saline. Twenty-four hours later, the rats
were sacrificed; the frontal cortex was harvested and subsequently prepared for
binding by homogenization and differential centrifugation so as to isolate a crude,
well-washed synaptosomal fraction.
3H-MK-801
binding was estimated in the
presence of maximally activating concentrations of NMDA and the co-agonist,
glycine. Under these conditions, both acute and sub-chronic PCP produced a
robust increase in agonist stimulated 3H-MK-801 binding compared to saline.
Inhibition studies with selective NR2A and NR2B antagonists were performed to
determine the subunit composition of the NMDAR species responsible for its
enhanced function. Because of the disruption by Hurricane Ike, this series of
experiments was not completed and at this time it is difficult to interpret these
results. Nevertheless, these data are presented in their current form in hopes that
these experiments will be completed before finalization of this dissertation
project. At this point, 3H-MK-801 binding in the presence of glutamate and
glycine confirms that PCP treatment increases NMDAR function, most likely as a
result of an increase in receptor density. In summary, the use of radioligand
67
binding as a measure of open channel function has been partially successful in
elucidating the relationship between PCP-induced up-regulation of the NMDAR
and its functionality.
Introduction
PCP is a drug of abuse known to cause neurological effects, such as a
reversible psychotic state which includes the full range of positive and negative
symptoms of schizophrenia in healthy individuals (Mechri et al. 2001, Javitt &
Zukin 1991), a disease that is thought to be associated with a developmental
insult (Weinberger 1996). The NMDAR hypofunction theory of schizophrenia is
supported by the selectivity of PCP for the NMDAR as well as the recent findings
linking several putative schizophrenia genes to glutamatergic neurotransmission
and the NMDAR (Harrison & Weinberger 2005).
Glutamate, the primary
mediator of excitatory neurotransmission at central nervous system synapses,
plays an important role in synaptic plasticity, predominantly through its actions at
the NMDAR.
The NMDAR is a ligand-gated ion channel which requires the binding of
glutamate and the co-agonist glycine, as well as removal of a voltage-dependent
Mg2+ block, in order to be functionally active (Johnson & Jones 1990, MacDonald
et al. 1987). Cloning studies have identified several NMDA receptor subunits:
the obligatory NR1, NR2A-D, and NR3A-B (McBain & Mayer 1994). The NR1
subunit forms a heteromeric complex with NR2 or NR3 subunits.
The most
abundant receptor combinations in the forebrain are thought to be diheteromeric
(NR1/NR2A, NR1/NR2B) or triheteromeric (NR1/NR2A/2B) with the physiology
and pharmacology of the NMDAR dependent on its subunit composition and
localization (Cull-Candy et al. 2001). PCP is an open channel blocker of the
NMDAR, inhibiting the channel in a use and voltage-dependent manner. MK-801
is a high-affinity, noncompetitive NMDAR open-channel antagonist which has
proven to be an effective tool to examine the functional properties of the NMDAR
68
as well as the effects of PCP or ketamine in animal models of drug abuse and
mental diseases (Vanderschuren et al. 1998, Beardsley et al. 1990).
Noncompetitive NMDA antagonists, including PCP and MK-801, are
routinely used to model schizophrenia in adult and young animals (Wang et al.
2001, Hargreaves & Cain 1995, Jentsch et al. 1997, Moghaddam et al. 1997,
Ogren & Goldstein 1994, Ikonomidou et al. 1999). Blockade of the NMDAR
results in increased release of glutamate in the frontal cortex (Moghaddam et al.
1997), which leads to the subsequent overactivation of either AMPA or KA
receptors, resulting in excitotoxic cell death (Large 2007). Additionally, blockade
of trophic NMDAR function with MK-801 or PCP causes widespread neurotoxicity
in various brain regions in developing rodents, including the frontal cortex,
hippocampus, and striatum (Lei et al. 2008, Xia et al. 2008, Ikonomidou et al.
1999). This laboratory has also reported the presence of apoptotic (TUNELpositive) neurons in the cortex of perinatal rats, following sub-chronic PCP (10
mg/kg) administration on postnatal day (PN)7, 9, and 11; however, neurotoxicity
is not evident in the striatum or hippocampus, thereby suggesting developmental
tolerance or limited susceptibility in these regions (Wang et al. 2001, Wang &
Johnson 2005).
Thus, further investigation of PCP-induced regulation of the
NMDAR in conjunction with neurotoxicity may shed light on the molecular
mechanisms underlying the psychotomimetic properties of PCP, particularly
those
mediating
the
schizophrenia-like
symptoms
following
perinatal
administration.
The consequences of PCP-induced neurotoxicity could be multifaceted,
involving various compensatory and signaling mechanisms, including but not
limited to altering the expression levels of the NMDAR in the membrane of
surviving neurons.
Several studies have reported that NMDA antagonists
including PCP, MK-801 and ethanol are able to regulate the synaptic membrane
expression levels of the NMDAR through changes in protein synthesis or
trafficking of the NMDAR complex from intracellular compartments to the
69
membrane (Suvarna et al. 2005, Dong et al. 2004, Sircar et al. 1996, Anastasio &
Johnson 2008b). Specifically, we have discovered that in developing rats, the
membrane expression of NMDAR is up-regulated and that the composition of the
receptor subunits is dependent on the treatment paradigm, i.e. PCP
administration on PN7 causes NR1/NR2B to be up-regulated while PCP
administration on PN7, 9, & 11 results in up-regulation of NR1/NR2A receptor
protein (Anastasio & Johnson 2008b). These results parallel studies using 3Hglutamate binding to show increases in NMDAR density in the cingulate cortex,
striatum, and hippocampus in PN7 rats 24 hrs following MK-801 (1mg/kg)
administration (Gao & Tamminga 1994, McDonald et al. 1990b).
Glutamate and glycine which promote opening and activation of the
NMDA channel stimulate radioligand binding in well washed membrane
preparations and inhibition by noncompetitive NMDAR antagonists is dependent
on the presence of both agonists (Johnson et al. 1989, MacDonald et al. 1987).
Therefore, since MK-801 binds deep within the NMDAR channel, which is
influenced by the open state of the channel, it is possible to study the regulation
and function of the NMDAR by measuring the binding of radiolabeled MK-801
(Johnson et al. 1989). Postnatal PCP administration (1 and 5 mg/kg on PN5-15)
increased maximal agonist stimulated (100 µM glutamate + 100 µM glycine) 3HMK-801 binding in the hippocampus (~2 fold) and in the frontal cortex (~1.5 fold)
compared to the saline control (Sircar 2003). Ethanol (2 g/kg x 5 days) increased
the cortical expression of NR2B and agonist stimulated (100 µM glutamate + 100
µM glycine) 3H-MK-801binding in the frontal cortex, but not the hippocampus
(Sircar & Sircar 2006).
Further, the PCP-induced alterations in NMDAR
expression and function may play a role in the development of adverse behaviors
later in life, such as impairing the acquisition of spatial learning or the
development of locomotor sensitization in response to PCP challenge (Anastasio
& Johnson 2008a, Sircar 2003).
70
The exact mechanisms underlying the differential regulation of the
NMDAR following acute or sub-chronic PCP administration and the relationship
between NMDAR density and function are unknown in this animal model of
schizophrenia. Therefore, the present study seeks to determine whether the
apparent
treatment
dependent
up-regulation
of
the
NMDAR
subunits
corresponds to an increase in its functionality using a 3H-MK-801 binding assay.
This study also sought to determine the subunit composition of the NMDAR
responsible for changes in its function by measuring the inhibition of 3H-MK-801
binding by NR2A and NR2B selective inhibitors in saline and PCP-treated rats.
Materials and methods
Animals.
Timed, day 14 pregnant female Sprague-Dawley rats were obtained from
Charles River Laboratories (Wilmington, MA).
The dams were housed
individually with a regular 12h light-dark cycle (lights on 0700, off at 1900) with
food and water ad libitum. Following parturition, male and female pups from four
dams were combined and randomly cross-fostered to one of the four lactating
dams. Each litter consisted of ten to twelve pups. Pups were treated with 10
mg/kg PCP on PN7 or on PN7, 9 and 11 and killed by decapitation in
experiments described below. All experiments were conducted in accordance
with the NIH and the University of Texas Medical Branch at Galveston
Institutional Animal Care and Use Committee.
Drugs.
PCP was acquired from the National Institute on Drug Abuse (NIDA,
Rockville, MD) and dissolved in 0.9% NaCl. NVP-AAM007 was a generous gift
provided to us by Dr. Yves Auberson (Novartis Institute for Biomedical Research,
Basel, Switzerland).
[[[(1S)-1-(4-Bromophenyl) ethyl] amino] (1, 2, 3, 4-
tetrahydro-2, 3-dioxo-5-quinoxalinyl) methyl] phosphonic acid tetrasodium
71
hydrate (PEAQX) and ifenprodil (+)-tartrate salt were purchased from SigmaAldrich (St. Louis, MO) and dissolved in 0.9% NaCl.
Injections were
administered s.c. in 1 mL/kg of vehicle. 3H-MK-801 was purchased from Perkin
Elmer (Waltham, MA) with a specific activity of 22 Ci/mmol.
Experimental design.
Male and female rat pups were treated on either PN7 (acute) or on PN 7,
9, and 11 (sub-chronic) with 10 mg/kg subcutaneous (s.c.) PCP or saline. In the
acute studies, pups were sacrificed by decapitation 24 hours following saline or
PCP on PN7. In the sub-chronic studies, pups were sacrificed by decapitation 24
hours following the last injection of the aforementioned drug regimens on PN7, 9
and 11. The frontal cortex was dissected from 2 mm sections corresponding to
4.7 to 2.7 mm anterior to Bregma.
3H-MK-801
binding.
The buffy coat preparation exhibits pharmacological characteristics similar
to the electrophysiologically defined NMDAR; therefore, agonist stimulated 3HMK-801 binding represents a biochemical assay that allows a partial
characterization of functional properties of the NMDAR complex (Jones et al.
1989). Since the glutamate and glycine binding sites exist on the NR2 and NR1
subunits, respectively, it was anticipated that increased functionality (increased
channel opening associated with increased effects of either glutamate or glycine
on ligand binding) will reflect changes in NR1 and NR2 subunit function.
Buffy coat membranes from the frontal cortex were prepared according to
previous publications with minor modifications (Jones et al. 1989, Johnson et al.
1993). The tissue was homogenized in 20 vol 0.32 M sucrose and centrifuged at
1000 x g for 10 min. The supernatant was collected and centrifuged at 20,000 x
g for 10 min. The P2 pellet was resuspended in 20 vol ice cold dH 2O and
centrifuged at 8000 x g for 10 min.
The supernatant and buffy coat were
72
collected and centrifuged at 48,000 x g for 20 min.
The P4 pellet was
resuspended in 20 vol of ice cold dH2O and centrifuged at 48,000 x g for 20 min.
This pellet (P5) was frozen at -20oC and stored for 1-7 days. On the day of use,
the pellet was thawed and resuspended in 40 vol dH2O, incubated at 37oC for 20
min and centrifuged at 48,000 x g for 10 min. This pellet (P6) was resuspended
in 40 vol dH2O and centrifuged at 48,000 x g for 10 min two more times before
final resuspension in 10 mM HEPES.
For the 3H-MK-801 binding assay, aliquots of the homogenate (40 µg
protein/tube) were added to tubes containing 5 nM 3H-MK-801 (Perkin Elmer,
Boston, MA) and the appropriate concentrations of test drugs in 10 mM HEPES
(pH 7.4, 25oC) for a total volume of 1mL per tube. Basal binding was determined
in the presence of 30 µM 7-chlorokynurenic acid (7-Cl KYNA, a glycine
antagonist) and 1 mM (2R)-amino-5-phosphonovaleric acid (APV, a specific
NMDAR antagonist); nonspecific binding was determined in the presence of 100
µM PCP. Samples were incubated for 2 hrs at 25 oC and the binding reaction
was terminated by vacuum filtration over GF/C glass fiber filters. The filters were
soaked in 0.03% polyethylenimine to reduce filter binding. Filtration was followed
by two 5 mL washes of ice-cold buffer and retained radioactivity was measured
by liquid scintillation spectrometry. Total protein concentrations were determined
using the BCA (bicinchoninic acid) protein assay© (Pierce Chemical, Rockford,
IL). Experiments were repeated a minimum of three times. Possible differences
in specific binding values between saline and treatment were determined by
Student’s t-test; statistical values of p<0.05 were considered significant.
Preliminary studies determined that it was necessary to pool two frontal
cortices from PN8 or PN12 pups in order to obtain adequate CPM (counts per
minute) and binding levels similar to that obtained from adult cortical
synaptosomal preparations. Further, tissue preparation involved multiple washes
to facilitate removal of endogenous glutamate, Mg2+, and glycine so that the
effects of exogenously added glutamate and glycine could be detected.
73
Specificity of radioligand binding was verified through MK-801 and PCP
competition assays. The concentration of 3H-MK-801 used is near the Kd value
most commonly reported (4.92 nM) and as such, changes in binding could be the
result of changes in the Kd or Bmax for the MK-801 binding site. Under these
conditions, this, in turn, could be influenced by changes in the K d or Bmax of either
the glutamate or glycine receptor sites.
Results
In order to determine potential changes in NMDAR functionality following
PCP administration, 3H-MK-801 binding was studied under different conditions
and with different inhibitors, selective for either NR2A or NR2B. In rats acutely
treated with PCP, maximal 3H-MK-801 binding following agonist stimulation (100
µM NMDA + 30 µM glycine) was significantly higher (approximately three-fold)
than in saline treated controls (Figure 4.1).
following 100 µM NMDA + 30 µM glycine
Maximal
3H-MK-801
binding
stimulation was also significantly
greater in the sub-chronic PCP treated animals compared to their saline controls
(1113.93 ± 121.79 pmol/mg protein versus 760.14 ± 75.57 pmol/mg protein,
respectively), albeit to a lesser extent than in the acute treated animals (Figure
4.2).
74
Figure 4.1. Effects of agonist stimulation (100 µM NMDA + 30 µM glycine)
on 3H-MK-801 binding in the frontal cortex of saline and PN7 PCP treated
rat pups. PCP treatment resulted in a significant increase in agonist stimulated
3H-MK-801
binding in the frontal cortex of PN7 treated rat pups compared to
saline treated animals. Animals were sacrificed 24 hours after treatment.
Samples were run in triplicate, N=4/treatment group. *p<0.05 vs. saline
stimulated (Student’s t-test)
75
Figure 4.2. Effects of agonist (100 µM NMDA + 30 µM glycine) stimulation
on 3H-MK-801 binding in the frontal cortex of PN7, 9, & 11 PCP treated rat
pups. PCP treatment resulted in a significant increase in agonist stimulated 3HMK-801 binding in the frontal cortex of PN7, 9, & 11 treated rat pups compared to
saline control animals. Animals were sacrificed 24 hours after treatment.
Samples run in triplicate, N=7/treatment group. *p<0.05 vs. saline stimulated
(Student’s t-test)
76
To determine the subunit composition which underlies the observed
increase in maximal 3H-MK-801 binding, we performed inhibition studies with
selective NR2B (ifenprodil, 10 nM-100 µM) and NR2A antagonists [NVP-AAM007
(1 nM-10 µM) and PEAQX (10 nM-100 µM)]. The rationale for this experiment
was that if increased binding following treatment was mostly due to increases in
either NR2A or NR2B, then the 3H-MK-801 binding would be more susceptible to
either a selective NR2A or NR2B antagonist. No difference in ifenprodil IC50
values due to acute PCP treatment was measured (Figure 4.3); however,
specific 3H-MK-801 binding in the presence of 10 nM ifenprodil shows that there
is a significant decrease (~57%) in specific binding compared to control (no
inhibitor) experiments in the animals administered PCP on PN7, while no effect
on specific binding was observed in the saline treated animals compared to
control (Figures 4.3).
For the sub-chronic PCP treated animals, maximal 3H-
MK-801 binding was not different between saline or PCP treated animals
compared to control experiments; also, no difference in IC 50 values (saline=10.32
µM; PCP=15.87 µM) was measured (Figures 4.4).
Inhibition of 3H-MK-801
binding by the NR2A-preferring antagonist NVP-AAM007 caused no significant
difference in specific binding in saline treated animals compared to control, but
did cause a ~34% decrease in inhibition in maximal binding in the PCP treated
group compared to control binding. Since there was not a dose response, an
IC50 could not be determined (Figure 4.5). Similar results were obtained with
PEAQX inhibition, an additional NR2A-preferring antagonist (Figure 4.6).
77
Figure 4.3. 3H-MK-801 binding inhibition by the NR2B selective antagonist
ifenprodil in acute PCP treated animals. Agonist stimulated (100 µM NMDA +
30 µM glycine) 3H-MK-801 binding detected no significant difference in apparent
affinity for ifenprodil between saline and acute PCP treatment (IC50=10.71 µM
and 8.05 µM, respectively). Control binding was determined in the presence of
100 µM NMDA + 30 µM glycine without ifenprodil.
N=4/treatment group.
78
Samples run in triplicate,
Figure 4.4.
3H-MK-801
binding inhibition by the NR2B selective antagonist
ifenprodil in sub-chronic PCP treated animals. Agonist stimulated (100 µM
NMDA + 30 µM glycine) 3H-MK-801 binding detected no significant difference in
apparent affinity for ifenprodil between saline and sub-chronic PCP treatment
(saline IC50=10.32 µM; PCP IC50=15.87 µM). Control binding was determined in
the presence of 100 µM NMDA + 30 µM glycine without ifenprodil. Samples
were run in triplicate, N=4/treatment group.
79
Figure 4.5.
3H-MK-801
binding inhibition by the NR2A selective antagonist
NVP-AAM007. Agonist stimulated (100 µM NMDA + 30 µM glycine) 3H-MK-801
binding detected no NVP-AAM007 dose response in both saline and sub-chronic
PCP treatment. Control binding was determined in the presence of 100 µM
NMDA + 30 µM glycine without NVP-AAM007. Samples were run in triplicate,
N=4/treatment group.
80
Figure 4.6.
PEAQX.
3H-MK-801
displacement by the NR2A preferring antagonist
Agonist stimulated (100 µM NMDA + 30 µM glycine) 3H-MK-801
binding detected no PEAQX dose response in either saline or sub-chronic PCP
treated rat pups. Control binding was determined in the presence of 100 µM
NMDA + 30 µM glycine without PEAQX. Samples run in triplicate, N=4/treatment
group.
81
Discussion
Glutamatergic neurotransmission during early postnatal development is
primarily mediated by the NMDAR in the frontal cortex (Ben-Ari et al. 1997).
Blockade of the NMDAR during this stage of development with PCP causes a
robust increase in cortical neurotoxicity (Wang et al. 2001, Wang & Johnson
2005), presumably resulting in a compensatory response to maintain synaptic
transmission in the surviving neurons, such as an increase in synaptic
expression of the NMDAR.
We have previously observed that acute PCP
treatment increased membrane levels of NR1 and NR2B protein in the frontal
cortex, while no effect on NR2A levels was detected (Anastasio & Johnson
2008b). Conversely, sub-chronic PCP treatment stimulated an increase in NR1
and NR2A expression in membrane fractions of the frontal cortex, while no effect
on NR2B expression was measured (Anastasio & Johnson 2008b) . Here we
investigated whether this apparent up-regulation in receptor density corresponds
to an alteration in receptor function as measured by glutamate and glycinestimulated 3H-MK-801 binding. We discovered that upon agonist stimulation,
there was a significant increase in the maximal binding 3H-MK-801 binding in the
frontal cortex of both acute (~ 3 fold) and sub-chronic PCP (~1.5 fold) treated rat
pups.
The difference between 3H-MK-801 binding levels between the day 8
controls and day 12 controls is most likely due to maturation of the brain and/or
the NMDAR, e.g. changes in the probability of channel opening. Nonetheless,
the increase in binding in the PCP treated animals is consistent with acute PCPinduced up-regulation of membrane NR1/NR2B protein expression as well as the
increase in NR1/NR2A membrane protein levels measured following sub-chronic
PCP administration (Anastasio & Johnson 2008b).
Thus, this supports the
hypothesis that the newly inserted NMDAR subunits in the membrane following
PCP administration are functionally active.
Next, to determine the NMDAR subunit composition responsible for the
increase in receptor function and to verify previous Western blot experiments
82
(Anastasio & Johnson 2008b), we performed inhibition studies with selective NR2
subunit antagonists. Ifenprodil is a neuroprotectant which acts as an NMDAR
antagonist by blocking the polyamine site on the NR2B subunit (Williams 2001,
Reynolds & Miller 1989). Ifenprodil has been shown to inhibit 3H-MK-801 binding
in a biphasic fashion (Reynolds & Miller 1989). The high affinity phase (Ki ~
15nM) represents binding to the polyamine site and accounts for 50-60% of total
3H-MK-801
binding in the presence of 100 µM glutamate; however, the addition
of 30 µM glycine reduces total binding due to the high affinity phase to 20%
without altering ifenprodil affinity for the polyamine site (Reynolds & Miller 1989).
The low affinity phase (Ki ~ 20-40 µM) is regulated by binding to the Zn2+ site on
the NR2B subunit (Reynolds & Miller 1989).
Inhibition of 3H-MK-801 binding by
the lowest concentration of ifenprodil tested (10nM) decreased maximal binding
in the PCP treated animals to 43% of control experiments. The design of this
experiment limits our interpretation of these data such that we cannot definitively
determine if this effect is due to alterations in the polyamine site or the Zn 2+ site;
therefore, experiments which address these concerns are currently ongoing and
are designed to test a greater range of ifenprodil concentrations with the goal of
elucidating the mechanism of ifenprodil inhibition in this assay. Nevertheless,
due to the stark difference in ifenprodil inhibition in the acutely treated animals,
this study may provide preliminary evidence that acute PCP administration alters
the high affinity polyamine binding site on the NR2B for ifenprodil such that the
newly inserted NR1/NR2B receptor function is more sensitive to ifenprodil
inhibition.
Additionally, this study may provide preliminary evidence for the
presence of an increase in the triheteromeric NMDAR following a single injection
of PCP. The presence of a single copy of NR2B (or NR2A) is adequate for
ifenprodil binding (or selective NR2A antagonists) albeit to a lesser extent than in
the presence of two copies of the NR2B subunit; therefore, no change in
ifenprodil IC50 values following PCP administration does not signify that the
83
NR2B subunit is absent or nonfunctional, but suggests that it may have coassembled with NR2A (Hatton & Paoletti 2005).
Interestingly, in the sub-chronic paradigm, ifenprodil maximally inhibited
3H-MK-801
binding in both the saline and PCP treated animals with no difference
in IC50 values.
This suggests that the increase in sub-chronic PCP specific
binding is not mediated through NR2B-containing receptors and is most likely
through the NR2A-containing receptors, since this is the most commonly
expressed NMDAR composition in the frontal cortex (Cull-Candy et al. 2001).
This is supported by our previous study which reported that sub-chronic PCP
administration up-regulates the membrane levels of NR1/NR2A subunit in the
cortex three-fold, but does not alter the protein expression levels of NR2B
(Anastasio & Johnson 2008b).
Further, inhibition by the NR2A antagonist
should produce a lower IC50 in the sub-chronic PCP treated animals compared to
saline (Anastasio & Johnson 2008b); however, no dose response was observed
with the NR2A antagonists, NVP-AAM007 or its diastereomer PEAQX; therefore,
no IC50 values could be determined. NVP-AAM007 and PEAQX are considered
to be competitive antagonists for the glutamate site on the NR2A subunit;
therefore, the lack of a dose response may in part be due to the concentration of
NMDA applied in this assay (Neyton & Paoletti 2006). Future experiments are
currently ongoing which tests a lower concentration of NMDA that still activates
the receptor, but does not compete with and inhibit the actions of NVP-AAM007
or PEAQX as well as experiments which are designed to test a greater range of
NR2A antagonist concentrations. We also plan to determine the effects of NR2A
inhibition on 3H-MK-801 binding in the cortex of animals treated with PCP on
PN7. We anticipate no effect of NR2A inhibition on maximal binding because
acute PCP selectively up-regulates NR1/NR2B membrane protein levels
(Anastasio & Johnson 2008b).
We must also acknowledge that while NVP-AAM007 and PEAQX are
thought to be potent inhibitors of the NR2A-containing NMDARs, their ability to
84
discriminate between NR2A and NR2B-containing receptors in an in vitro assay
is actually much smaller than originally reported by Liu et al., (2004b). In fact,
several more recent studies have reported that the NVP-AAM007 IC50 values for
NR2A and NR2B differ by approximately 9-fold and that PEAQX is ~12-fold more
selective for NR2A than NR2B, far from the original 130-fold difference originally
described (Feng et al. 2004, Neyton & Paoletti 2006, Frizelle et al. 2006, Liu et
al. 2004b). The reason for this discrepancy is most likely due to differences in
the level of glutamate occupancy of both NR2A and NR2B. Glutamate has a
higher affinity for NR1/NR2B compared to NR1/NR2A (Cull-Candy et al. 2001,
Laurie & Seeburg 1994, Neyton & Paoletti 2006), thus, to achieve a similar level
of activation a higher level of glutamate should be applied at NR1/NR2A
receptors than at NR1/NR2B (Neyton & Paoletti 2006). To study the effects of
NVP-AAM007, a competitive and irreversible inhibitor of the glutamate site
(Frizelle et al. 2006), Liu et al. (2004b) stimulated NR1/NR2A receptors with 2 µM
glutamate and NR1/NR2B receptors with 5 µM glutamate resulting in their report
of a greater subunit selectivity for NR2A than NR2B. However, NR1/NR2A
receptors were most likely not fully activated in that experiment; therefore, the
interpretation of the results is biased. Thus, precaution must be taken in to
account when interpreting results from experiments obtained with these
incompletely understood pharmacological tools. The development of a highly
selective and potent NR2A antagonist is desperately needed and would
exponentially advance this field.
In conclusion, postnatal PCP administration results in a compensatory
increase in functional NMDARs in the synaptic membrane in the frontal cortex of
developing rat pups. The functional consequences of this up-regulation and role
it plays in altering cortical function, as well as the development of abnormal
schizophrenic-like behaviors will be addressed in subsequent studies.
85
Chapter 5: Atypical Antischizophrenic Drugs Prevent
Changes in Cortical NMDARs and Behavior Following
Sub-chronic PCP Administration
We sought to determine the relationship between PCP-induced alterations
in behavior and NMDAR expression in the cortex by examining the effect of
antischizophrenic drug treatment on both.
Sprague-Dawley rat pups were
pretreated with risperidone or olanzapine 30 min prior to treatment with PCP on
PN7 or sub-chronically on PN7, 9, & 11. Pre-pulse inhibition (PPI) of acoustic
startle was measured on PN24-26 and following a challenge dose of 4 mg/kg
PCP, locomotor activity was measured on PN28-35. PCP treatment on PN7 did
not cause a deficit in PPI, but did cause locomotor sensitization. This was
prevented by both antipsychotics. Further, PCP treatment on PN7 caused an upregulation of
NR1 and NR2B, but this was not affected by either
antischizophrenic drug. On the other hand, PCP treatment on PN7, 9, & 11
caused a deficit in PPI and a sensitized locomotor response to PCP challenge as
well as an up-regulation of NR1 and NR2A, all of which were prevented by both
atypical antischizophrenic drugs. These data support the hypothesis that
subchronic, but not single injection PCP treatment to developing rats results in
behavioral alterations that are sensitive to antipsychotic drugs and these
behavioral changes observed could be related to up-regulation of cortical NR1
and NR2A receptors.
Introduction
Schizophrenia is a severe neuropsychiatric disorder which afflicts
approximately 1% of the population worldwide and shows strong genetic
tendencies, with symptoms first presenting in early adulthood (Lewis &
Lieberman 2000, Bromet & Fennig 1999). The disease is characterized by the
presence of both positive, e.g. paranoia, hallucinations, delusions, and negative
86
symptoms, including but not limited to emotional and social withdrawal,
anhedonia, and depression, as well as cognitive impairments such as memory
and attention deficits (McGlashan 1996a).
PCP intoxication in humans has been shown to mimic both the positive
and negative symptoms of schizophrenia as well as exacerbate psychosis in
schizophrenics. These psychotomimetic properties led researchers to examine
the effects of PCP in animals (Luby et al. 1962, Javitt & Zukin 1991). PCP
administration to rats results in increased locomotor activity, stereotypy, ataxia,
head-weaving, circling and walking backwards (Steinpreis 1996, Castellani &
Adams 1981, Braff & Geyer 1990, Martinez et al. 2000b, Geyer & Ellenbroek
2003). In general, alterations in motoric behaviors such as these are thought to
model the positive symptoms of schizophrenia.
N-methyl-D-aspartate receptor (NMDAR) antagonists (PCP and MK-801)
have been shown to reproducibly disrupt PPI of acoustic startle in animals and
are routinely used to model the sensorimotor gating deficits of schizophrenia
(Geyer et al. 2001, Rasmussen et al. 2007).
PPI of acoustic startle is a gauge
for information processing in specific pathways that are known to be abnormal in
schizophrenia (Bunney et al. 2000, Adams & Moghaddam 1998, Castellani &
Adams 1981, Wang et al. 2001, Ellenbroek & Cools 2000). This deficit may
contribute to the thought disorder and cognitive fragmentation characteristic of
this disease (Braff & Geyer 1990). It is a measure of the reduction of the startle
response when a smaller non-startling acoustic stimulus (pre-pulse) is presented
80-120 ms prior to the startling stimulus (pulse) (Swerdlow et al. 1994).
The
etiology
of
schizophrenia
has
been
described
as
a
neurodevelopmental disorder (Weinberger 1987, Weinberger 1996, Pilowsky et
al. 1993).
Neurodegeneration during early stages of development has been
shown following PCP or MK-801 treatment in the cortex, hippocampus, and
striatum, all of which are regions of the brain implicated in schizophrenia (Wang
& Johnson 2005, Wang & Johnson 2007, Ikonomidou et al. 1999). In addition,
87
PCP treatment on PN7, results in neurodegeneration (positive silver staining) in
the frontal cortex, striatum, and hippocampus within 9 hours of treatment (Wang
& Johnson 2005, Wang & Johnson 2007). Accompanying increases in caspase3 immunoreactivity and terminal dUTP nick-end labeling (TUNEL) of broken DNA
show this degeneration to be apoptotic in nature. Furthermore, administration of
MK-801 and/or PCP to the neonate has been shown to produce aberrant
behaviors later in development (Beninger et al. 2002, Wang et al. 2001)
specifically, locomotor sensitization and deficits in PPI of acoustic startle were
reported in postnatal (PN24-28) rats treated with PCP on PN7, 9, & 11 (Wang et
al. 2001).
While it is generally acknowledged that the schizophrenic “break” does not
often occur before puberty, there is evidence that this does occur in some cases
(Ross et al. 2006b, Nicolson et al. 2000).
Further, when examined
retrospectively, there is ample evidence of abnormalities such as poor academic
performance and poor social interaction skills in children who are eventually
diagnosed as schizophrenic. This laboratory and others have demonstrated that
this model (PCP treatment on PN7, 9, & 11) has face validity in that it results in
behavioral deficits in rats similar to those seen in schizophrenia, including deficits
in spatial learning, PPI of acoustic startle and social behavior (Wiley et al. 2003a,
Wiley et al. 2003b, Wang et al. 2001, Rasmussen et al. 2007, Harich et al.
2007a).
Further, pretreatment with olanzapine was able to prevent both
behavioral and cytotoxic indices of postnatal PCP administration, suggesting that
this treatment paradigm is a suitable model of schizophrenia in rats (Wang et al.
2001)
Up-regulation of the NR1 subunit of the NMDAR has been reported to be
associated with PCP-induced behavioral changes (Wang et al., 2001); however,
it is not known whether this is accompanied by commiserate increases in NR2
subunits, which would be necessary for altered receptor function (Kutsuwada et
al. 1992, Monyer et al. 1994, Paoletti & Neyton 2007). Further, it is not known
88
whether behavioral changes following single dose PCP treatment on PN7
administration are associated with NMDAR up-regulation, and if so, whether
these changes can also be modulated by antischizophrenic drugs. Therefore, in
this study we determined the effect of PN7 and PN7, 9, 11 PCP administration on
two behaviors thought to model different aspects of schizophrenia as well as on
the levels of the three most abundant cortical and striatal NMDAR subunits (NR1,
NR2A and NR2B).
Finally, the relevance of the changes in behavior and
NMDAR subunit protein as a model of schizophrenia was challenged by
determining the effect of pretreatment with two anti-schizophrenic drugs,
risperidone and olanzapine.
Materials and Methods
Animals.
Timed, day 14 pregnant female Sprague-Dawley rats were obtained from
Charles River Laboratories (Wilmington, MA).
The dams were housed
individually with a regular 12h light-dark cycle (lights on 0700, off at 1900) with
food and water ad libitum. Following parturition, male and female pups from four
dams were combined and randomly cross-fostered to one of the four lactating
dams.
Each litter consisted of ten to twelve pups with approximately equal
numbers of each gender. All experiments were conducted in accordance with
the NIH and the Institutional Animal Care and Use Committee.
Drugs.
Phencyclidine was acquired from the National Institute on Drug Abuse
(NIDA, Rockville, MD) and dissolved in 0.9% NaCl. Risperidone was obtained as
a solution from Janssen Pharmaceutica (Titusville, NJ) and dissolved in 0.9%
NaCl. Olanzapine was a generous gift from Eli Lilly and Company (Indianapolis,
IN) and was dissolved in 0.1 N HCl and titrated to pH 7.0 with 0.1 N NaOH and
finally diluted with 0.9% NaCl. Doses were chosen based on prior experiments
89
that addressed PCP-induced regulation of the NMDAR (Wang et al. 2001) and
our own preliminary experiments.
Experimental design.
Male and female rat pups were treated on either PN7 or on PN 7, 9, and
11 (sub-chronic) with 10 mg/kg PCP or saline vehicle (s.c).
Olanzapine (1
mg/kg, s.c.) or risperidone (0.25 mg/kg, s.c.) were administered 30 minutes prior
to PCP or saline administration on PN7 or on PN 7, 9, 11. Pups were sacrificed
by decapitation on PN7 at 24 hours following saline/vehicle (control, time=0
hours), PCP/vehicle, antagonist/vehicle, or antagonist/PCP treatment.
In a
separate experiment, pups were sacrificed by decapitation 24 hours following the
last injection of the aforementioned drug regimens on PN7, 9 and 11 (on PN12).
For biochemical studies, the frontal cortex or striatum was dissected as
described below and used for Western blot analysis. In behavioral experiments,
animals were assessed for PPI of acoustic startle on PN24-26 and then tested
for locomotor activity following a 4 mg/kg PCP (i.p.) challenge on PN28-35.
We chose to investigate the frontal cortex and striatum because of the prominent
role these two brain regions play in the pathophysiology of schizophrenia.
Furthermore, the frontal cortex and striatum are part of the brain circuitry involved
in the expression of both PPI (Koch & Bubser 1994, Swerdlow et al. 1995,
Ellenbroek et al. 1996) and locomotor sensitization (Pierce & Kalivas 1997).
Sub-cellular fractionation.
Protein extracts were prepared from the frontal cortex or striatal brain
tissue as previously described with some modifications (Wang et al. 2001,
Anastasio & Johnson 2008b). Briefly, 2 mm sections corresponding to 4.7 to 2.7
mm anterior to Bregma for the frontal cortex and 0.7 mm to -1.3 mm for the
striatum (Paxinos & Watson 1986) were cut with the aid of an aluminum brain
mold.
Cortical brain sections were homogenized in 500 μL of lysis buffer with
90
the aid of an automatic tissue grinder (Kontes Pellet Pestle Motor, Kimble /
Kontes, Vineland, New Jersey). The lysis buffer consisted of 10 mM HEPES (pH
7.4), 1 mM EDTA, 2 mM EGTA, and 500 μM DTT. Just prior to use, protease
inhibitor cocktail [4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), pepstatin
A, E-64, bestatin, leupeptin, and aprotinin without metal chelators (Sigma-Aldrich,
St Louis, MO)] at a concentration of 10 μL/mL was added to the lysis buffer. The
homogenate was then centrifuged at 1000 x g at 4oC for 10 minutes to pellet the
nuclear protein fraction (P1).
The supernatant (S1) was collected and
centrifuged at 8000 x g at 4oC for 30 minutes to pellet the membrane protein
fraction (P2).
The membrane fraction was re-suspended in homogenization
buffer and centrifuged at 20,000 x g at 4oC for 30 minutes; the resultant pellet
was re-suspended in lysis buffer + 1% SDS, boiled for 10 minutes and stored at 80oC.
Western blot analysis.
Equal amounts of protein were separated on 10% Bis-Tris gels
(Invitrogen, NY) using SDS-PAGE with a MES-SDS running buffer system, pH
7.4. Following electrophoresis (110 V for 2 hours), proteins were transferred to
polyvinylidene difluoride (PVDF) membranes (0.2 μm) in a Mini Electrotransfer
Unit (Bio-Rad, Hercules, CA) overnight.
The membrane was blocked in 5%
nonfat milk, followed by incubation with the primary antibody in 1% milk for 2
hours at room temperature. Following washes (3 x 10 minutes) in TBS+0.1%
Tween 20 (TBST), the membrane was incubated with horseradish peroxidase
conjugated secondary antibodies for 1 hour at room temperature. Analysis was
carried out using the enhanced chemiluminescence (ECL) plus Western blotting
detection reagents (Amersham Biosciences, Piscataway, NJ).
corresponding to the various proteins of
The bands
interest were scanned and
densitometrically analyzed by using an automatic imaging analysis system
(Alpha Innotech Corporation, San Leandro, CA). All quantitative analyses were
91
normalized to β-actin (after stripping [Reblot mild, Chemicon International,
Temecula, CA]).
Antibodies.
The monoclonal anti-NR1, anti-NR2A, and anti-NR2B were purchased
from BD Biosciences (San Jose, CA). Anti-actin antibody was obtained from
Chemicon International (Temecula, CA). Primary antibody dilution was 1:5001:1000.
Secondary antibodies were purchased from Zymed (Invitrogen
Corporation, Carlsbad, CA) and used at a concentration of 1:5000.
Pre-pulse inhibition (PPI) of acoustic startle.
Measurement of PPI of acoustic startle was performed according to
previously published procedures with minor modifications (Wang et al. 2001,
Wang et al. 2003). Testing was performed between 0900 and 1600 hours as
described below. Male and female rat pups (PN24-26) were transferred into a
small sound-attenuated, dedicated behavior room on the day of testing and
allowed to acclimate to the room for 20 minutes. Animals were then placed into
one of three startle chambers (SR-Lab, San Diego Instruments, San Diego, CA)
with a background noise level of 65 dB.
Following a 10 minute acclimation
period, rats were exposed to three randomly administered stimuli: no stimulus, a
73 dB 20 ms pre-pulse 100 ms prior to a 120 dB pulse, or a 120 dB 40 ms pulse
alone with a variable inter-trial interval (5-20 sec) for a total of 63 trials (21 no
stimulus, 21 pulse alone, and 21 pre-pulse + pulse). % PPI of acoustic startle
was calculated as the [pulse-(pre-pulse + pulse)]/pulse x 100.
Locomotor Activity.
On the day of testing animals were placed in locomotor chamber boxes
and allowed to habituate for 30 minutes prior to a 4 mg/kg challenge dose of PCP
(i.p.).
Locomotor activity was measured for an additional 90 minutes via an
92
open-field activity system (San Diego Instruments, San Diego, CA) which
consisted of a square enclosure with Plexiglas walls (40 x 40 x 40 cm).
Horizontal activity was measured with a 16 x 16 photobeam matrix which
recorded both central and peripheral activity in 5 min bins.
Statistical analysis.
Group comparisons were specifically defined before the beginning of each
experiment; therefore, planned comparisons were performed instead of an
overall F test in a multifactorial ANOVA (Keppel 1982). Statistical comparisons
for each experiment were conducted using a one-way ANOVA. All values are
presented as mean ± SEM. The null hypothesis was rejected at p<0.05.
Results
PCP administration in adult rats causes substantial impairments in normal
behavior (Hanania et al. 1999, Phillips et al. 2001, Geyer et al. 2001). In perinatal
rats, 10 mg/kg PCP results in significant reduction of normal behaviors, most
notably, a reduction of suckling behavior. This lasts approximately 6-8 hours and
causes a concomitant weight loss, especially in male pups. While females regain
the lost weight within 24 hrs, the males do not; however, at the time of behavioral
testing, there is no significant difference in weight between saline and PCP
treated animals of either gender (Table 5.1). All experimental groups consisted
of equal numbers of males and females.
93
Table 5.1 Effects of PCP (10 mg/kg) on weight (g) gain in developing rats
PN7
PN8
PN9
PN10
PN11
PN12
PN28
14.3±0.7
19.7±0.2
17.4±1.2
23.7±0.5
20.9±1.5
29.1±0.7
81.1±2. 8
14.7±0.8
17.3±0.6*
16.5±0.8
21.4±0.4*
19.9±1.3
24.9±1.5
79.2±4
16.2±0.6
21.2±0.4
20.7±0.8
26.4±0.6
25.5±1.1
31.5±0.6
91.2±3.5
15.9±0.8 17.7±0.5^ 17.5±0.8^ 21.9±0.7^
Data presented as mean (g) ±SEM
*p<0.05 vs. saline female N=6-11/group (Student’s t-test)
^p<0.05 vs. saline male N=6-13/ group (Student’s t-test)
20.6±1.1^
23.8±0.3^
85.6±4.6
Saline
female
PCP
female
Saline
male
PCP
male
94
PCP treatment on PN7 had no significant effect on PPI (F 3, 31=2.264, NS),
suggesting that acute neurotoxicity previously observed (Wang & Johnson 2007)
is not sufficient to alter this behavior (Figure 5.1). Further, PCP treatment on
PN7 had no effect on the startle amplitude (data not shown). However, we found
that PCP treatment on PN7 caused a significantly sensitized locomotor response
to PCP challenge on PN28-35 that was prevented by either olanzapine
(F3,31=8.071, p<0.05) or risperidone (F3,31=10.001, p<0.05) (Figure 5.2).
As with PCP treatment on PN7, treatment on PN7, 9, & 11 produced no
significant effect on startle amplitude (data not shown). On the other hand, PCP
treatment on PN7, 9, & 11 resulted in a significant deficit in PPI of acoustic startle
measured on PN24-26, which was blocked by pretreatment with either
olanzapine (F3,31=4.194, p<0.05) or risperidone (F3,31=2.852, p<0.05) (Figure
5.3).
Previous studies from this laboratory reported that the atypical
antipsychotic drug, olanzapine, prevented sensitization to the locomotor
activating effects of PCP challenge with 2 mg/kg on PN42 as well as the
inhibition of baseline pre-pulse inhibition of acoustic startle on PN24-28 in female
pups (Wang et al. 2001). This experiment was repeated using the present design
which also included male pups in order to exclude any gender related
differences. Further, this design was extended to risperidone, which has some
atypical properties at low doses.
For these studies, animals treated sub-
chronically with PCP and then challenged with 4 mg/kg PCP on PN28-35,
showed enhanced locomotor activity compared to saline treated animals (Figure
5.4).
p<0.05)
Both olanzapine (F3,31=5.920, p<0.05) and risperidone (F3,31=5.044,
pretreatment on PN7, 9, & 11 prevented PCP-induced locomotor
sensitization measured on PN28-35 with no effect of their own (Figure 5.4).
95
Figure 5.1. Effects of PCP administration (PN7 only) on PPI of acoustic
startle. PCP (10 mg/kg) treatment on PN7 does not produce a significant deficit
in PPI of acoustic startle as measured on PN24-26. N=8 in both control and
experimental groups.
96
Figure 5.2. Effects of PCP administration (PN7 only) the development of
locomotor sensitization. Locomotor sensitization induced by a 4 mg/kg
challenge to PCP treatment on PN7 treated animals as measured on PN28-35.
Pretreatment on PN7 with olanzapine (1 mg/kg) or risperidone (0.25 mg/kg)
inhibits the development of locomotor sensitization N=8/group (top).
Time-
course of locomotor activity (bottom) *p<0.05 vs. saline (one-way ANOVA with
Bonferroni’s post hoc test). N=8 in both control and experimental groups.
●-saline; ○-10 mg/kg PCP; ▼-1 mg/kg olanzapine; ∆-olanzapine + PCP; ■-0.25
mg/kg risperidone; □-risperidone + PCP
97
Figure 5.3. Effects of subchronic PCP administration (PN7, 9 & 11) on PPI
of acoustic startle.
PCP treatment (10 mg/kg) treatment on PN7, 9, & 11
results in a deficit in PPI of acoustic startle as measured on PN24-26.
Pretreatment on PN7, 9, & 11 with olanzapine (1 mg/kg) or risperidone (0.25
mg/kg) inhibits the PCP-induced deficit in PPI of acoustic startle. *p<0.05 vs.
saline (one-way ANOVA with Bonferroni’s post hoc test). N=8 in both control and
experimental groups.
98
Figure 5.4. Effects of subchronic PCP administration (PN7, 9 & 11) on the
development of locomotor sensitization. Locomotor sensitization induced by
a 4 mg/kg challenge to PCP treatment on PN7, 9, & 11 treated animals as
measured on PN28-35. Pretreatment on PN7, 9, & 11 with olanzapine (1 mg/kg)
or risperidone (0.25 mg/kg) inhibits the development of locomotor sensitization.
(top) Time course of locomotor activity (bottom) *p<0.05 vs. saline (one-way
ANOVA with Bonferroni’s post hoc test). N=8 in both control and experimental
groups. ●-saline; ○-10 mg/kg PCP; ▼-1 mg/kg olanzapine; ∆-olanzapine + PCP;
■-0.25 mg/kg risperidone; □-risperidone + PCP
99
In order to delineate one possible biochemical mechanism underlying the
above mentioned behavioral changes, we determined the effects of PCP
treatment on PN7 or on PN7, 9, & 11 on the density of NR1, NR2A, and NR2B in
the synaptosomal membrane fraction of the frontal cortex and striatum.
Quantitative western analysis revealed that PCP treatment on PN7 produced a 3fold increase in membrane bound NR1 and NR2B subunits in the frontal cortex
24 hours after PCP administration, while no effect on NR2A was observed
(Anastasio & Johnson 2008b). In another experiment, perinatal animals were
treated with either olanzapine (1 mg/kg) or risperidone (0.25 mg/kg) 30 minutes
prior to PCP treatment on PN7. Twenty-four hours after the PCP injection, the
rats were killed and the density of NMDARs was determined in the cortical
membrane fraction. Pretreatment with either olanzapine or risperidone did not
inhibit the up-regulation of membrane cortical NR1 or NR2B induced by PCP
treatment on PN7 (Figure 5.5).
Furthermore, treatment with olanzapine or
risperidone alone had no effect on the concentration of membrane NR1 or NR2B
in the frontal cortex compared to saline controls (data not shown).
PCP treatment on PN7, 9, & 11 treatment caused a 3-fold increase in
membrane NR1 and a 10-fold increase in NR2A protein levels in the frontal
cortex with no effect on NR2B protein expression (Anastasio & Johnson 2008b).
To analyze the actions of atypical antipsychotics on PCP-induced changes in the
membrane protein levels of NMDAR subunits in the frontal cortex, animals were
treated with olanzapine or risperidone 30 minutes prior to PCP on PN7, 9, & 11
and sacrificed 24 hours after the last of 3 injections. Each of the antipsychotics
investigated inhibited PCP-induced up-regulation of the NR1 subunit in the
membrane protein fraction of the frontal cortex (Figure 5.6), while having no
effect on NR1 subunit expression on their own (data not shown). Pre-treatment
with either olanzapine or risperidone also was able to completely block the
increase in membrane cortical NR2A protein levels caused by PCP treatment on
PN7, 9, & 11 treatment (Figure 5.6) with no effect on their own (data not shown).
100
PCP treatment on PN7 had no significant effect on NR1 or NR2B protein
expression levels in the striatum at 24 hours following treatment (Figure 5.7).
The effects of pretreatment with the antipsychotics on NR1 and NR2B protein
levels in the striatum were also investigated following a single injection of PCP.
Treatment with olanzapine or risperidone did not significantly alter expression of
NR1 or NR2B protein levels in the striatum prior to PCP on PN7 at 24 hours
following treatment (Figure 5.7) or when administered alone (data not shown).
Similarly, treatment with either PCP or atypical antipsychotics did not alter striatal
NR2A protein expression levels (data not shown).
Unlike the up-regulation of NR1 observed in the frontal cortex, Western
blot analysis of membrane bound protein showed that levels of striatal NR1
protein were significantly decreased following sub-chronic PCP treatment on
PN7, 9, & 11 (Figure 5.8), with no effect on regulation of striatal NR2A (Figure
5.8) or NR2B (data not shown). Pretreatment with the anti-schizophrenic drug
olanzapine, but not risperidone, prevented the down-regulation of NR1 protein
expression in the striatum caused by sub-chronic PCP administration (Figure
5.8). Administration of these drugs alone had no effect on NR1 or NR2A protein
levels in the striatum (data not shown).
We also measured the levels of NR1, NR2A, and NR2B in the frontal
cortex of animals following PCP challenge on PN28-35. No alterations in the
protein levels of any of the subunits in the frontal cortex were evident in the
animals treated sub-chronically with PCP (data not shown).
101
Figure 5.5. Pharmacological antagonism of PCP treatment on PN7 effects
on NR1 and NR2B in the frontal cortex. Quantitative analysis reveals that
antipsychotics pretreatment has no effect on PCP treatment on PN7-induced upregulation of NR1 or NR2B protein levels in the frontal cortex 24 hours following
PCP (10 mg/kg, N=5-15/treatment). *p<0.05 vs. SAL NR1 (one-way ANOVA
with Bonferroni's post hoc test) ^p<0.05 vs. SAL NR2B (one way ANOVA with
Bonferroni’s post hoc test).
102
Figure 5.6. Pharmacological antagonism of PCP treatment on PN7, 9, & 11induced up-regulation on NR1 and NR2A in the frontal cortex. Quantitative
analysis reveals that pretreatment with antipsychotics prevents PCP-induced (10
mg/kg, N=5-15/treatment) up-regulation of NR1 or NR2A protein in the frontal
cortex. *p<0.05 vs. SAL NR1 (one-way ANOVA with Bonferroni's post hoc test)
^p<0.05 vs. SAL NR2A (one way ANOVA with Bonferroni’s post hoc test).
103
Figure 5.7. Effects of a single injection of PCP on PN7 on the membrane
protein expression of NR1 and NR2B in the striatum. Quantitative analysis
reveals that PCP treatment on PN7 has no effects on the expression of striatal
NR1 or NR2B (10 mg/kg, N=5-15/treatment).
104
Figure 5.8. Antagonism of PCP treatment on PN7, 9, & 11-induced downregulation on NR1 in the striatum.
pretreatment
with
olanzapine
prevents
Quantitative analysis reveals that
PCP-induced
(10
mg/kg,
N=5-
15/treatment) down-regulation of NR1 in the striatum. PCP treatment had no
effect on protein levels of NR2A *p<0.05 vs. SAL NR1 (one-way ANOVA with
Bonferroni's post hoc test)
105
Discussion
A number of genes related to glutamatergic function have been
discovered that are significantly associated with schizophrenia, though in as yet
undefined ways. Included in this list is NRG1 (neuregulin-1), which is known to
regulate NMDAR expression (Chong et al. 2008, Craddock et al. 2005).
DTNBP1 (dystrobrevin binding protein 1) has been shown to modulate glutamate
function through up-regulation of presynaptic proteins and neurotrophic effects
mediated by the Akt signaling pathway (Duan et al. 2007, Numakawa et al.
2004).
DAOA (D-amino acid oxidase activator) activates NMDA receptors
through a series of reactions involving the glycine-like molecule, D-serine
(Korostishevsky et al. 2004, Ross et al. 2006a).
RGS4 (regulator of G-protein
signaling 4) is under-expressed in schizophrenic prefrontal cortex in postmortem
microarray studies and has been reported to regulate G proteins in glutamate
neurons, thereby dampening the effects of neurotransmitter interactions at Gprotein coupled receptors (Chowdari et al. 2002, Chowdari et al. 2008).
In
addition to the glutamate related genes above found with linkage analysis,
several other genes putatively associated with the NMDA synapse have been
discovered to be associated with schizophrenia, including GRM3 (mGluR3 a
member of the group II metabotropic receptor family, which also includes
mGluR2 (Egan et al. 2004); mGluR3 is known to modulate glutamate release.
Thus, genetic evidence supports a role for alterations in various glutamatergic
genes including those specific to NMDAR function in schizophrenia. Importantly,
these data are strongly supported by pharmacological data that also imply a role
for altered NMDAR function. However, the complexities of these data do not
allow a direct comparison between the data in this model to what may occur in
the human brain in schizophrenia. Our view is that animal models may reveal the
gross structural features underlying behavioral alterations in rats that are similar
to schizophrenia and possibly point to novel pharmacological approaches that
may be helpful in treatment of the disease.
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It is well known that acute PCP treatment produces a disruption in PPI in
adult rats, similar to that seen in schizophrenic patients (e.g. (Martinez et al.
2000b, Mansbach & Geyer 1989). Typical antipsychotics, such as haloperidol,
are not able to reverse deficits in PPI caused by acute PCP treatment in adult
rats (Geyer et al. 2001) or in pre-pubertal rats (Martinez et al. 2002), but in adult
rats, they can reverse the effects of dopamine agonists (Geyer et al. 2001).
Atypical antipsychotics, including clozapine, olanzapine, and quetiapine, are
effective at alleviating acute PCP inhibition of PPI in adult rats (Johansson et al.
1995, Martinez et al. 2002, Geyer et al. 2001, Ballmaier et al. 2001, Johansson et
al. 1994), but not in pups (PN16-19) or pre-pubertal (PN45) rats (Martinez et al.
2002). However, olanzapine is effective at preventing the deficits observed in PPI
in PN24-28 pups following PCP treatment on PN7, 9 and 11 (Wang et al. 2001).
In addition, both olanzapine and risperidone are able to increase PPI in NR1 -/mice (Duncan et al. 2006). The current study shows that both olanzapine and
risperidone pretreatment also blocks the PPI deficit observed in both male and
female PN24-26 pups following PCP pretreatment on PN7, 9 and 11. In contrast,
PCP treatment on PN7 administration did not produce a deficit in PPI in
developing rat pups. It is then reasonable to postulate that PCP treatment on
PN7, 9, & 11 produces a chronic deficit in NMDA receptor function compared to a
single injection of PCP and that this more closely models the disease and the
developmental NMDA hypofunction theory of schizophrenia (Duncan et al. 2006).
Like other psychomotor stimulants, repeated administration of PCP
causes a progressive augmentation of locomotor activity (Xu & Domino 1994),
referred to as sensitization. The neuroadaptations associated with sensitization
may be linked to the mechanisms underlying addiction (Robinson & Berridge
1993). Sensitization is also thought to be an important index related to psychosis
as well as movement and thought disorders in schizophrenia (Robbins 1990).
PCP-induced sensitization is blocked by haloperidol and risperidone (Kitaichi et
al. 1995) and cross-sensitizes with MK-801 (Pechnick & Hiramatsu 1994), but not
107
with amphetamine (Balster 1989, Balster 1986).
In the current study, PCP
treatment on PN7 or on PN7, 9, & 11 produced locomotor sensitization in rats at
PN28-35 that was blocked by pretreatment with either olanzapine or risperidone.
Furthermore, the neurotoxicity evident in the developing frontal cortex following
either a single injection or multiple injections suggests a role for altered cortical
function in the development of this aberrant behavior (Wang & Johnson 2007,
Wang & Johnson 2005, Wang et al. 2001).
Our group and others have demonstrated that a single administration of
PCP, MK-801 or ketamine to PN7 pups induces widespread neuronal apoptosis
(Ikonomidou et al. 1999, Scallet et al. 2004, Young et al. 2005, Wang & Johnson
2007, Wang & Johnson 2005).
Other laboratories have also reported that
transient NMDAR blockade by acute PCP, MK-801, ketamine, and ethanol to
rodents during development causes behavioral, structural, and molecular
abnormalities in adulthood (Fredriksson & Archer 2004, Fredriksson & Archer
2003, Fredriksson et al. 2004, Wozniak et al. 2004, Harris et al. 2003). For
example, Harris et al (2003) found that PN7 female rat pups administered MK801 (0.5 mg/kg, twice, 8 hours apart, s.c.) showed PPI deficits and increased
locomotor activity accompanied by a reduction of brain volume and neuronal
number within the hippocampus and altered hippocampal NR1 subunit
expression.
Several studies from this laboratory have investigated a possible
mechanism by which PCP may elicit its neurotoxic effects and produce
alterations in behavior in rats. In developing rats, PCP treatment on PN7, 9, & 11
of pups resulted in increased expression of NR1 mRNA in the frontal cortex,
striatum, nucleus accumbens and olfactory cortex that was inhibited by
pretreatment with the atypical antipsychotic olanzapine (Wang et al. 2001).
These data are consistent with our previous report demonstrating that chronic
PCP treatment resulted in increased NR1 immunoreactivity in the frontal cortex
and striatum of adult rats treated chronically with PCP (Hanania et al. 1999). In
108
addition, recently we reported that PCP treatment on PN7 produces an upregulation of cortical NR1 and NR2B subunits via an increase in trafficking from
intracellular compartments to the membrane, while subchronic PCP treatment
results in an up-regulation of cortical NR1 and NR2A subunits by increasing new
protein synthesis of these subunits (Anastasio & Johnson 2008b). We extended
these experiments in this study and show that olanzapine and risperidone pretreatment is unable to prevent the up-regulation of cortical NR1 and NR2B
subunits following PCP treatment on PN7 administration. These data suggest
that NMDAR up-regulation in the cortex and subsequent locomotor sensitization
following a single injection of PCP are independent of each other. This suggests
that expression of sensitized behavior is dependent on mechanisms requiring
activation of D2 and/or 5-HT2A/2C receptors.
Thus, even though a sensitized
behavioral response to acute PCP challenge appears to involve activation of D2
and/or 5-HT2 receptors, the increase in NMDAR trafficking appears to be
independent of these receptors. Furthermore, the DA and 5-HT mechanisms
involved in locomotor sensitization may be downstream of receptor regulation. If
so, antipsychotics would be ineffective at preventing PCP-induced changes in
receptor trafficking, but still capable of preventing PCP-induced sensitization and
other behaviors.
In contrast, both olanzapine and risperidone are able to prevent upregulation of NR1 and NR2A subunits in the frontal cortex following sub-chronic
PCP treatment.
These data suggest that up-regulation of NR1 and NR2A
subunits (rather than NR1 and NR2B) may underlie the development of deficits in
PPI following PCP treatment on PN7, 9, & 11, though the relative role of NMDAR
subunits in locomotor behavior and PPI are not completely understood. Several
studies have reported that blockade of NR2B subunits with selective antagonists
(eliprodil and Ro63-1908) does not disrupt PPI (Depoortere et al. 1999, Wiley
1998, Higgins et al. 2003) and that it is the combined blockade of NR2A and
NR2B subunits which is necessary to induce deficits in PPI as well as
109
hyperactivity in rodents (Spooren et al. 2004). Administration of the selective
NR2A antagonist, NVP-AAM007, to adult rats did not disrupt PPI and resulted in
hypolocomotor activity, while the NR2B antagonist, Ro 25-6981disrupted PPI and
produced a dose-dependent hyperlocomotion (Chaperon et al. 2003). Thus, the
role of NR2A and NR2B subunits in the detrimental behavioral effects of PCP
merits further investigation.
Neither the mechanisms underlying PCP-induced alterations in behavior
nor NMDA receptor upregulation nor those responsible for the ability of
olanzapine and risperidone to prevent these alterations are clear. Clozapine,
olanzapine, and M100907, but not haloperidol, have been reported to prevent the
blockade of NMDA responses in the medial prefrontal cortex caused by acute
PCP administration (Arvanov & Wang 1999, Wang & Liang 1998). These data
suggest a prominent role for 5-HT2A receptors in preventing the acute effect of
PCP.
Furthermore, clozapine has been shown to inhibit hypersensitive
responses to NMDA following subchronic PCP (Arvanov & Wang 1999),
suggesting that the expected increase in NMDAR function following PCP upregulation of NR1 and NR2A may also be prevented by olanzapine treatment. It
is possible that the ability of olanzapine to inhibit PCP-induced neurotoxicity,
NMDAR up-regulation and deficits in PPI following PCP treatment stems from its
blockade of both DA and 5-HT2A receptors.
Additionally, if PCP-induced
neurotoxicity and the subsequent loss of these cortical neurons results in a hypoglutamatergic state, then the ability of antipsychotics to prevent this loss of
glutamatergic tone through blockade of DA and 5-HT receptors may be related to
their effectiveness in alleviating both positive and negative symptoms of
schizophrenia (Jardemark et al. 2000). Understanding the mechanism by which
olanzapine and risperidone prevent PCP-induced neuronal apoptosis could
provide insights into the molecular and cellular mechanisms involved in the
behavioral effects of PCP in rats, which in turn could provide insight into the
etiology and pharmacotherapy of schizophrenia.
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PCP treatment on PN7 had no effect on the expression of either PSD-95
(data not shown) or any of the NMDA receptor subunits in the striatum. PSD-95
interacts with the intracellular tail of NR2 subunits and is thought to be
responsible for anchoring the functional NR1/NR2 receptor complex in the
membrane (Wenthold et al. 2003).
Thus, it is possible that the lack of an
observable effect on NMDA receptor expression is related to the lack of effect on
PSD-95. This suggests that the striatal neurotoxicity previously reported (Wang
and Johnson, 2005) is unrelated to alterations in the expression of NMDAR
protein. Similarly, the locomotor sensitization measured following a single
injection of PCP must also be due to mechanisms other than changes in striatal
NMDAR expression levels. However, this does not rule the possible role that
altered NMDAR expression in the frontal cortex may play in the sensitization
response.
Pretreatment with olanzapine was able to prevent the down-regulation of
striatal NR1 polypeptide that was caused by sub-chronic PCP administration;
however, pretreatment with risperidone did not. We have recently observed that
neither SCH23390 (selective D1 antagonist), sulpiride (selective D2 antagonist),
nor M100907 (selective 5-HT2A antagonist) were able to prevent the downregulation of NR1 in the striatum (Anastasio and Johnson, unpublished
observations). Thus, this effect of olanzapine may require blockade of both DA
and 5-HT receptors. In addition to affinity for DA and 5-HT receptors, olanzapine
possesses high affinity for muscarinic ACh receptors (Ki = 1.89 nM), H1
histamine receptors (Ki=7.14 nM), as well as α1 noradrenergic receptors (Ki=19
nM) (Arnt & Skarsfeldt 1998, Raggi et al. 2004); therefore, it is possible that in
the striatum, an area rich in muscarinic ACh receptors, olanzapine’s effect of
blocking down-regulation of NR1 caused by sub-chronic PCP administration to
postnatal rats could also involve an action at these receptors.
PN7-11 is a critical stage in brain development, with ample evidence of
neurite growth and synapse formation, and is referred to as the brain growth
111
spurt (Olney et al. 2002). Thus, it is reasonable that even temporary changes
during this critical period could result in behavioral changes as these animals
mature. No variation in the expression of cortical NR1, NR2A, or NR2B was
observed in either saline or sub-chronically PCP treated pups as measured
immediately following PCP challenge and behavior measurement on PN28-35.
Thus, it could be argued that the behavioral effects observed at this time are
independent of the effects of PCP challenge on the expression of the NMDAR
and that are more likely due to changes in NMDAR expression during an earlier,
critical period of development and the subsequent maturation of the brain.
Additionally, these results are consistent with the neurodevelopment theory of
schizophrenia, which hypothesizes that damage during this early, critical stage of
brain development may account for the later manifestation of mental disorders
(du Bois & Huang 2007).
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Chapter 6: Modeling the Negative Symptoms of
Schizophrenia
Disruptions in the glutamate system are thought to play a causative role in
the pathogenesis of schizophrenia. PCP or “angel dust” is a drug of abuse,
which blocks the NMDA receptor in several corticolimbic structures responsible
for emotions, memory, and higher thought processes. PCP administration in
humans has been shown to mimic both the positive and negative symptoms of
schizophrenia. PCP administration in immature rats has been shown to cause
neuronal cell death and as the rat matures, irregular behaviors become evident.
The purpose of this study was to determine the effect of perinatal PCP treatment
in rat models of the negative symptoms of schizophrenia. We incorporated two
behavior models to accomplish this, the social interaction test and the social
discrimination task. Male and female Sprague-Dawley rats were treated with 10
mg/kg PCP on PN7, 9, & 11 and were tested for social interaction on PN28 (prepubescent) and again on PN54 (post-pubescent) or were tested for social
discrimination on PN58-60. Pre-pubescent males showed a significant reduction
in social interaction, which was no longer evident when measured two weeks
after the onset of puberty (PN54). PCP treatment of perinatal females had no
effect on social interaction at either age.
Both adult male and female rats
showed a deficit in social discrimination, as marked by a reduction in the novelty
discrimination index.
These data support the use of sub-chronic PCP
administration in perinatal rats as useful in modeling the negative symptoms of
schizophrenia.
Introduction
Schizophrenia is a severe neuropsychiatric disorder characterized by the
presence of both positive, e.g. paranoia, hallucinations, delusions, and negative
symptoms, including but not restricted to social and emotional withdrawal,
113
anhedonia, and depression, as well as cognitive impairments such as memory
and attention deficits (McGlashan 1996a, Koros et al. 2007).
Moreover, the
negative symptoms have a propensity to be more enveloping and relentless, and
less sensitive to antipsychotic medications (Fenton & McGlashan 1991). PCP is
well known to exacerbate psychoses in schizophrenics (Luby et al. 1962, Javitt &
Zukin 1991, Lahti et al. 2001). Importantly, clinical observations have shown that
PCP and ketamine intoxicated humans repeatedly present with symptoms which
mimic the positive as well as several negative symptoms of schizophrenia,
including social withdrawal. PCP animal models of this disease have been
developed in order to replicate the various symptoms of schizophrenia and serve
as investigatory tools for development of future therapeutic compounds.
Hypofunction in the prefrontal cortex is thought to underlie the origins of
the negative symptoms of the disease (Goff & Evins 1998). In addition, there is
anatomical
evidence
of
deficits
“hypoglutamatergic” in nature.
in
cortical
function
that
may
be
Glutamate promotes various phases of neuronal
development, including migration, differentiation, and synaptic plasticity (Komuro
& Rakic 1993).
In the perinatal rat, the NMDAR is the primary mediator of
glutamatergic excitatory neurotransmission in the brain and neurons expressing
the NMDAR undergo a period of hypersensitivity to NMDAR blockade in which
they are highly prone to excitotoxicity and neurodegeneration (Ben-Ari et al.
1997). Transient inactivation of NMDARs can be lethal for many neurons;
administration of PCP or other noncompetitive NMDA antagonists, such as MK801 (dizocilpine), in the adult rat resulted in restricted neurotoxicity and structural
damage in brain regions similar to those in which damage is present in
schizophrenics (Olney et al., 1989; Olney and Farber, 1995b). PCP-mediated
neurotoxicity in the prefrontal cortex, may underlie the aberrant schizophreniclike behaviors reported in various animal models of the disease.
This
laboratory
has
previously
reported
that
sub-chronic
PCP
administration to developing rat pups can model the positive symptoms of
114
schizophrenia (enhanced locomotor activity and development of locomotor
sensitization following PCP challenge) as well as deficits in pre-pulse inhibition of
acoustic startle (Anastasio & Johnson 2008a, Wang et al. 2001), both of which
can be alleviated by treatment with atypical antipsychotics, such as olanzapine
and risperidone. Acute and repeated PCP, but not amphetamine treatment of
rats, also results in deficits in social interaction (Sams-Dodd 1999, Sams-Dodd
1998) and cognitive deficits as measured by the delayed spatial alternation task
(Koros et al. 2007, Wang et al. 2001, Wiley et al. 2003a). Chronic pre-treatment
(21 days) with the atypical antipsychotic, clozapine, but not the typical
antipsychotic haloperidol reversed the PCP (2 mg/kg x 3 days)-induced deficits in
social behavior (Sams-Dodd 1996).
An additional symptom characteristic of
schizophrenia is a deficit in selective attention and information processing, i.e.
the inability to differentiate relevant from irrelevant information (Goldberg & Gold
1995). The capacity of an adult rat to differentiate between a familiar and a novel
juvenile in a social environment has been suggested to be a suitable animal
model of selective attention deficits (Engelmann et al. 1995).
Acute PCP
administration to adult rats has been reported to produce a reduced novelty
discrimination index [time spent investigating the familiar juvenile compared to
time spent investigating novel juvenile] (Terranova et al. 2005). Further, adult
rats which received PCP during the perinatal stage (PN7, 9, & 11) also present
with a deficit in the novelty discrimination index (Depoortere et al. 2005,
Terranova et al. 2005, Harich et al. 2007a). These deficits could be reversed
with post-treatment with the atypical antipsychotic clozapine (Terranova et al.
2005), supporting this behavioral test as a suitable model of the attention
processing deficits present in schizophrenia.
There is a paucity of studies investigating the PCP perinatal model and its
ability to mimic the negative symptoms of schizophrenia; therefore, the purpose
of this study was to explore whether this specific sub-chronic PCP treatment
paradigm models the negative symptoms of schizophrenia. Specifically, the
115
effect of perinatal PCP administration on social interaction was measured in preand post-pubescent rats and its effect on social discrimination was also
determined when the pups were adults.
Materials and Methods
Animals.
Timed, day 14 pregnant female Sprague-Dawley rats were obtained from
Charles River Laboratories (Wilmington, MA).
The dams were housed
individually with a regular 12h light-dark cycle (lights on 0700, off at 1900) with
food and water ad libitum. Following parturition, male and female pups from four
dams were combined and randomly cross-fostered to one of the four lactating
dams.
Each litter consisted of ten to twelve pups.
All experiments were
conducted in accordance with NIH regulations and with approval of the University
of Texas Medical Branch at Galveston Institutional Animal Care and Use
Committee.
Drugs.
Phencyclidine was acquired from the National Institute on Drug Abuse
(NIDA, Rockville, MD) and dissolved in 0.9% NaCl.
Experimental design.
Male and female Sprague-Dawley rat pups were treated on PN 7, 9, and
11 (sub-chronic) with 10 mg/kg PCP or saline vehicle (s.c). On PN22, littermates
were weaned and housed 2 per cage according to treatment and sex. Animals
were then tested for deficits in social interaction on PN28 (pre-pubescent) and
PN54 (post-pubescent) or deficits in the social discrimination task on PN58-60
(adult).
116
Social Interaction Test.
Social interaction is best defined as the time a pair of animals spends in
an active social interaction (sniffing and grooming its partner, close following,
mounting and crawling over or under its partner) with or without direct physical
contact (Mouri et al. 2007). Testing was conducting during the dark cycle in
order to decrease anxiogenic behavior and aggressive/territorial behavior as well
as increase basal social behavior. The test was performed in open arena (40 x
40 x 40 cm) with bottom and sides made of clear Plexiglass placed in a sound
attenuated chamber equipped with video recording capabilities.
The light
intensity was 30 lx and the bedding had been pre-exposed to other rats in order
to maintain a constant odor level in the arena. On the day of testing, unfamiliar
rat pairs that were matched for treatment, weight and sex were placed
simultaneously in the arena 20 cm apart. Each testing session lasted for 10
minutes.
Videos of the testing session were analyzed for % time of direct
physical contact as well as types of social interacting behaviors. The types of
social behavior measured were: investigative sniffing (anogenital, snout, other
body parts), climbing over or under (climbing over the back or pushing head and
forepart of body beneath), grooming the partner, following (rat moves after its
partner without direct physical contact), avoiding (rat actively turns away or
freezes when approached by its partner) (Sams-Dodd 1999, Sams-Dodd 1995).
No aggressive or territorial behaviors were noted in any of the test groups.
Social Discrimination Task.
Social discrimination was measured as described previously with minor
modifications (Harich et al. 2007a). All tests were performed during the light
period of the night-day cycle under dimmed illumination (30 lx). Animals were
treated as previously described with saline or PCP, allowed to age and on the
day of testing (PN58-60), they were brought to a designated behavior room and
allowed to acclimate for 30 min.
They were then placed in an open field (40 x
117
40 x 40 cm) together with a same sex familiar juvenile (40-55 g) for 30 min. At
the end of this period, a second unfamiliar juvenile, termed the novel juvenile,
was placed in the chamber for an additional 5 min. The entire session was
video-recorded and the time of investigative behavior of or by the adult
(anogenital exploration, sniffing with direct contact, crawling over each other,
grooming, play behaviors) towards each of the two juveniles was analyzed by a
trained observer. The time spent investigating the familiar juvenile divided by the
time spent investigating the novel juvenile is presented as the novelty
discrimination index.
Statistical Analysis.
Group comparisons were specifically defined before the beginning of each
experiment; therefore, planned comparisons were performed instead of an
overall F test in a multifactorial ANOVA (Keppel 1982). All values are presented
as mean ± SEM. The null hypothesis was rejected at p<0.05.
Results
The social interaction test we employed was based on previous studies
with some modifications and has been shown repeatedly to model the negative
symptoms of the disease with face and predictive validity (Snigdha & Neill 2008,
Sams-Dodd 1996, Koros et al. 2007).
Perinatal PCP treatment of young males
(PN28) caused an overall significant 25 % decrease in total time spent in social
interaction (from 6.7 min in saline treated pups to 5 min in PCP treated pups).
Concomitant with this, we observed that PCP treated pups showed a significant
91% increase in total (central and peripheral) locomotor activity compared to the
saline treated controls (Figure 6.1 left). Both the deficit in social interaction and
increase in locomotor activity disappeared in mature males aged PN54 (Figure
6.1 right). No deficit in social interaction or alterations in locomotor activity was
118
measured in either pre-or post pubertal (aged PN28 and PN54, respectively)
females (Figure 6.2).
In order to extend previous work which suggested that PCP treatment can
also model the selective attention and information processing deficits seen in
schizophrenia (Goldberg & Gold 1995) Deficits in social novelty discrimination,
i.e. the inability of an adult rat to discriminate between a familiar and a novel
juvenile, is thought to serve as a suitable animal model of this symptom of
schizophrenia (Engelmann et al. 1995). Deficits in novelty discrimination have
been shown to occur after acute PCP (Terranova et al. 2005) and in adult rats
that received PCP treatment during development (Depoortere et al. 2005,
Terranova et al. 2005, Harich et al. 2007a).
In this study, it was observed that
both male and female rats treated on PN7, 9, & 11 with PCP have significant and
long-lasting deficits in novelty discrimination as measured on PN58-60 (Figure
6.3).
119
Figure 6.1. Effects of neonatal PCP administration on social interaction
and locomotor activity in pre-and post-pubescent male pups. Quantitative
analysis of the % total time of social interaction (left) and the total locomotor
activity (right) in pre- and post-pubescent saline and PCP-treated males.
N=6/treatment group. *p<0.05 vs. saline PN28 male (Student’s t-test).
120
Figure 6.2. Effects of neonatal PCP administration on social interaction
and locomotor activity in pre- and post-pubescent females.
Neither the
percent of total time spent in social interaction (left) nor the total locomotor
activity (right) in either pre-pubescent (PN28) or post-pubescent (PN54) was
different between saline and PCP-treated females. N=6/treatment group.
121
Figure 6.3. Effects of perinatal PCP treatment on PN7, 9, & 11 on social
discrimination in adult (PN56-58) animals. The novelty discrimination index is
defined by the time spent investigating the novel juvenile divided by time spent
investigating the familiar juvenile) of both adult male and female Sprague-Dawley
rats treated during development on PN7, 9 and 11.
*p<0.05 vs. saline (Student’s t-test).
122
N=8/treatment group.
Discussion
It has been observed in the clinical setting that NMDAR antagonists, but
not d-amphetamine, repeatedly and reliably induce several negative symptoms of
schizophrenia, specifically social isolation and withdrawal (Sams-Dodd 1999).
Further, stereotyped behaviors and locomotor hyperactivity, but not social
interaction, relates to the positive symptoms of schizophrenia and may also serve
as a marker of the psychotomimetic effects of a drug (Mouri et al. 2007). The
present study shows that sub-chronic PCP administration to the perinatal rat
results in long-lasting deficits in behavior which are thought to represent the
negative symptoms of schizophrenia.
Repeated PCP administration has been shown to induce deficits in social
interaction, some of which have been shown to be persist for at least 28 days
after withdrawal from treatment (10 mg/kg/day for 14 days) (Sams-Dodd 1999,
Qiao et al. 2001, Sams-Dodd 1996). Here, we report that PCP treatment causes
a deficit in the social interaction test in pre-pubertal, but not post-pubertal males.
Further, stereotyped behavior, ataxia, and immobility were absent on the day of
testing suggesting a specific effect on social behavior and not due to the
intoxicating effects of PCP administration. The PCP pharmacokinetic differences
between male and female rats are well known and are a significant parameter
which must be acknowledged when interpreting PCP-induced behavioral
differences between genders. Further, there are important sex differences in
social interaction, such that social interaction may play a different role in females
compared to males; hence, caution should be exercised when interpreting results
from females (File & Seth 2003). The present study reports that PCP treatment
to females on PN7, 9, & 11 has no effect on social interaction in either the prepubescent or post-pubescent female. An analogous study which tested social
interaction 10 weeks after PCP treatment of females on PN7, 9, & 11 also saw
no deficit (du Bois et al. 2008). Alternatively, a study by Snigdha and Neill (2008)
treated adult female rats with PCP (2 mg/kg, 2x/day for seven days) reported
123
enduring deficits in social interaction which persisted for at least six weeks after
treatment. The discrepancies are most likely due to differences in drug treatment
schedules, ages and species of rats used, and/or the test parameters employed.
In these experiments the observed hyperlocomotion in pre-pubertal males
represents the total locomotor activity of both animals; therefore, it is not possible
to draw unequivocal conclusions concerning the PCP treated rats. However, it is
reasonable to postulate that the observed hyperactivity in response to the novel
environment in the perinatal PCP-treated pre-pubertal males was due to perinatal
PCP treatment of the subjects rather than to an unlikely difference in the
untreated pups. PCP and MK-801 induce spontaneous hyperactivity in laboratory
animals in a novel environment (Ford et al. 1989, Verebey et al. 1981). PCPinduced locomotor activity and the positive symptoms of the disease can be
blocked by typical antipsychotics such as haloperidol, a potent and high affinity
dopamine D2 antagonist (Moghaddam 1994, Verma & Moghaddam 1996).
Synaptic interaction between dopaminergic and glutamatergic systems in the
mesocorticolimbic system may underlie spontaneous exploration of a novel
environment, a complex behavior which is affected by sensory processing,
environmental cues, the animals’ state of arousal and interactions with other
animals (Kelley et al. 1989, Clements & Greenshaw 2005). Thus, because of the
ability of PCP to block the NMDA glutamate receptor subtype and increase
activity in mesolimbic, mesocortical and nigrostriatal dopaminergic pathways
(Jentsch et al. 1998, French & Ceci 1990), it is reasonable to expect that
perinatal PCP treatment during development could alter exploratory behaviors
mediated by these pathways later in life.
An additional purpose of this study was to investigate whether sub-chronic
PCP treatment of rat pups could model multiple aspects of the deficits in social
function characteristic of schizophrenia, specifically by also measuring the effects
of PCP on selective attention and information processing deficits. The social
discrimination task used in the current study is an animal behavior model for
124
selective attention capacities (Harich et al. 2007a, Terranova et al. 2005). In this
task, the adult animal must focus on the relevant juvenile, the novel juvenile,
which requires a high level of selective attention since both juveniles move
around rapidly playing with each other, i.e. the spatial position of the two stimuli
changes constantly. Terranova et al. (2005) determined that this test is not likely
to be a measure of impaired short term memory or social recognition abilities
since the adult rat’s competence for social discrimination was maintained
subsequent to each juvenile being placed into a small mesh cage thereby
preventing it from freely moving around in the chamber.
Previously it has been established that PCP administration on PN7, 9 and
11 to male rats results in a diminished novelty discrimination index, a deficit that
was effectively treated with novel and potential antipsychotics, including a glycine
transport inhibitor and mGluR2/3 agonists (Depoortere et al. 2005, Harich et al.
2007a). Further, others have extended our model by showing that a 5-HT1A
agonist/D2 antagonist dulled the effects of perinatal PCP on novelty
discrimination in a social context (Terranova et al. 2005). Here we demonstrate
that sub-chronic PCP administration to both developing male and female rat pups
results in long-lasting deficits in the novelty discrimination index, i.e. following
treatment with PCP during development, the young adult animals that were
treated with PCP appear to lack the ability to focus their attention on the novel
juvenile. Similar to Harich et al (2007a), PCP treated animals spent more time
investigating the familiar juvenile compared to the saline treated adults,
suggesting an incomplete familiarization by the adult PCP treated animal within
the first 30 minutes of the test, thereby increasing the exploratory time of the
familiar juvenile during the last 5 min of the task.
Taken in context with other reductions in information processing as
evidenced by deficits in pre-pulse inhibition (Anastasio & Johnson 2008a, Wang
et al. 2001) this study further supports the use of perinatal PCP treatment as a
125
useful tool in characterizing and designing pharmacotherapeutics approaches to
the multiple symptoms of schizophrenia.
126
Chapter 7: Conclusions and Future Directions
General Conclusions
Clinical use of PCP, a potent dissociative anesthetic, was abandoned as a
result of reports of post-operative hallucinations and disoriented behavior. Due
to its hallucinogenic effects, PCP appeared on the drug of abuse scene in the
mid-1960’s known as “angel dust”; nevertheless, illicit use of PCP has subsided
because of its psychotomimetic properties. In addition to the psychotomimetic
symptoms produced by PCP, several parallels exist between PCP administration
and schizophrenia.
PCP has been reported to increase cerebral metabolic
activity in several limbic brain regions including the entorhinal cortex,
retrosplenial and anterior cingulate cortex, the subiculum and the nucleus
accumbens (Hargreaves et al. 1993, Weissman et al. 1989). Ketamine has also
been shown to increase cerebral blood flow in the anterior cingulate cortex (Lahti
et al. 1995).
In addition, acute PCP administration was reported to cause
neurotoxicity in the posterior cingulate/retrosplenial cortex (Olney et al. 1989b).
Chronic administration of MK-801 or PCP caused neurodegeneration in
additional brain regions including the anterior cingulate, parietal, temporal,
piriform, and entorhinal cortices, hippocampus, and amygdala (Olney & Farber
1995b, Olney & Farber 1995a). These brain regions in which PCP and other
noncompetitive NMDA antagonists alter cerebral blood flow and glucose
utilization as well as produce neurotoxicity are some of the same brain regions
that are thought to be affected in schizophrenia (Strous & Javitt 1996).
It was postulated that since symptoms of schizophrenia do not appear
until early adulthood that the etiology of the disease may be developmental in
nature (Weinberger 1987), which led to the investigation of the neurotoxic effects
of acute PCP or MK-801 during stages of rodent development (Ikonomidou et al.
1999, Wang et al. 2001, Wang & Johnson 2005).
127
As evidenced by several
measures of neurotoxicity (TUNEL positive, cleaved caspase-3, and silver
staining) PCP administration on postnatal day (PN)7 causes a robust loss of
neurons in layers II-IV of the cingulate, parietal, and retrosplenial cortices, but
also in the subiculum/CA1 region, anterior ventral nucleus of the thalamus and
striatum (Wang & Johnson 2007, Wang & Johnson 2005). On the other hand,
PCP administration on PN7, 9, & 11 shows a neurotoxic regional specificity for
the frontal cortex only (Wang et al. 2001, Wang & Johnson 2005). Interestingly,
PCP administration on PN7 results in a loss of parvalbumin (PV)-containing, but
not calretinin or calbindin-containing neurons in the superficial layers (II–IV) of
the primary somatosensory, motor, and retrosplenial cortices, but not in the
striatum or hippocampus in PN56 rats (Wang et al. 2008).
The effects of PCP
on administration on PN7, 9, & 11 on PV expression in the adult animal have yet
to be determined. The developmental dependence and neuronal specificity of
this effect of PCP is remarkably similar to the damage observed in schizophrenic
brains. These data strongly support the importance of determining the precise
mechanisms of PCP-induced neurotoxicity and the functional consequences of
this neurotoxicity in this model of schizophrenia.
The overall purpose of this study is to address the hypotheses that
neurotoxicity during a critical stage in brain development and subsequent
alterations in cortical function may underlie the expression of aberrant
schizophrenic-like behaviors in the developing rat.
In summary, this work
demonstrates that PCP induces neurotoxicity in developing pups in a manner
that is highly regulated affecting the distribution, subunit composition, number,
and function of NMDAR and that this loss of cortical neurons and altered cortical
landscape is associated with behavioral deficits that are similar to those
observed in schizophrenia.
Specifically, PCP on PN7 causes cortical
neurotoxicity by blocking NR1/NR2A receptors and induces a compensatory upregulation of NR1/NR2B in the synapse and the subsequent activation of calpain.
Up-regulation of NR1/NR2B is most likely due to an increase in trafficking of
128
NR1/NR2B from ER to membrane.
Further, the simultaneous increase in PSD-
95 most likely protects NR2B from calpain mediated cleavage; therefore, no
internalization of the receptor occurs and membrane expression levels remain
high. PCP on PN7, 9, & 11 also induces neurotoxicity in the frontal cortex by
inhibiting NR1/NR2A-containing receptors resulting in a compensatory upregulation of NR1/NR2A receptors at the synaptic membrane, most likely through
an increase in new NMDAR protein synthesis via NFκB nuclear translocation
(Wang et al. 2001).
We had hypothesized that an increase in NMDAR
expression would result in intracellular Ca2+ overload and the activation of the
apoptotic cell death cascade (McInnis et al. 2002, Wang et al. 2001, Wang et al.
2003). However, we no longer believe this to be true, since the NMDAR protein
measured in these studies must come from live cells and the time course of upregulation of the receptor does not correspond to the time course of cell death.
Caspase-3 is activated by 2 hrs in the frontal cortex; furthermore, TUNEL
staining appears 6 hrs after treatment, peaks at 16 hrs and is completely cleared
by 24 hrs (Wang & Johnson 2005, Wang & Johnson 2007). Therefore, we now
propose that the compensatory mechanism of up-regulation occurs in surviving
neurons in response to the death of neighboring cells and may serve to “protect”
these neurons from PCP-induced neurotoxicity and re-establish NMDARmediated neurotransmission.
Further, the newly inserted NMDARs are
functionally active receptors and following agonist stimulation those from PCP
treated animals (both acute and sub-chronic) function at a higher level compared
to their saline counterparts. This change in function and loss of neurons may
alter the architecture of the cortex as well as cortical glutamatergic and
GABAergic neurotransmission (Olney & Farber 1995a, du Bois et al. 2009),
which results in deficits in PPI of acoustic startle, social interaction, social
discrimination as well as the development of locomotor sensitization.
129
Specific Aim 1
Specific Aim 1 was designed to determine the mechanism of PCP-induced
regulation of the NMDAR in the frontal cortex.
Acute PCP treatment (single
injection on PN7) increased membrane levels of NR1 and NR2B protein in the
frontal cortex, while no change in the expression of NR2A was detected. We
next examined the effect of PCP on the expression of PSD-95, a member of the
NMDAR post-synaptic density complex. Acute PCP administration produced an
increase in membrane cortical protein levels of PSD-95, which paralleled the
observed increase in membrane NR1 and NR2B expression in the frontal cortex.
On the other hand, NR1 and NR2B protein levels in the ER fraction were
decreased by acute PCP, suggesting an increased trafficking of NR1/NR2B from
the ER to the membrane. Pretreatment with various antipsychotics and selective
D1, D2 or 5-HT2A receptor antagonists had no effect on up-regulation of NMDAR
protein expression induced by acute PCP. In distinction from the effects of acute
PCP administration, sub-chronic (PN7, 9, & 11) PCP treatment caused an
increase in NR1 and NR2A expression in both the ER and membrane fractions of
the frontal cortex, while no effect on NR2B expression was evident in either
fraction. We also determined that sub-chronic PCP administration did not alter
levels of PSD-95 in the cortical membrane fraction. The increase in NR1/NR2A
in the membrane fraction was blocked by pretreatment with antipsychotics and
selective 5-HT2A, D1, or D2 receptor antagonists.
The mechanism(s) of trafficking of NR1, NR2A, and NR2B subunits from
intracellular compartments to the membrane and the role of calpain-mediated
trafficking in the regulation of the NMDAR was also examined. We examined the
role of calpain, a calcium-dependent neutral cysteine protease which is thought
to regulate NMDAR trafficking, in both the acute and sub-chronic treatment
paradigm. Calpain activation has been shown to play a role in excitotoxicity
(Saido et al. 1994, Brorson et al. 1995, Araujo et al. 2004, Zhou & Baudry 2006)
in several neurodegenerative disorders including Parkinson’s disease as well as
130
Alzheimer’s disease (Etienne & Baudry 1987, Vanderklish & Bahr 2000, Crocker
et al. 2003).
Calpain-mediated enzymatic cleavage of cytoskeletal proteins
results in the loss of structural integrity and disturbances in axonal transport
(Yamashima 2004).
Additional substrates for calpain include αII-spectrin
(cleavage position Tyr1176 & Gly1177), PSD-95, the NR2A subunit (cleavage
position Phe1279; Ser1330), and the NR2B subunit (cleaved fragments of 140,
130, & 115/120 kDa) (Bi et al. 1998a, Bi et al. 1998b, Guttmann et al. 2001). To
measure calpain activation, we performed Western blots on membrane cortical
tissue from acute or sub-chronic PCP treated animals in order to examine the
protein expression levels of αII-spectrin (a member of the post-synaptic density
that is enzymatically cleaved directly by calpain) and its calpain specific
breakdown products (SBP) which migrates at 145 and 150 kDa. Acute PCP
treatment resulted in an increase in the αII-spectrin calpain SBP while subchronic PCP treatment had no effect on αII-spectrin calpain SBP expression
levels.
In order to add validity to the role of calpain in acute PCP-induced
trafficking of the NR1/NR2B, we determined the effect of administering 10 mg/kg
MDL28170 (a selective calpain inhibitor every 2 hrs for 8 hrs after PCP on PN7)
on the membrane expression levels of NR1/NR2B in the frontal cortex.
MDL28170 was able to prevent the up-regulation of NR1and NR2B protein levels
and it also prevented cleavage of calpain itself.
These data support our
hypothesis that a treatment dependent mechanism of calpain-mediated
trafficking may underlie the regulation of the NMDAR subunit composition
following acute PCP administration to the perinatal rat.
The NR1 subunit contains eight splice variants, one in the N terminus and
two in the C terminus that possess distinguishing features. The splice variants
differ in regard to their regional pattern of expression, their regulation by
phosphorylation, polyamines and protons, their electrophysiological properties,
and their affinity for cytoskeletal proteins (Cull-Candy et al. 2001). The C terminal
splice variant composition affects trafficking of the NR1 subunit. It has been
131
suggested that the C2’ cassette present in four of the NR1 splice variants
possesses a PDZ binding domain (STVV) (Wenthold et al. 2003). In addition, the
NR1 subunit contains an ER retention signal (RXR) in the C1 cassette that must
be masked for the polypeptide to be released from the ER (Wenthold et al.
2003).
Thus, an alternative explanation underlying the mechanism of PCP-
induced alterations in trafficking of the receptor may exist. It is reasonable to
suggest that following acute PCP administration, binding of PSD-95 to the PDZ
binding domain of the C2’ cassette of the NR1 subunit may mask the RXR motif
in the C1 cassette, resulting in the exit of the NR1 subunit from the ER and
insertion in the membrane (Standley et al. 2000); therefore, the robust increase
in cortical membrane levels of PSD-95 caused by acute PCP treatment could be
due to its persistent binding to the C2’ cassette of the NR1 subunit in conjunction
with its presence in the post-synaptic density complex. This suggests that PSD95 interacts early in secretory pathways, perhaps functioning as an export protein
that overrides the ER retention signal for the NR1 subunit in addition to serving to
anchor the functional, assembled receptor complex in the synapse by binding to
the C-terminus of the NR2 subunit (Wenthold et al. 2003).
PCP blockade of the NMDA receptor results in the efflux of glutamate in
the frontal cortex (Adams & Moghaddam 2001) leading to the subsequent
activation of either AMPA/KA receptors or metabotropic glutamate receptors
(mGluRs).
Investigation of the interactions between mGluRs and NMDARs
suggests that stimulation of mGluRs potentiates NMDAR function (Aniksztejn et
al. 1991, Bleakman et al. 1992, Harvey & Collingridge 1993). Modulation of
NMDAR function by mGluRs is thought to be limited to Group I; both mGluR1
(Lan et al. 2001) and mGluR5 (Pisani et al. 2001) have been implicated. Group I
mGluRs are G-protein coupled receptors that are positively coupled to
phospholipase C (PLC) resulting in the formation of diacylglycerol (DAG) and
activation of protein kinase C (PKC), leading to the release of Ca 2+ from
intracellular stores (Conn & Pin 1997).
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Potentiation of NMDAR function by
mGluR5 is dependent on G-protein second messenger signaling cascades,
which includes PKC and Src kinase (Benquet et al. 2002). PKC phosphorylation
of sites near the RXR motif of the NMDAR NR1 subunit masks the ER retention
signal resulting in the trafficking of the subunit to the membrane (Scott et al.
2003).
Thus, activation of mGluRs may be an additional compensatory
mechanism in response to acute PCP blockade of the NMDAR.
Next, we tested the role of protein synthesis in the regulation of the
NMDAR. Cycloheximide (CHX) has been shown to cause transient (~12 hrs)
suppression of brain protein synthesis in rats (Pavlik & Teisinger 1980). Since,
protein synthesis would be intermittently released from inhibition in our subchronic paradigm we anticipated that the rats would tolerate the CHX posttreatment well (Liu et al. 1997). Although the chosen dose and dosing schedule
of CHX had been reported to be well tolerated in adult animals (Liu et al. 1997), it
was toxic in our experimental paradigm using perinatal rats. Animals treated with
PCP (10 mg/kg) + CHX (0.25 mg/kg) on PN7, 9, & 11 died on PN12. Therefore,
we administered CHX only on PN7 and PN9 three hours after PCP treatment.
No animals in this paradigm died; however, a significant difference in their weight
was observed.
Since we were not comfortable with the level of toxicity, we
decided to not pursue this project further.
The ability of atypical antipsychotics as well as selective DA and 5-HT
receptor antagonists to block sub-chronic induced alterations in the NMDAR
subunits suggests that up-regulation of NR1 and NR2A in the frontal cortex
following sub-chronic PCP administration may be due to increased synthesis of
protein and mediated in part by D1, D2, and 5-HT2A receptors, which may have
been activated by the effects of PCP on dopaminergic and serotonergic neuronal
populations (Adams & Moghaddam 2001, Verma & Moghaddam 1996). PCPinduced release of DA in the prefrontal cortex (Verma & Moghaddam 1996) could
lead to the activation of D1 receptors resulting in increased levels of cAMP and
increased activation of PKA, which would be expected to increase the
133
phosphorylation of the transcription factor CREB (cAMP response element
binding protein) (Lau et al. 2004).
The NR1 gene contains a CRE (cAMP
response element) binding site upstream of its transcription start site (Lau et al.
2004). PCP-induced phosphorylation of CREB by PKA or from activation of the
MAP kinase pathway could then lead to increased binding of P-CREB to the NR1
CRE site, resulting in increased transcription of NR1 subunit protein in the frontal
cortex. However, preliminary data indicates that the expected increase in cortical
levels of P-CREB following both acute and sub-chronic PCP administration is not
quite significant (Robeson and Johnson, unpublished observation).
In addition, the NR1 gene contains a binding site for the transcription
factor NF-κB (Liu et al. 2004a). Liu et al. (2004a) reported that binding to the
NR1 NF-κB site up-regulates the NR1 promotor and subsequent transcription of
the gene through interactions with Sp1/Sp3 factors. Wang et al. (2001) reported
that sub-chronic PCP treatment increased nuclear translocation of NF-κB
subunits p50 and p65 as evidenced by electrophoresis mobility shift assay
(EMSA); this was prevented by olanzapine pretreatment.
Therefore, PCP-
induced activation of NF-κB transcription factors may be an alternative
explanation for the observed increase in synthesis of NR1 protein.
Specific Aim 2
Specific Aim 2 attempted to determine the function of the NMDAR in the
frontal cortex using NMDA and glycine stimulated 3H-MK-801 binding.
PCP
blockade of the NMDAR results in a loss of cortical GABAergic PV-containing
interneurons
(Wang
et
al.
2008)
and
disruption
in
glutamatergic
neurotransmission, resulting in the compensatory up-regulation of the NMDAR in
surviving neurons.
This aim was designed to test the hypothesis that up-
regulation of the NMDAR in the frontal cortex by acute or sub-chronic PCP
treatment of perinatal rats results in a hyper-functional receptor and is dependent
on the relative NR1/NR2B versus NR1/NR2A composition of the receptor. We
134
determined that both acute and sub-chronic PCP administration causes the
insertion of functionally active NMDARs in to the cortical synaptic space as
evident by an increase in maximal NMDA and glycine stimulated 3H-MK-801
binding, which paralleled the increase in NMDAR receptor density (Anastasio &
Johnson 2008b).
However, we were not able to definitively determine the
subunit composition responsible.
A flaw in the experimental design may
underlie the inability to draw conclusions from the ifenprodil and NR2A antagonist
(both NVP-AAM007 and PEAQX) inhibition studies; i.e. the dose range tested for
these compounds was too narrow and did not produce a full dose response
inhibition curve. Experiments are currently ongoing which address this concern.
Additionally, it would be useful to perform electrophysiological experiments to
verify the binding results as well as to determine whether any changes in
synaptic plasticity are evident, such as NMDA-dependent LTP which would
underlie the proposed altered cortical function. It has been hypothesized that the
NR2A subunit is the main mediator involved in delivering the AMPA GluR1
subunit to the synaptic membrane (via Ras-ERK activation), a critical step in
NMDAR-dependent LTP (Kim et al. 2005); therefore, it would be instructional to
measure the effect of acute and sub-chronic PCP treatment on synaptic GluR1
content as well as NMDAR-dependent LTP.
Specific Aim 3
The subunit composition of the NMDAR that mediates PCP-induced
neuronal neurotoxicity is currently unknown; therefore, we designed specific aim
3 to show that the synaptic or extrasynaptic localization of NR2A and 2B
receptors may determine function.
Over the last several years substantial
evidence indicates that activation of synaptic NMDARs promotes neuronal
survival, but activation of extrasynaptic receptors leads to cell death (Hardingham
et al. 2002, Hardingham & Bading 2002). Thus, we proposed that PCP damages
neurons by blocking synaptic NMDA receptors, most of which are thought to
135
contain NR1 and NR2A (Hardingham & Bading 2003, Hardingham et al. 2002).
To investigate the role of synaptic and extrasynaptic NMDARs in PCP-induced
neurotoxicity in vitro and in vivo we used antagonists at various doses for NR2A
(NVP-AAM007 and PEAQX) and NR2B (ifenprodil). NVP-AAM007 and ifenprodil
were administered to organotypic corticostriatal slice cultures as well as
developing rat pups and apoptosis was estimated by measurement of caspase-3
enzymatic activity and histochemical assessment of TUNEL labeling of apoptotic
neurons. In corticostriatal slices and in vivo, we found that both NVP-AAM007
and PEAQX are neurotoxic in the cortex, while ifenprodil is not. These data
suggest that neurotoxicity in the frontal cortex of developing rat pups following
acute or sub-chronic PCP administration is due to blockade of NR1/NR2A
receptors, but not NR1/NR2B receptors. These results implicate synaptic NMDA
receptors, rather than non-synaptic receptors as mediators of PCP-induced
neuronal death. Although NVP-AAM007 and PEAQX may have a minor capacity
for inhibition of NR2B-containing receptors (Frizelle et al. 2006, Neyton & Paoletti
2006), the lack of a neurotoxic effect by ifenprodil (selective NR2B antagonist)
strongly argues against a role for the NR2B subunit at this stage of development
in this PCP neurotoxicity model of schizophrenia.
Cell death in the striatum following a single injection of PCP was induced
by high doses of PEAQX, but not ifenprodil. These data suggest that acute PCPmediated neurotoxicity in the striatum is also mediated through blockade of
NR1/NR2A receptors. Sub-chronic PCP does not induce noticeable neurotoxicity
in the striatum after the last of three doses, but sub-chronic blockade of NR2A
receptors in the striatum with PEAQX resulted in neurotoxicity at the highest
dose of PEAQX administered. It has been postulated that at high doses this
compound will block both NR2A and NR2C subunits (Feng et al. 2004).
Therefore, it is reasonable to postulate that PCP selectively blocks NR2A
subunits in the frontal cortex inducing neurotoxicity; however blockade of both
136
NR2A and NR2C receptors in the striatum may be necessary to result in
neurotoxicity.
The differential “synaptic” localization of NR2A and NR2B following either
acute or sub-chronic PCP administration in vivo is uncertain, and was thus
verified experimentally.
All members of the MAGUK family (e.g. PSD-95,
SAP102, SAP97) contain three PDZ domains, a Src homology domain 3 (SH3)
and a C-terminal guanylate kinase-like (GK) domain.
These post-synaptic
proteins play key roles in NMDAR trafficking to the membrane, clustering at the
membrane, and stabilization of the NMDAR complex organization in the postsynaptic architecture (Mauceri et al. 2007, Wu et al. 2007, Lu et al. 2000). PSD95 is thought to bind to the PDZ domain in the C-terminal region of synaptic
NR2A or NR2B subunits (Lu et al. 2000, Wu et al. 2007). SAP102 is linked to
extrasynaptic NR2B subunits (Wu et al. 2007, Sans et al. 2003) while SAP97
plays a role in the trafficking and insertion of NR2A subunits in the synaptic
membrane (Mauceri et al. 2007). The expression level of NMDARs, their subunit
composition as well as postsynaptic linkers can be altered following
administration of NMDA antagonists including MK-801, ethanol and PCP (Dong
et al. 2004, Anastasio & Johnson 2008b, Sircar et al. 1996, Suvarna et al. 2005)
We performed co-immunoprecipitation studies to determine the localization of
cortical NMDARs in a crude synaptoneurosomal preparation from postnatal
cortex and demonstrated that following PCP treatment on PN7, 9, & 11, there is
an apparent compensatory up-regulation in the surviving neurons of NR1/NR2A,
but not NR1/NR2B in the synapse. This response to PCP-induced neurotoxicity
by neighboring neurons may represent a protective mechanism to prevent further
damage to the brain and/or restore cortical synaptic communication. An increase
in the association of both NR2A and NR2B with SAP102 following PCP
administration was also observed, suggesting a change in the expression levels
of extrasynaptic NR2A and NR2B subunits as well as providing further evidence
137
that the localization of NR2A to the synaptic space is not necessarily true in all
instances (Cull-Candy et al. 2001) .
We have hypothesized that aberrant behaviors induced by PCP are due to
its neurotoxic effects; therefore, an additional aspect of specific aim 3 was to
show a relationship between NR2-blockade mediated neurotoxicity and the
development of locomotor sensitization.
As anticipated, PCP challenge to
animals treated during development (PN7, 9, & 11) with PCP or PEAQX (an
NR2A selective antagonist) resulted in locomotor sensitization associated with
the neurotoxicity induced by these agents during a critical age of brain
maturation.
PEAQX or ifenprodil challenge did not result in locomotor
sensitization, suggesting that the expression of this behavioral phenomenon
following PCP challenge is not entirely due to blockade of the NMDAR, instead
expression of this behavior may be in part due to PCP’s indirect effects on DA
transmission (Verma & Moghaddam 1996, Johnson & Jones 1990, Lodge &
Johnson 1990).
Specific Aim 4
Finally, specific aim 4 was designed to test the validity of our treatment
paradigm as a model of schizophrenia.
Sub-chronic PCP administration to
perinatal rats resulted in deficits of PPI of acoustic startle and the development of
locomotor sensitization, which was prevented by olanzapine and risperidone pretreatment. To determine the pharmacological properties of the protective effects
of olanzapine and risperidone, we tested the ability of selective receptor
antagonists for D1 (SCH23390), D2 (sulpiride), and 5-HT2A (M100907) to prevent
the behavioral effects of PCP. Interestingly, pre-treatment on PN7, 9, & 11 with
SCH23390 and sulpiride prevented PCP-induced deficits in PPI, while M10907
was ineffective, suggesting that D1 and D2 receptors, but not 5-HT2A receptors
may play a role in PCP-induced deficits in PPI.
Further, we developed a
methodology in our laboratory based on others (Snigdha & Neill 2008, Sams138
Dodd 1996, Koros et al. 2007, Harich et al. 2007a) with the purpose to identify
PCP-induced deficits in social behavior, a key component of the negative
symptoms of schizophrenia. Here we were able to effectively model the negative
symptoms of schizophrenia with our treatment paradigm and report that subchronic PCP administration causes a deficit in social interaction and social
discrimination, lending additional credibility to our treatment paradigm as a viable
model of schizophrenia. In the future, we propose testing the effects of posttreatment with various antipsychotics in addition to DA and 5-HT receptor
antagonists on the PCP-induced deficits in PPI, social behaviors, and
development of locomotor sensitization so as to more closely mirror the clinical
setting.
Future Directions
The dose and treatment paradigm employed in this study was designed to
induce robust neurotoxicity, and while the this level of cortical neuropil loss does
not occur in schizophrenia, the ability of perinatal PCP to kill PV-containing
interneurons in cortical layers II-IV (Wang et al. 2008) as well as other neurons in
regions similar to those affected in schizophrenia (Wang et al. 2001, Wang &
Johnson 2005) is a significant finding.
This is made even more important
because of the concomitant deficits in social behavior, sensorimotor gating and
working memory caused by postnatal PCP administration (Anastasio & Johnson
2008a, Wang et al. 2001, Harich et al. 2007b). However, the detailed molecular
signaling mechanism(s) of neuronal death are still largely unknown as are the
mechanisms responsible for the ability of antipsychotics and other compounds to
provide protection against both the behavioral and neurotoxic effects of PCP
(Anastasio & Johnson 2008a, Wang et al. 2001, Wang et al. 2003).
The results from the abovementioned neurotoxicity studies are in general
agreement with earlier studies which proposed that stimulation of synaptic
NMDARs resulted in increased CREB activity, BDNF production and neuronal
139
survival, while stimulation of extrasynaptic receptors leads to a CREB shutoff
pathway that prevents BDNF activation resulting in the subsequent neuronal
death (Hardingham et al. 2002). It is important to note that some argue that the
subunit composition not the localization of the NMDAR underlies the
neurotoxicity (Liu et al. 2007). In contradiction, a recent publication by Martel et
al. (2009) reports that NR2B subunits can mediate both neuronal survival and
survival death.
Hence, deciphering this debate between subunit composition
versus localization and which determines how a neuron responds to toxic stimuli
merits further investigation. To address this controversy, we will perform the
following experiments in the organotypic slice culture system because it
maintains the cortico-striatal inputs which are present in the whole animal and
also allows us to perform many more experimental conditions (e.g. large drug
concentration range), which would be difficult to do in whole animal experiments.
In organotypic slice cultures synaptic transmission will be activated by blocking
GABAA receptors and K+ channels
with 50 µM bicuculline and 25 µM 4-
aminopyridine, respectively (Lei et al. 2008, Hardingham et al. 2002, Ivanov et al.
2006). A low concentration of NMDA (10 µM) which is thought to be selective for
synaptic versus extrasynaptic receptors (Soriano et al. 2006) in the presence of
PCP will also be added and neurotoxicity measured with the caspase-3 activity
assay.
We have previously shown that this concentration of NMDA protects
against PCP-induced neurotoxicity in a dissociated neuronal culture (Lei et al.
2008). The addition of a NR2A antagonist (NVP-AAM007 or PEAQX) or NR2B
antagonist (ifenprodil) would then determine which subunit was responsible for
the synaptic response to PCP-mediated neurotoxicity.
Blockade of synaptic
NMDARs with a low concentration of PCP and a moderate concentration of
NMDA would stimulate extrasynaptic NMDARs and produce cell death.
The
post-treatment with NR2A antagonists or NR2B antagonists would allow one to
distinguish the subunit composition responsible for the extrasynaptic response
allowing us to clarify the controversy in our system.
140
PCP addition to organotypic slice cultures results in a long-lasting
inhibition of ERK1/2 activity most likely through blockade of Ca 2+ influx and
inactivation of ERK1/2 signaling (Xia et al. 2008). Therefore, the actions of PCP
can be thought of as inhibiting synaptic activity and inducing cell death by
uncoupling of the NMDAR and ERK1/2 signaling which, in turn, leads to a
decrease in transcription and translation of the pro-survival proteins CREB and
BDNF.
As mentioned previously, NR2A-containing receptors are thought to
primarily localize to the synaptic space, while NR2B-containing receptors have
been found in both the synaptic (possibly in heterotrimeric form) and
extrasynaptic milieu, most likely as a heterodimeric NR1/NR2B receptors (CullCandy et al. 2001). Ivanov et al. (2006) reported that extrasynaptic NMDARs
inactivate the ERK1/2 pathway, while synaptic NMDARs favor activation of the
ERK1/2 signaling cascade. Importantly, it is thought that NMDAR localization,
rather than subunit composition alone determines the physiological and
pharmacological properties of the receptors. That is, synaptic NR2B has been
shown to be responsible for ERK1/2 phosphorylation and activation, while in
contrast the extrasynaptic NR2B contributes to ERK1/2 dephosphorylation and
inactivation (Ivanov et al. 2006).
This laboratory has also demonstrated that activation of synaptic NMDARs
prevents both PCP-induced neurotoxicity as well as inhibition of the PI3K/Akt/GSK-3β pathway in primary forebrain cell culture, corticostriatal slice
cultures and in neonatal rats (Lei et al. 2008, Xia et al. 2008). This pathway is an
additional and well established pro-survival pathway and promotes cell survival
by phosphorylation and inhibition of pro-apoptotic proteins such as caspase-9
and GSK-3β (Cardone et al. 1998, Cross et al. 1995). Activating the PI-3K/Akt
pathway by enhancing synaptic NMDAR strength prevented PCP-induced
apoptosis (Lei et al. 2008). Further, PCP causes neuronal death in the layers IIIV of the cortex by dephosphorylation and activation of GSK-3β (Xia et al. 2008);
unphosphorylated GSK-3β is known to activate the pro-death protein, caspase-3
141
(Rubinfeld et al. 1996). Therefore, the actions of PCP can be thought of as
inhibiting the neuroprotective effects of Akt signaling as well as promoting the
toxic effects of GSK-3β signaling.
We propose that this bidirectional control of the ERK1/2 and the PI3K/Akt
pathways by different synaptic or extrasynaptic pools of the NMDAR is an early
signaling event responsible for neuronal survival or neuronal death following PCP
administration and thus, is an important subject for future studies. To test this
hypothesis we would first determine the time course of ERK1/2 activation
following PCP administration in vivo. Next, we would administer NR2A or NR2B
preferring antagonists to perinatal rat pups and measure the effects on ERK1/2
as well as the PI3K/Akt/GSK3-β pathways. We would also perform the reverse
experiment in which we administer selective inhibitors for ERK1/2 or the
PI3K/Akt/GSK3-β pathway and measure their effects on the expression of
synaptic/extrasynaptic NR2A or NR2B subunits as well as neurotoxicity in the
frontal cortex of developing rats.
Recently, we have demonstrated that activation of D1 receptors in the
frontal cortex protects against PCP-induced neurotoxicity by enhancing synaptic
strength
through
synergistic
activation
of
PI3K/Akt/GSK3-β pathways (Lei et al. 2009).
PKA/Src
kinase
and
the
Interestingly, stimulation of D1
receptors diminishes PCP-induced deficits in PPI of acoustic startle (Lei et al.
2009). These studies demonstrate a novel mechanism for protection against
PCP-induced neurotoxicity through enhancement of synaptic NMDAR strength
by activation of D1 receptors as well as activation of the MEK/ERK and PI3K/Akt/GSK-3β pathways.
Enhancing synaptic strength following PCP administration is thought to be
the next step in treatment of schizophrenia. Co-administration of antipsychotics
with compounds which enhance NMDAR function, such as glycine, d-cycloserine
or glycine transporter 1 (GlyT1) inhibitors, have been somewhat successful in
treating schizophrenics (Coyle & Tsai 2004, Lane et al. 2006). Thus, we believe
142
that answers to the aforementioned processes may lead to the discovery of new
therapeutic targets for schizophrenia either through elucidating the molecular
signaling cascades and the NMDAR subunit composition/localization involved in
PCP-induced neurotoxicity or determination of the best mechanism to enhance
synaptic NMDAR strength.
143
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Vita
Noelle Anastasio was born on December 16, 1978 to Philip and Barbra
Anastasio in Metairie, Louisiana. After college, she worked as a research
assistant for Dr. Kenneth M. Johnson in the Pharmacology and Toxicology
department at University of Texas Medical Branch in Galveston, Texas. In
August, 2003, she enrolled in the M.S. program in the department of
Pharmacology and Toxicology at UTMB and completed her degree in August,
2005. In 2006, she was awarded a Ruth L. Kirschstein NRSA F31 Fellowship
from the National Institute on Drug Abuse to pursue her doctoral studies. During
the 2007-2008 academic year, she was chosen as a Bromberg Scholar which
entailed serving as a scientific research mentor to a local high school student,
Zoe O’Connor. In 2008, she was awarded the James E. Beall II Memorial Award
in Anatomy and Neurosciences.
Education
B.A. 2001, University of Texas at Austin, Austin TX
M.S. 2005, University of Texas Medical Branch, Galveston TX
Publications in Peer-Reviewed Journals
Anastasio NC, Xia Y, O’Connor ZR, and Johnson KM (2009) NR2A-containing
Synaptic NMDA Receptors Mediate Phencyclidine-Induced Neurotoxicity and the
Subsequent Development of Schizophrenia-Like Behaviors in the Rat
Neuroscience in revision
Lei G, Anastasio NC, Fu Y, Neugebauer V, and Johnson KM (2008) Activation
of Dopamine D1 Receptors Prevents Phencyclidine-Induced Neurotoxicity by
Enhancing Synaptic NMDA Receptor Strength. Journal of Neurochemistry
accepted 2009 Feb 26
Xia Y, Wang CZ, Liu Jie, Anastasio NC, and Johnson KM (2008) Lithium
Protection of Phencyclidine-Induced Neurotoxicity in Developing Brain: The Role
of PI-3 Kinase and MEK/ERK Signaling Pathways. Journal of Pharmacology and
Experimental Therapeutics 326(3):838-48
Anastasio NC and Johnson KM (2008b) Atypical Antischizophrenic Drugs
Prevent Changes in Cortical N-methyl-D-Aspartate Receptors and Behavior
Following Sub-Chronic Phencyclidine Administration in Developing Rats.
Pharmacology, Biochemistry and Behavior 90(4):569-77
Anastasio NC and Johnson KM (2008a) Differential Regulation of the N-methylD-Aspartate Receptor by Acute and Sub-chronic Phencyclidine Administration in
the Developing Rat. Journal of Neurochemistry 104(5):1210-8
Wang C, Anastasio NC, Popov V, LeDay A, and Johnson KM (2004) Blockade
of N-Methyl-D-Aspartate by Phencyclidine Causes the Loss of Corticostriatal
Neurons. Neuroscience 125:473-483
Wang C, McInnis J, West J, Bao J, Anastasio NC, Guidry J, Ye Y, Salvemini D,
and Johnson KM (2003) Blockade of Phencyclidine-Induced Cortical Apoptosis
and Deficits in Prepulse Inhibition by M40403, a Superoxide Dismutase Mimetic.
Journal of Pharmacology and Experimental Therapeutics 304(1):266-271
McInnis J, Wang C, Anastasio NC, Hultman M, Ye Y, Salvemini D, and Johnson
KM (2002) The Role of Superoxide and Nuclear Factor-B Signaling in NMethyl-D-Aspartate Induced Necrosis and Apoptosis. Journal of Pharmacology
and Experimental Therapeutics 301(2):478-487
Abstracts
Anastasio NC, Xia Y, Wang CZ and Johnson KM (2008) The role of NMDAR 2A
and 2B in PCP-induced neurotoxicity and development of locomotor
sensitization. College on Problems of Drug Dependence. San Juan, Puerto Rico
Anastasio NC, Xia Y, Wang CZ and Johnson KM (2008) Synaptic versus
extrasynaptic NMDA receptors: Role in PCP-induced neurotoxicity and
development of locomotor sensitization. Experimental Biology/ASPET. San
Diego, CA
Xia Y, Wang CZ, Anastasio NC and Johnson KM (2008) BDNF protects against
phencyclidine neurotoxicity in neonatal brain via activation of the PI-3K/Akt/GSK3β and MEK/ERK pathways. Experimental Biology/ASPET. San Diego, CA
Anastasio NC, Xia Y, and Johnson KM (2007) PCP-treatment dependent
differential regulation of the NMDAR is dependent on PSD-95 expression and
mediated by calpain.
Canada
College on Problems of Drug Dependence. Quebec City,
Xia Y, Wang CZ, Anastasio NC and Johnson KM (2007) Lithium protection of
phencyclidine neurotoxicity in neonatal brain is mediated by brain-derived
neurotrophic factor activation of MEK/ERK signaling.
Experimental
Biology/ASPET. Washington, D.C.
Xia Y, Wang CZ, Anastasio NC and Johnson KM (2006) Lithium chloride
protection of phencyclidine-induced neurotoxicity in corticostriatal slices. The
NIMH, Division of Intramural Research Programs, the first Pre-doctoral Research
Festival. Washington, D.C.
Anastasio NC and Johnson KM (2006) PCP-induced regulation of the NMDAR
and development of locomotor sensitization. College on Problems of Drug
Dependence. Scottsdale, AZ
Anastasio NC and Johnson KM (2006) Effects of typical and atypical
antipsychotics in a PCP-induced behavioral model of schizophrenia.
Experimental Biology/ASPET. San Francisco, CA
Anastasio NC and Johnson KM (2005) Pharmacological analysis of
phencyclidine-induced regulation of the NMDA receptor. American Society for
Neurochemistry. Madison, WI
Anastasio NC and Johnson KM (2005) Pharmacological analysis of PCPinduced regulation of the NMDA receptor. National Student Research Forum.
Galveston, TX
Anastasio NC and Johnson KM (2004) Effect of olanzapine on PCP-induced
changes in the N-Methyl-D-Aspartate receptor (NMDAR).
Society for
Neuroscience. San Diego, CA
Anastasio NC and Johnson KM (2004) Role of NF-B in NMDA-induced cell
death in cultured neurons. International Society of Neurochemistry: Advanced
School of Neurochemistry. Avignon, France
Wang C, Anastasio NC, Geng T, LeDay A, and Johnson KM (2003)
Phencyclidine-induced cortical apoptosis is associated with reduced PSA-NCAM
and synaptophysin in the striatum. Society for Neuroscience. New Orleans, LA
Johnson KM, McInnis J, Wang CZ, Anastasio NC, Bao J, Ye Y, and Wang C
(2003) Regional specificity and time course of phencyclidine-induced apoptosis
in rat brain. Experimental Biology/ASPET. San Diego, CA
Wang C, Bao J, Guidry J, West J, Anastasio NC, Ye Y, Robeson N, McInnis J,
and Johnson KM (2002) Association of phencyclidine-induced cortical apoptosis
with reduced PSA-NCAM expression in the nucleus accumbens. Society for
Neuroscience. Orlando, FL
Guidry J, Bao J, Wang C, Anastasio NC, Ye Y, Robeson N, and Johnson KM
(2002) NMDA-induced neuronal apoptosis is mediated by an early increase in
nuclear NF-B p50 and p65 proteins. Society for Neuroscience. Orlando, FL
Permanent address:
1750 League City Pkwy Apt 223
League City, Texas 77573
This dissertation was typed by Noelle C. Anastasio.