APPROACHES TO MODELING SCHIZOPHRENIA FN THE RAT by ...

APPROACHES TO MODELING SCHIZOPHRENIA FN THE RAT
by
JOHN GEORGE HOWLAND
B A. (Hons.), University of Saskatchewan, 1999
M.A., The University of British Columbia, 2001
A THESIS SUBMITTED IN PARTIAL FULFILMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
in
THE FACULTY OF GRADUATE STUDIES
(Psychology)
THE UNIVERSITY OF BRITISH COLUMBIA
October 2005
© John George Howland, 2005

ABSTRACT
Schizophrenia is a complicated and variable disorder that is notoriously difficult
to study. Converging lines of evidence support the hypothesis that schizophrenia is
characterized by a diverse array of distributed changes in limbic and cortical areas of the
brain involving abnormalities in dopamine and glutamate transmission. Furthermore,
genetic and behavioral studies indicate that abnormalities in normal development
contribute to the etiology of the disorder. The present dissertation used two general
strategies in an attempt to model some of the basic characteristics of the disorder. In
Chapter Two, experiments were conducted that demonstrate short periods of higher
frequency stimulation applied to the ventral, but not dorsal, hippocampus in adult rats
reversibly reduce prepulse inhibition, a pre-attentive processing mechanism that is
disrupted in schizophrenic patients. In Chapters Three and Four, the behavioral effects of
reversible pharmacological manipulation of glutamate receptors early in development on
prepulse inhibition and locomotor activity were assessed both before and after puberty.
Additional experiments tested the putative role of dopaminergic abnormalities following
these manipulations. Results from Chapter Three demonstrate that administration of a
convulsive dose of the glutamate receptor agonist kainic acid to neonatal rats on postnatal
day seven results in the delayed emergence of PPI deficits in early adulthood. Levels of
locomotor activity were not reliably altered in a novel environment or following
amphetamine administration. The experiments conducted in Chapter Four were designed
to assess alterations in prepulse inhibition and locomotor activity following
administration of the NR2B-subunit selective NMDA antagonist Ro25-6981.
Unexpectedly, Ro25-6981 administration resulted in behavioral convulsions when

administered during the first postnatal week; however, no consistent behavioral
abnormalities were revealed in rats treated with the drug. Although the present results
are somewhat mixed, the experiments were successful at providing novel insights into the
symptoms and etiology of schizophrenia. In general, they support the assertion that short
periods of altered activity in the limbic system, and hippocampus in particular, at
different points during development may underlie the expression of some of the most
basic symptoms of schizophrenia. These data also suggest that the nature and anatomical
location of these alterations critically determines their long-term functional effects.
iii

TABLE OF CONTENTS
ABSTRACT ii
TABLE OF CONTENTS iv
LIST OF TABLES viii
LIST OF FIGURES ix
ACKNOWLEDGMENTS xi
CO-AUTHORSHD? STATEMENT xii
CHAPTER ONE: GENERAL INTRODUCTION 1
Schizophrenia 2
Brief Overview of the Disorder 2
Etiological Theories 7
Animal Models 8
Genetic Models 11
Adult Pharmacological Models 12
Adult Lesion Models 14
Developmental Animal Models 16
Maternal Separation and Social Isolation Models 16
Neonatal Lesion Models 18
Neonatal Pharmacological Models 22
References 24
CHAPTER TWO: ELECTRICAL STIMULATION OF THE HIPPOCAMPUS
DISRUPTS PREPULSE INHIBITION IN RATS: FREQUENCY AND SITE
DEPENDENT
EFFECTS 37
Introduction 37
Methods 41
Subjects 41
Surgery 41
Prepulse Inhibition Testing 42
Neurochemical Experiments 44
Histology 46
Data Analysis 46
Results 47
Immediate Behavioral Effects of Stimulation 47
Differential Effects of 20 Hz Stimulation of the vHip or dHip on PPI and Startle
Amplitude : 48
iv

Effects of 2 Hz Stimulation of the vHip on PPI and Startle Amplitude 50
Effects of Stimulation of the vHip and dHip on NAc DA Efflux 52
Histology 52
Discussion 55
Potent Disruption of Sensorimotor Gating Induced by 20 Hz Stimulation of the vHip,
but not dHip 55
Frequency Dependence of the vHip Stimulation-induced Disruption of PPI 57
Increases in NAc DA Efflux following Hip Stimulation are Site and Hemisphere
Specific 59
Potential Mechanisms Underlying vHip Stimulation-induced Disruption in PPI 61
Conclusion 62
References 63
CHAPTER THREE: DELAYED ONSET OF PREPULSE INHIBITION DEFICITS
FOLLOWING KAINIC ACID TREATMENT ON POSTNATAL DAY SEVEN IN
RATS 70
Introduction 70
Methods 72
Subjects 72
Kainic Acid Administration 73
Prepulse Inhibition 74
Locomotor Activity 75
Water Maze Testing 76
Histology ". 77
Data Analysis 77
Results ., 78
Immediate Behavioral Effects ofPND7 KA Administration 78
Effects of PND7 KA Administration on Body Weight 79
Prepulse Inhibition is Reduced in Post-Pubescent, but not Pre-pubescent Rats,
Following PND7 KA Administration 81
Locomotor Activity in Response to Novelty and Amphetamine in Rats Following PND7
KA Administration 84
Spatial Learning and Memory in the Morris Water Maze is not Altered in Rats
Following PND7 KA Administration 87
Histology 89
Discussion 91
Effects of Neonatal KA Administration on PPI 91
Effects of Neonatal KA on Locomotor Activity 93
Potential Mechanisms Underlying the Observed PPI Changes 94
Conclusion 97
References 99
CHAPTER FOUR: BEHAVIORAL CONVULSIONS INDUCED BY EARLY
POSTNATAL ADMINISTRATION OF THE NR2B ANTAGONIST R025-6981
FAIL TO AFFECT SENSORIMOTOR GATING OR LOCOMOTOR

BEHAVIOR IN PRE- AND POST-PUBESCENT
RATS..... : 105
Introduction 105
Methods 110
Experiment 1 - Incidence and Characteristics of Behavioral Convulsions in Postnatal
Rats Following Antagonism of NMDA Receptors Containing the NR2B Subunit.... 110
Subjects 110
Experimental Procedures 110
Experiment 2 - Long-term Behavioral Effects of Convulsions Resulting from Antagonism
of NMDA Receptors Containing the NR2B Subunit 1111
Subjects Ill
Ro25-6981 Administration Ill
Prepulse Inhibition 112
Spontaneous Locomotor Activity 113
Dopaminergic Challenges of PPI and Locomotor Activity 113
Data Analysis 114
Results 115
Experiment 1 - Behavioral Effects ofRo25-6981 Administration in the Early Postnatal
Period 115
Experiment 2 - Effects of Neonatal Ro25-6981 Administration on Body Weight 116
Neonatal Ro25-6981 Administration Does not Alter PPI Responding Either before
Puberty or in Early Adulthood 118
Apomorphine Challenge in Adulthood has Similar Effects on PPI in Neonatally
Saline- or Ro25-6981 -treated Rats 121
Neonatal Treatment with Ro25-6981 and Amphetamine-Induced Locomotor Activity in
Adult Rats 121
Discussion 125
Antagonism of NR2B-containing NMDA Receptors Induces Behavioral Convulsions
126
The Long-Term Behavioral Effects of Ro25-6981 on PPI and Locomotor Activity.. 129
Conclusion 131
References 132
CHAPTER FIVE: GENERAL DISCUSSION 138
The Role of the Hippocampus in the Regulation of Prepulse Inhibition and Locomotor
Activity 139
The Utility of Adult Animal Models of Schizophrenia 142
Developmental Models of Schizophrenia - Effects of Early Postnatal Glutamate
Manipulations 143
Developmental Models of Schizophrenia - The Time Course of Symptom Emergence
148
Criteria for Establishing Validity in Behavioral Models of Schizophrenia 149
Conclusion 152
References 153
vi

LIST OF TABLES
Table 4-1. The incidence of behavioral convulsions in rats administered
NR2B-selective NMDA antagonists at various postnatal ages 117
vii

LIST OF FIGURES
Figure 2-1. Effects of 20 Hz stimulation of the hippocampus on PPI and
acoustic startle amplitude 49
Figure 2-2. Effects of 2 Hz stimulation of the vHip on PPI and acoustic
startle amplitude. 51
Figure 2-3. Unilateral stimulation (20 Hz, 10 s) of either the vHip or dHip,
and its effect on NAc DA efflux in the ipsilateral or contralateral hemisphere 53
Figure 2-4. Schematic diagram of the placements of stimulating electrodes and
microdialysis probes in all experiments 54
Figure 3-1. Prepulse inhibition scores from rats in Group 1 treated on
postnatal day 7 with saline or kainic acid 80
Figure 3-2. Prepulse inhibition scores from rats in Group 2 treated on
postnatal day 7 with saline or kainic acid 83
Figure 3-3. Effects of pretreatment with vehicle or apomorphine on
average percent PPI scores in adulthood 85
Figure 3-4. Locomotor activity levels in response to novelty or amphetamine
of all rats tested on either postnatal day 36 or 57 86
Figure 3-5. Average latencies to locate the hidden platform in the water
maze of rats treated with saline or kainic acid on postnatal day 7 88
Figure 3-6. A representative cresyl violet stained section of the dorsal
hippocampus of a KA-treated and saline-treated rat 90
Figure 4-1. Cartoon illustrating an NMDA receptor within the cell membrane 107
Figure 4-2. Prepulse inhibition scores of rats in Group 1 treated on
postnatal day 6 and 7 with saline or Ro25-6981 119
Figure 4-3. Prepulse inhibition scores from rats in Group 2 treated on
postnatal day 6 and 7 with saline or Ro25-6981 120
Figure 4-4. Effects of pretreatment with vehicle or apomorphine on
percent PPI scores in adulthood 122
Figure 4-5. Locomotor activity levels in response to novelty or
amphetamine of rats tested on either postnatal day 36 or 57 in Group 1 123
viii

Figure 4-6. Locomotor activity levels in response to novelty or amphetamine of rats
tested on either postnatal day 36 or 57 in Group 2. 124

ACKNOWLEDGMENTS
The work contained within this dissertation would not have been completed without the
help of many people. Those who directly assisted me with these projects are co-authors
on my publications. Many of those who indirectiy assisted me in the lab are listed
alphabetically below. I would like to especially acknowledge the wisdom and expert
advise of Drs. Tony Phillips, Stanley B. Floresco, and Darren 'Twinkle-Toes' Hannesson
throughout my graduate career and Dr. Karen Brebner for providing many helpful
comments on the General Introduction and Discussion Sections of my dissertation.
Finally, I would like to acknowledge NSERC, CIHR, and the Michael Smith Foundation
for Health Research for funding.
Thank you:
Soyon Ahn, Steven Barnes, Alasdair Barr, Karen Brebner, Deanna Chavez, Christina
Cheng, Michael Corcoran, Carine Dias, Brennan Eadie, Rachel Genn, Rosie Gilham,
Natalia Gorelova, Lucy Greggorios-Pippas, Rebecca Harrison, Fred Lepiane, Sarah C.
Lidstone, Brandi Ormerod, Julie Pongrac, Kitty So, Aline Stephan, Christina Thorpe,
Giada Vacca, and liana Winrob.
To my friends and family who may not have helped directly with my academic life, but
in many other ways, don't worry, your thanks is coming (and you're likely not reading
this anyway). USB UBC.

CO-AUTHORSHIP STATEMENT
I am the first author on all manuscripts presented in this thesis. I was directly
involved in all stages of the research including, but not limited to, conception of the
ideas, designing the experiments, conducting the studies, analyzing the data, and writing
the manuscripts. Research assistants and other students helped in gathering some of the
data presented in this dissertation and they are included as authors on the manuscripts
with which they were involved. Dr. Phillips was also involved in all aspects of the
research included in this dissertation. He also acted as my PhD supervisor and is
included as an author on all manuscripts.

CHAPTER ONE: GENERAL INTRODUCTION
The present dissertation summarizes several novel approaches to the development
of rodent models of schizophrenia that are reliable and valid. As will become clear in the
introduction, schizophrenia is an incredibly complicated and variable disorder (or group
of disorders). Therefore, it is difficult to put forth a unitary set of criteria by which to
verify the success of putative models of schizophrenia. The following pages detail a
number of strategies aimed at reproducing some characteristics of the disorder. As the
experiments of Chapter Two demonstrate, acute changes in hippocampal activity in
adulthood reversibly alter behavior in a manner consistent with schizophrenia.
Additionally, Chapters Three and Four show that under some circumstances, transient
disruption of normal activity patterns in the developing brain is sufficient to subtiy
change adult behavior in a manner consistent with schizophrenia. Finally, the
implications of these studies and a number of directions for future research are discussed
in Chapter Five.
This dissertation is presented in a manuscript-based format, rather than the
traditional thesis format. Chapters Two through Four are stand-alone manuscripts,
containing their own Introduction and Reference sections, while Chapter One and Five
are the General Introduction and Discussion sections of the entire dissertation. While this
format has numerous practical advantages, it has two clear limitations. First, there is
inevitably a certain amount of repetition between chapters, and second, individual
chapters do not necessarily flow into the next. In an effort to address the second problem,
I will include a brief statement of the objectives of each chapter in the General
Introduction, and summarize the findings from all chapters in the General Discussion.
1

Schizophrenia
Brief Overview of the Disorder
Schizophrenia is a debilitating mental illness with a lifetime prevalence of 1.4-4.6
per 1000 people worldwide (Jablensky, 2000). Symptoms usually appear in late
adolescence or early adulthood, although cases with earlier and later onset certainly exist
(Wong and Van Tol, 2003). For many patients, schizophrenia has a chronic course with
unpredictable relapses that require repeated hospitalizations. Although antipsychotic
drugs effectively reduce symptom severity in many patients, their side effects are severe,
and the drugs often fail to improve many of the negative and cognitive symptoms of the
disorder (see below). It is certainly worth noting that the complexities and
inconsistencies of the disorder are far from understood. As a result, the behavioral
symptoms and neuropathological characteristics of the disorder described below have
been simplified. Within the following chapters, pertinent details related to relevant
aspects of schizophrenia are described where necessary.
As the pharmacology underlying antipsychotics drug action has been used to
support hypotheses related to the neural bases of the symptoms of schizophrenia, a brief
review of their main classes is required. Typical antipsychotic drugs, which antagonize
dopamine receptors, were discovered in the 1950's and revolutionized treatment of
schizophrenia (Wong and Van Tol, 2003). During the 1980's and 1990's, psychiatrists
began prescribing numerous atypical antipsychotic drugs to patients who did not respond
to typical antipsychotics (either due to symptom profiles, side effects, or unknown
reasons). Atypical antipsychotics have unique pharmacological effects, including
2

affinities for serotonin, adrenergic, dopamine, histamine, and muscarinic receptors, which
may underlie their effectiveness in some patients (Wong and Van Tol, 2003).
The behavioral symptoms of schizophrenia are varied and involve numerous
advanced functions of the human brain. Generally, symptoms can be divided into three
main categories: the classic psychotic or 'positive' symptoms, deficit or 'negative'
symptoms, and cognitive impairment (Wong and Van Tol, 2003). Positive symptoms are
those typically associated with the illness - disturbances such as hallucinations,
delusions, and thought disorder. Importantly, the appearance of positive symptoms in
late adolescence or early adulthood generally leads to the diagnosis of schizophrenia. A
number of researchers have argued that altered activity of the dopamine system is the
likely cause of positive symptoms, a hypothesis largely based on the effectiveness of
antipsychotic drugs, which are dopamine D2 receptor antagonists, in reducing positive
symptoms and dopamine agonists at inducing or aggravating positive symptoms (Snyder,
1973;Kapur and Mamo, 2003).
Whereas positive symptoms may be thought of as the presence of abnormal
behaviors, negative symptoms may be regarded as a reduction or loss in a number of
normally occurring behaviors. Symptoms such as social withdrawal, apathy, reduced
expression of affect, and the ability to experience pleasure are common examples of
negative symptoms. The causes and treatment of negative symptoms are more poorly
understood than the positive symptoms, although some atypical antipsychotic drugs, with
actions at receptor subtypes other than the D2 receptor, may be more effective than
typical antipsychotics at improving these symptoms (Meltzer et al., 1994;Wong and Van
Tol, 2003).
3

Finally, impairments in cognitive domains such as pre-attentive processing,
executive function, attention, learning, memory, and general intellectual function are an
important component of schizophrenia (Elvevag and Goldberg, 2000;Sharma and
Antonova, 2003;Lewis, 2004). Intense interest has focused recently on these symptoms
because of studies showing that the cognitive symptoms are the most enduring and
consistently debilitating of the disorder (Elvevag and Goldberg, 2000). Additionally,
impairments in cognition may precede the development of the positive symptoms of the
disorder, thereby providing a potential tool with which to identify those individuals at
risk for expressing full-blown psychosis in early adulthood (Walker, 1994;Walker et al.,
1994;Jones et al., 1994;Lewis, 2004).
In addition to the behavioral changes observed in those suffering from the
disorder, many alterations in the normal structure and function of the brain have been
reported. For example, an overwhelming array of neuroanatomical changes is reported to
exist in schizophrenia, many of which occur in the frontal and temporal lobes (McCarley
et al, 1999;Wright et al., 2000). Ventricular enlargement (Stevens, 1997;Harrison, 1999)
and reductions in temporal (Lawrie and Abukmeil, 1998;Nelson et al., 1998) or frontal
lobe volumes (Wright et al., 1999) are observed in the brains of schizophrenic patients.
Reduced volume of the hippocampus (Suddath et al., 1990;Noga et al., 1996;Bogerts,
1997;Heckers, 2001), reduced cell numbers in the hippocampus (Gothelf et al.,
2000;Heckers, 2001), nucleus accumbens (NAc; Pakkenberg, 1990), and thalamus
(Jones, 1997;Clinton and Meador-Woodruff, 2004), and altered neuronal size and density
in certain frontal cortical areas (Harrison, 1999) have been observed in patients with
schizophrenia. Abnormalities in patterns of hippocampal (Heckers et al., 1998;Benes,

2000;Heckers, 2001;Holt et al., 2005), striatal (Meyer-Lindenberg et al., 2002) and
prefrontal activation (McClure and Weinberger, 2001;Meyer-Lindenberg et al., 2002)
during the performance of a variety of cognitive tasks are also characteristic of
schizophrenic patients (Wong and Van Tol, 2003). Taken together, these
neuroanatomical changes may be considered an enduring trait for schizophrenia in
adulthood. Interestingly, relatives of patients with schizophrenia may also have reduced
cortical volumes (Cannon et al., 1993), altered hippocampi (Seidman et al., 2002) and
enlarged ventricles (Honer et al., 1994;Lawrie et al., 1999), thereby supporting the
assertion that an underlying genetic abnormality may predispose individuals to develop
the disorder, although this underlying 'trait' for schizophrenia may have to interact with
other environmental factors to result in the expression of a schizophrenic 'state' in early
adulthood. Such conceptions of the etiology of schizophrenia are termed 'two-hit'
hypotheses by some authors (McCarley et al., 1999;Bayer et al., 1999;Wong and Van
Tol, 2003;Ellenbroek, 2003).
The neurochemical changes present in schizophrenia are also complex and
incompletely understood. The affinity of typical antipsychotics for the D2 receptor and
psychotomimetic agents such as amphetamine and phencyclidine for the dopamine
transporter and the N-methyl-D-aspartate (NMDA) receptor, respectively, are suggestive
of the importance of dopamine and glutamate neurotransmission in schizophrenia
(Angrist et al., 1974;Jentsch and Roth, 1999). More direct evidence of abnormal
dopamine-glutamate interactions is gained from studies suggesting that alterations in
glutamate and glutamate receptor levels may be present in schizophrenia (for review, see
Goff and Coyle, 2001) and that schizophrenic patients show elevated synaptic levels of
5

dopamine (Abi-Dargham et al., 2000) and potentiated increases in striatal dopamine
release in response to amphetamine administration during episodes of acute psychosis
(Breier et al, 1997;Abi-Dargham et al., 1998;Laruelle et al., 1999;Laruelle et al., 2003)
and periods of relative remission (McGowan et al., 2004). Current theories of cortico-
striatal-limbic function and schizophrenia have begun to delineate mechanisms by which
these neurochemical changes and developmental abnormalities may manifest themselves
as the symptoms of schizophrenia. One such example is the tonic-phasic model of
ventral striatal dopamine release proposed by Grace (Grace, 1991). The tonic-phasic
model posits that in schizophrenia, reduced activity from cortical glutamatergic afferents
to the ventral striatum reduces basal (or tonic) levels of dopamine in the ventral striatum.
Tonic levels of dopamine are thought to regulate the responsivity of the ventral striatum
to short-lasting (or phasic) changes in dopamine efflux in response to environmental
stimuli. As a result, patients with schizophrenia are predicted to exhibit increased
dopaminergic responsivity to salient environmental stimuli, which may be a partial
explanation of the positive symptoms (Grace, 1991). This model, along with others like
it (Carlsson et al., 1999), illustrates the importance of dopamine-glutamate interactions in
the neurobiology of schizophrenia.
Additionally, the therapeutic efficacy of atypical antipsychotic drugs (Emsley and
Oosthuizen, 2003; A wad and Voruganti, 2004) and a variety of additional
neuropathological findings (Costa et al., 2004;Lewis et al., 2005) underlie the importance
of other neurotransmitter systems in schizophrenia. In particular, alterations in the
GABA and serotonin systems may exist in schizophrenia (Costa et al., 2004;Roth et al.,
2004), although they will not be the focus of the present review.
6

Etiological Theories
Currently, the cause(s) of schizophrenia remain poorly defined. Twin and gene
linkage studies provide convincing evidence that a genetic predisposition to develop
schizophrenia exists, although environmental factors must also contribute as concordance
rates between monozygotic twins are approximately 50 percent, whereas for dizygotic
twins and siblings, the risk is approximately 10 percent (Kennedy et al., 2003;Owen et
al., 2004). One particularly prominent etiological theory of the last 15 to 20 years is the
neurodevelopmental theory (Weinberger, 1987;Lewis and Levitt, 2002;Church et al.,
2002;Eastwood, 2004;Rapoport et al., 2005). In its original form, the theory suggests that
genetic abnormalities may alter early brain development, or make the brain more
susceptible to disruption. This 'compromised brain' then interacts with environmental
factors or later occurring events of brain development and ultimately results in the
expression of the symptoms typically observed in schizophrenic patients (Benes,
2000;Benes et al., 2000;McClure and Weinberger, 2001;Lewis and Levitt, 2002).
The neurodevelopmental theory is supported by evidence suggesting that people
who develop schizophrenia have an increased incidence of adverse gestational and
perinatal events, cognitive and behavioral abnormalities before diagnosis, congenital
physical abnormalities, and alterations in brain morphology (for reviews see McClure and
Weinberger, 2001;Lewis and Levitt, 2002). Further support is gained from evidence
suggesting that the neuroanatomical changes observed in schizophrenia are present from
the first episode diagnosis and their severity usually does not increase over time
(Weinberger, 1987), although recent data challenges this notion (Weinberger,
1996;Rapoport et al., 1999;Church et al., 2002;Rapoport et al., 2005). Additionally, signs
7

of active neurodegeneration, such as gliosis, are generally absent from the brains of
schizophrenic patients (Roberts et al., 1986;McClure and Weinberger, 2001), although
such observations do not exclude the possibility that active neurodegenerative processes
such as increased rates of apoptosis occur in patients with schizophrenia (Church et al.,
2002).
Animal Models
Animal models have been used extensively in medicine to simplify complex and
poorly understood pathologies. In psychiatry, and specifically in regards to
schizophrenia, the development of reliable and valid animal models has proven difficult.
Obviously, many symptoms of psychiatric disorders are inherently human, and thus can
never be fully modeled in animals. As a result, attempts at modeling disorders such as
schizophrenia in animals have been met with skepticism. Recently, a number of authors
(Geyer and Markou, 1995;Lipska and Weinberger, 2000;Robbins, 2004) have advanced
the notion that valuable and unique insights can be gained from modeling psychiatric
disorders in animals, including rodents. Paramount to this undertaking is a realistic
notion of the goal of the animal model, and the criteria which will be used to verify
whether or not this goal has been achieved.
With respect to schizophrenia, animal models have been developed for at least
three main goals: furthering our understanding of its etiology, advancing our
understanding of the symptoms (both behavioral and biological), and finally, aiding in the
development and understanding of pharmaceutical therapies (Lipska and Weinberger,
2000;Robbins, 2004). The evaluation criteria (or dependent measures) used to validate
models of schizophrenia are quite varied, mostly due to the inherent difficulties of
8

measuring the disorder in animals. Generally, models are designed to replicate the
abnormal neurochemistry/neuropathology or behavioral changes described for
schizophrenia. Within the present thesis, two behavioral tests - prepulse inhibition and
locomotor behavior - are used extensively to validate the experimental manipulations
used to model schizophrenia.
Prepulse inhibition (PPI) of the acoustic startle response is an operational measure
of sensorimotor gating, and is a normal pre-attentive behavioral response in which a weak
sensory event (or prepulse) inhibits, or gates, the motor response to a starding stimulus
(or pulse) (Fig. 1-1; Swerdlow et al., 2000a). PPI is disrupted in patients with
schizophrenia or schizotypal personality disorder (Braff et al., 2001;Hamm et al.,
2001 ;Ludewig et al., 2003) and in the first degree relatives of patients with schizophrenia
(Cadenhead et al., 2000). These data suggest that PPI deficits are not a result of acute
psychosis (Braff et al., 2001; but see also Meincke et al., 2004). Typical antipsychotic
drugs do not ameliorate PPI deficits in schizophrenic patients (Mackeprang et al.,
2002;Duncan et al., 2003a;Duncan et al., 2003b), although some beneficial effects of
atypical antipsychotic drugs have been reported (Braff et al., 2001;Leumann et al.,
2002;Kumari et al., 2002). More sophisticated longitudinal designs are necessary to
further address the efficacy of antipsychotic drugs in reversing the PPI deficits observed
in patients with schizophrenia (Kumari and Sharma, 2002). Importantly, PPI deficits are
correlated with cognitive and behavioral symptoms such as distractibility, social
perception, and thought disorder (Perry and Braff, 1994;Karper et al., 1996;Braff et al.,
1999;Perry et al., 1999;Swerdlow et al., 2000a;Meincke et al., 2004;Wynn et al., 2005).
Deficits in information processing capabilities, such as those measured by PPI, are core
9

cognitive symptoms of schizophrenia and may relate to deficits in stimulus filtering
commonly proposed to exist in the disorder (Swerdlow et al., 2000a).
As PPI is easily and reliably measured in animals, it has been extensively used in
cross-species work as valid behavioral test with relevance to schizophrenia (Swerdlow et
al., 2000a). The use of PPI as an "index" of schizophrenia in animals is validated by
pharmacological experiments demonstrating that DA agonists such as apomorphine and
NMDA antagonists such as phencyclidine alter PPI responding in animals and healthy
humans, and antipsychotic drugs ameliorate these deficits in animals (Braff et al.,
2001;Geyer et al., 2001). Extensive experimentation has been conducted to understand
the neural substrates underlying the dopaminergic and glutamatergic regulation of PPI
(Swerdlow et al., 2001). These experiments reveal a distributed circuitry in cortical,
limbic, striatal, and pallidal areas important in the regulation of PPI. The pertinent details
of this literature are reviewed in Chapter Two, and therefore will not be elaborated upon
here.
Locomotor responses to a variety of arousing stimuli such as novel environments
or stimulant drugs are commonly measured in rodents as a measure of mesolimbic
dopamine activity (Kelly et al., 1975;Castall et al., 1977;Porrino et al., 1984). More
vigorous locomotor behavior in response to these treatments correlates positively with
elevated ventral striatal dopamine levels (Kelly et al., 1975;Castall et al., 1977;Hooks et
al., 1992). As previously reviewed, ventral striatal dopamine levels are higher in patients
with schizophrenia than controls (Laruelle et al., 2003). As a result, locomotor activity
levels are often used as an indirect measure of ventral striatal dopamine levels in animal
models of schizophrenia (Lipska and Weinberger, 2000;Bast and Feldon, 2003;Boksa,
10

2004). Additionally, some authors have argued that increased locomotor activity may be
homologous to some cognitive abnormalities in schizophrenia patients during acute
episodes (Bast and Feldon, 2003), although these accounts are debatable (Marcotte et al.,
2001).
In the present thesis, PPI and locomotor activity levels were used as the main
endpoints to assess whether the novel experimental manipulations employed would
produce effects resembling aspects of schizophrenia. These tests were selected for the
following reasons: (1) Both tests have been used frequendy to assess previous preclinical
models of schizophrenia. Thus, comparisons could easily be rnade between existing
models and those developed within the present work. (2) These tests can be reliably
conducted before and after puberty, enabling the developmental course of the behavioral
effects of the neonatal manipulations performed in Chapters Three and Four to be
assessed. (3) Pharmacological challenges are easily conducted in conjunction with these
tests thereby allowing the neurochemical mechanisms of observed behavioral changes to
be evaluated.
Given the multi-dimensional etiology of schizophrenia, researchers have
advanced numerous strategies to model the disorder in animals, each with its own
strengths and limitations. A brief review of the models pertinent to the present discussion
of PPI and locomotor activity will now be presented.
Genetic Models
As previously discussed, the etiology of schizophrenia is at least partly dependent
on complex genetic risk factors, likely involving a number of different genes. Therefore,
there have been numerous attempts to model the disorder with genetic strategies.
11

Knockout and knockdown strategies typically target genes for neurotransmitter systems
implicated in schizophrenia. One well-known example includes dopamine transporter
knockout mice, which show hyperlocomotion and deficits in PPI that are reversed with
antipsychotics (Gainetdinov et al., 2001). Similar results have been gained with the NR-1
knockdown mouse, which has reduced NMDA receptor function (Mohn et al.,
1999;Duncan et al, 2004).
Genetic models targeting genes involved in neurodevelopment and implicated in
schizophrenia have also been developed. Reduced expression of reelin, a protein
important for normal cell positioning in the brain, has been described in schizophrenia
(Impagnatiello et al., 1998). Heterozygous reeler mice, which exhibit a down regulation
of reelin similar to levels observed in schizophrenic patients, also have a variety of
behavioral abnormalities similar to those in schizophrenia (Costa et al., 2002). These
genetic strategies are useful in understanding the impact of genetic alterations on
behavior, neurochemistry, and histology at a systems level, although these single gene
approaches only model part of the complex etiology of schizophrenia. Clearly, the future
challenge for genetic strategies lies in modeling the complex multi-gene abnormalities
that will likely be implicated in schizophrenia (Wong and Van Tol, 2003). Additionally,
combining genetic approaches with environmental factors in a 'two-hit' approach may be
especially valuable in the future (Ellenbroek, 2003).
Adult Pharmacological Models
These models generally rely on producing acute or long-lasting behavioral
changes relevant to schizophrenia by administering psychoactive drugs to animals.
Dopamine agonists such as amphetamine and NMDA antagonists such as MK-801,
12

phencyclidine or ketamine are most commonly used. Acute administration of these drugs
induces deficits in PPI and increased locomotor activity (for review see Marcotte et al.,
2001). Not surprisingly, the changes induced by dopamine agonists are reversed by
typical antipsychotics (Marcotte et al., 2001). Additionally, sensitization with
amphetamine, but not phencyclidine, in rats induces long-lasting deficits in PPI, which
may more faithfully model the stable PPI deficits in schizophrenia (Tenn et al., 2005).
Interestingly, administration of NMDA antagonists may more accurately model
schizophrenia by inducing a broader range of symptoms, including cognitive deficits and
negative symptoms (Jentsch and Roth, 1999). The effects of NMDA antagonism are also
reversed by atypical antipsychotics, thereby improving the potential of the model to
identify novel antipsychotic compounds.
The data gained from adult pharmacological models is limited for a number of
reasons. First, the effects are typically induced in normally developed adult animals. A
wide range of evidence indicates that people who develop schizophrenia have
abnormalities before adulthood. Importantly, combining neonatal perturbations of brain
development with pharmacological challenges has provided valuable data, and will be
discussed in the next section. Secondly, the changes in the activity of neurotransmitters
such as dopamine are also more regionally specific than can be modeled with systemic
administration of drugs (Kilts, 2001). Nevertheless, some treatment paradigms have
proven to model more faithfully the neurochemical and behavioral changes in
schizophrenia. For example, chronic exposure to phencyclidine in non-human primates
results in cognitive deficits and reduced dopamine turn-over in the prefrontal cortex, both
of which persist after drug treatment in discontinued (Jentsch et al., 1997). These effects
13

can also be reversed following treatment with the atypical antipsychotic clozapine
(Jentsch et al, 1997). Finally, some researchers argue that the development of novel
antipsychotic drugs is precluded by dopamine agonist models because the therapeutic
drugs that have emerged from these models block dopamine receptors, thereby leading to
a circular pattern of discovery (Lipska and Weinberger, 2000).
Adult Lesion Models
Adult lesion models focus on the effects of lesioning limbic and cortical areas
commonly implicated in both subcortical dopamine regulation and schizophrenia, such as
the prefrontal cortex, hippocampus, and thalamus (Lipska and Weinberger,
2000;Marcotte et al., 2001). Obviously, the construct validity of adult lesion models is
compromised by their adult initiation and large degree of damage that occurs (at least
compared to that observed in schizophrenic patients); however, such models do provide
unique insights into the neural substrates that may underlie many of the behavioral and
neurochemical abnormalities in schizophrenia.
Adult lesions of the medial prefrontal cortex in rodents result in hyper-
responsiveness to stress (Jaskiw et al., 1990b), increases in spontaneous locomotor
activity (Braun et al., 1993), increased activity in response to amphetamine (Jaskiw et al.,
1990a), and deficits in PPI after challenge with the dopamine agonist apomorphine
(Swerdlow et al, 1995). Similarly, lesions of the ventral, but not dorsal, hippocampus
result in increased spontaneous activity and hyperactivity and PPI deficits following
administration of dopamine agonists (Lipska et al., 1991 ;Lipska et al., 1992;Swerdlow et
al., 1995;Mittleman et al., 1998). These data enable a detailed understanding of the
neural substrates mediating PPI and locomotor activity to be developed, and will be
14

eturned to in greater detail in Chapter Two. Importantly, they can be effectively
integrated with neuroanatomical and neurochemical data, leading to new hypotheses
regarding the mechanisms underlying the behavioral effects of the lesions. For example,
the connectivity of the ventral, but not dorsal, hippocampus with areas such as the
prefrontal cortex, amygdala, nucleus accumbens, and ventral tegmental area of the
midbrain may be linked causally to the behavioral effects observed following its removal
(Bast and Feldon, 2003).
OBJECTIVE ONE: Numerous well-documented shortcomings of permanent lesions
exist, such as compensatory alterations in brain areas other than the lesioned area
(Schoenfeld and Hamilton, 1977;Stein, 1979;Bast and Feldon, 2003). Additionally,
animals may develop strategies that aid in coping with the permanent loss of function
normally carried out by the lesioned area (Lomber, 1999;Bast and Feldon, 2003).
Specifically in the context of PPI and locomotor behavior, differences between
permanent and temporary manipulations of the hippocampus have been described
(Swerdlow et al., 1995;Pouzet et al., 1999;Swerdlow et al., 2000b;Zhang et al., 2002).
Therefore, in Chapter Two, I examine the effect of brief stimulation of discrete sub-
regions of the hippocampus on PPI. Given the important role of the hippocampus in
schizophrenia, these experiments may provide further insights into the pathophysiology
of the disorder.
15

Developmental Animal Models
As previously discussed, the neurodevelopmental hypothesis of schizophrenia has
received considerable attention. Therefore, it is not surprising that numerous strategies
have been developed to model schizophrenia using manipulations at distinct periods from
embryological development until the time of weaning. As the present experiments focus
on manipulations in the early neonatal period, most of the studies detailed below will
focus on this time point. Numerous excellent reviews of experiments examining the
effects of prenatal stress, obstetric complications, prenatal infection, and other
developmental manipulations are available (Van den Buuse et al., 2003;Boksa, 2004).
Before discussing developmental models, it is important to note that rodents are
born at later stage of development than humans. Thus, the first week of postnatal life in
rodents is typically described as corresponding to the third trimester of human fetal brain
development (Lipska and Weinberger, 2000). Additionally, the surge of gonadal
hormones that coincide with the onset of puberty occurs during the 5 th
and 6 th
postnatal
weeks of the rodent life span. Thus, assessments of prepubertal function are generally
conducted at approximately postnatal day 35 and assessments of adult function are
performed after postnatal day 56.
Maternal Separation and Social Isolation Models
These two types of models involve removal of developing rats from their normal
sources of social interaction, whether it be from their mothers early in development
(maternal separation) or from their peers immediately following weaning (social
isolation). Stress likely plays a major role in producing the behavioral changes observed
in both models (Hall, 1998;Lipska and Weinberger, 2000;Weiss and Feldon, 2001 ;Van
16

den et al., 2003). In one example of a maternal separation paradigm, a 24 hour separation
period on postnatal day three, six, or nine is sufficient to produce deficits in PPI and
increased sensitivity to dopamine agonists that occur only after puberty (Ellenbroek et al.,
1998;Husum et al., 2002). Interestingly, these deficits can be reversed with typical or
atypical antipsychotics (Ellenbroek et al., 1998). Strain differences are also observed,
with effects present in Wistar rats, but not the Lewis or Fisher 344 strains (Ellenbroek
and Cools, 2000). These data provide important support for the assertion that early life
experiences interact with genetic background to alter adult patterns of behavior with
relevance to schizophrenia (Lipska and Weinberger, 2000;Ellenbroek and Cools, 2000).
The social isolation model involves rearing weanling rat pups individually
without access to their peers, thereby depriving them of social contact and considerable
sensory stimulation (Hall, 1998;Weiss and Feldon, 2001 ;Van den et al., 2003). This
model is distinct from maternal separation discussed above in that early development of
the animals is not disturbed. Considerable research has been conducted using the social
isolation model showing fairly consistent reductions in PPI, alterations in spontaneous
locomotor activity, and locomotor responses to dopamine agonists and NMDA
antagonists (Hall, 1998;Lipska and Weinberger, 2000;Weiss and Feldon, 2001).
Parametric studies of the duration of rearing reveal that the pups must be in isolation for
greater than three weeks starting at weaning (postnatal day 21 to 28) for behavioral
alterations to be observed (Bakshi and Geyer, 1999;Varty et al., 1999). The behavioral
effects are also sensitive to the strain of rats used (Weiss and Feldon, 2001), and are
usually reversed with either typical or atypical antipsychotic drug treatment (Varty and
Higgins, 1995;Bakshi et al., 1998). Thus, the social isolation model is an important
17

nonpharmacological tool that can be used to identify novel antipsychotic compounds
(Weiss and Feldon, 2001).
With respect to both the maternal separation and isolation rearing paradigms,
reliability is a concern. For example, some groups have failed to observe PPI deficits in
maternally separated rats using similar, but not identical, methods (Lehmann et al.,
2000;Weiss and Feldon, 2001). Factors such as the temperature at which the pups are
maintained and the exact conditions under which they are separated (i.e. separated and
kept in a group versus kept individually) may help explain the conflicting results (Weiss
and Feldon, 2001). With regard to isolation rearing, the robustness of the behavioral
effects has been questioned, as some reports indicate that the model is quite sensitive to
handling or other testing procedures (Weiss and Feldon, 2001). However, a long-term
evaluation of the reliability of PPI deficits in the social isolation model indicate that PPI
deficits occur in approximately 85 percent of groups tested against matched controls
(Cilia et al., 2005).
Neonatal Lesion Models
A great deal of work in the last ten to fifteen years has examined the utility of
performing neonatal lesions of cortical and limbic areas in modeling schizophrenia
(Lipska and Weinberger, 2000). This procedure has a number of distinct advantages over
those discussed above: (1) The normal maturation of specific brain areas is altered and
this simplifies the search for mechanisms underlying observed changes in the model. (2)
The consequences of the lesion can be examined over the course of development thereby
allowing the timing of any changes to be compared to the normal course of the disease.
(3) Manipulating the brain at an early age is consistent with the neurodevelopmental
18

theory of schizophrenia. (4) Further manipulations can be performed later in development
to test the 'two-hit' hypothesis of schizophrenia.
Most prominent among these models is the ventral hippocampal lesion model
initially described by Lipska and colleagues (Lipska et al., 1993). In this model, rats are
subjected to bilateral lesions of the ventral hippocampus with the excitotoxin ibotenic
acid on postnatal day seven. The animals are then allowed to develop normally and can
be tested before or after puberty on an array of behavioral tests relevant to schizophrenia.
Numerous reports indicate that rats with neonatal ventral hippocampal lesions display
PPI deficits and increased spontaneous locomotor activity (Lipska et al., 1993;Lipska et
al., 1995). The PPI deficits are potentiated by a low dose of apomorphine (Lipska et al.,
1995) and reversed by atypical antipsychotic drugs, but not by haloperidol (Le Pen and
Moreau, 2002). Locomotor activity is also increased following stress (Lipska et al.,
1993), dopamine agonist administration (Lipska et al., 1993), or treatment with NMDA
antagonists (Al Amin et al., 2000;A1 Amin et al., 2001). The alterations in locomotor
behavior can be normalized with haloperidol, but not atypical antipsychotic drugs (Al
Amin et al., 2000). Treatment with ionotropic glutamate receptor antagonists (Al Amin
et al., 2000) and increasing glycine availability (Le Pen et al., 2003) also reverses some
of the behavioral impairments, thereby indicating a role of glutamate transmission in the
underlying effects of neonatal ventral hippocampal lesions.
Consistent with the typical course of schizophrenia, the PPI deficits and
locomotor activity changes are not expressed until after the rats reach puberty and persist
at least until mid-adulthood (Lipska and Weinberger, 1993;Lipska et al., 1995;Lipska and
Weinberger, 2000;A1 Amin et al., 2001). Interestingly, lesioned rats show reduced social
19

interaction before and after puberty, a test which may have some relevance to the
abnormalities observed in schizophrenic patients before their first episode (Sams-Dodd et
al., 1997). Parametric experiments also suggest that the delayed onset of behavioral
changes in the animals only occurs if the lesions are created during the first postnatal
week of life (Wood et al., 1997), which corresponds to the third trimester in human
development (Lipska and Weinberger, 2000). Finally, in an important paper, Lipska and
Weinberger (Lipska and Weinberger, 1995) demonstrated that the effects of the neonatal
lesion depend on the genetic background of the animals. Rats highly responsive to stress
(the Fischer 344 strain) showed exaggerated effects of the lesion, whereas rats hypo-
responsive to stress (the Lewis strain) showed reduced lesion effects. Thus, this model
supports the theory that schizophrenia may develop from an interaction between genetic
predisposition to the disorder and adverse environmental events (Lipska and Weinberger,
1995;Benes, 2000;Benes et al., 2000;McClure and Weinberger, 2001;Ellenbroek, 2003).
The results gained from the ventral hippocampal model have led others to
examine the effects of neonatally lesioning other areas such as the medial prefrontal
cortex (Flores et al., 1996;Schneider and Koch, 2005), amygdala (Hanlon and Sutherland,
2000;Daenen et al., 2001;Daenen et al., 2002a;Daenen et al, 2002b;Daenen et al.,
2003a;Daenen et al., 2003b) and thalamus (Lipska et al., 2003) on a similar range of
behaviors. Many of these experiments have yielded results similar to, or less consistent
than, those already discussed. As a result, they will not be elaborated on further.
While it is clear that many characteristics of schizophrenia have been modeled
with great success using neonatal ventral hippocampal lesions, the severity and
permanent nature of the hippocampal lesion limits the construct validity of this model for
20

schizophrenia (Lipska and Weinberger, 2000). As a result, Lipska and colleagues (2002)
demonstrated that reversible temporary inactivation of the ventral hippocampus with
tetrodotoxin on postnatal day seven is sufficient to induce significant changes in
locomotor behavior in adulthood, but not before puberty. This manipulation produces
limited gross morphological damage to the ventral hippocampus, and is short-lasting (24-
48 hr) (Lipska et al., 2002). These data clearly highlight the fragility of the hippocampal-
cortical circuit during this developmental period. Additional data demonstrate that a
single infusion of the NMDA antagonist (±)-2-amino-5-phosphonopentanoic acid (APV)
into the rat medial prefrontal cortex on postnatal day seven results in exaggerated
amphetamine-induced locomotor activity in adulthood (Lafleur et al., 2001). These data
support the hypothesis that an abnormality in activity-dependent plasticity involving
glutamatergic hippocampal-prefrontal interactions may underlie some of the symptoms
observed following neonatal ventral hippocampal lesions (Lipska et al., 2002).
Additionally, these data support the hypothesis that the first postnatal week is a critical
period for the development of cortico-limbic circuits mediating behaviors such as
locomotor activity and PPI.
Although the neonatal ventral hippocampal lesion model provides unique insight
into the consequences of damaging a particular area early in development, it is unlikely
that schizophrenia results from damage to a single brain area. Thus, the efficacy of
altering the activity of the brain pharmacologically during the first postnatal week has
also been tested in an effort to model schizophrenia. These experiments are discussed in
the following section.
21

Neonatal Pharmacological Models
As previously discussed, alterations of the glutamatergic system are likely to be
involved in the expression of the symptoms of schizophrenia. A number of lines of
evidence support the hypothesis that the glutamatergic system may also be involved in
the development of the disorder. For example, adverse events during early development
such as febrile seizures, fetal alcohol exposure, and hypoxia/ischemia involve the
glutamatergic system (Morimoto et al., 1995;01ney et al., 1999;Zornberg et al.,
. 2000;Olney, 2004) and are risk factors for psychosis and/or schizophrenia (Cannon,
1997;Famy et al., 1998;01ney et al., 1999;Kanemoto et al., 2001;Vestergaard et al.,
2005). The normal maturation and development of limbic and cortical areas relevant to
schizophrenia is also dependent on normal glutamatergic function. For example,
processes such as cell birth, survival and synaptogenesis are critically dependent on
glutamate transmission, particularly at NMDA receptors (Rabacchi et al., 1992;Gould et
al., 1994;Fox et al., 1996;Vallano, 1998;Ikonomidou et al., 1999;Luthi et al., 2001).
Given the established role of glutamate neurotransmission in neonatal
development during periods implicated in schizophrenia, a number of studies have
examined the behavioral effects of manipulating glutamate transmission
pharmacologically early in life. Most of these experiments have tested the effects of
systemically administered NMDA receptor antagonists such as MK-801, ketamine or
phencyclidine during the neonatal period. A number of groups have demonstrated
deficits in PPI and alterations in locomotor behavior following treatment with these drugs
(Facchinetti et al., 1993;Wang et al., 2001;Ffarris et al., 2003;Fredriksson et al., 2004).
As will be elaborated further in Chapter Four of the present thesis, the long-lasting
22

morphological effects of administering non-competitive NMDA antagonists early in the
neonatal period are also relevant to schizophrenia (Wang et al., 2001 ;Harris et al., 2003).
OBJECTIVE TWO: Given that the end of the first postnatal week is a sensitive period for
the development of neural circuits mediating PPI and locomotor activity (Wood et al.,
1997;Wang et al., 2001;Lipska et al., 2002;Harris et al, 2003), the second objective of
this thesis was to extend these findings in two novel directions. In Chapter Three, a
series of experiments examined the long-term behavioral effects of administering the
glutamate receptor agonist kainic acid to postnatal day seven rats. Chapter Four
summarizes an independent set of experiments designed to test the effects of
administration of the highly specific NMDA receptor antagonist [(+/-)-(R*,S*)-alpha-(4-
hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-l-piperidine propanol] (Ro25-6981)
during the same time period. These pharmacological manipulations were designed to
subtly alter the normal development of the cortico-limbic circuitry involved in
schizophrenia - kainic acid by selectively activating the hippocampus and Ro25-6981 by
blocking NR2B subunit-containing NMDA receptors, which are highly expressed during
the first postnatal week in rodents.
23

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CHAPTER TWO: ELECTRICAL STIMULATION OF THE HIPPOCAMPUS
DISRUPTS PREPULSE INHIBITION IN RATS: FREQUENCY AND SITE
DEPENDENT EFFECTS. 1
Introduction
Prepulse inhibition (PPI) is the normal reduction in an acoustic startle response
produced when a brief, quieter stimulus or prepulse is presented 30-500 ms prior to
presentation of the starde-evoking stimulus (Koch, 1999;Swerdlow et al., 2000a). PPI
provides a measure of sensorimotor gating, the inhibitory process by which responses to
sensory stimuli are suppressed, thereby facilitating appropriate responding in complex,
sensory rich environments (Swerdlow, 1996). Interest in the neural mechanisms
underlying PPI has increased because it is often disrupted in humans diagnosed with a
variety of psychiatric disorders including schizophrenia (Weike et al., 2000;Braff et al.,
2001). As similar stimuli elicit PPI in rodents and humans, interventions in rodents that
disrupt PPI may reveal aspects of neuronal dysfunction that underlie disrupted
sensorimotor gating in schizophrenic patients (Swerdlow, 1996;Weike et al., 2000).
Similarities exist in the neuroanatomical and neurochemical substrates related to
both schizophrenia and PPI. A large body of research suggests that schizophrenic
patients show abnormalities in cortico-limbic-striatal circuitry, in areas such as the frontal
cortex, nucleus accumbens (NAc), and hippocampus (Pakkenberg, 1990;Bogerts,
1997;Harrison, 1999;Wright et al., 1999) likely involving alterations in dopamine (DA)
and glutamate (Glu) transmission (Breier et al., 1997;Carlsson et al., 1999;Laruelle et al.,
1999;Goff and Coyle, 2001;Meyer-Lindenberg et al., 2002). Consistent with these
1
A version of the chapter has been published: Howland JG, Mackenzie EM, Yim TT, Taepavarapruk P,
Phillips AG (2004). Electrical stimulation of the hippocampus disrupts prepulse inhibition in rats:
frequency and site dependent effects. Behavioural Brain Research 152:187-197.
37

findings is evidence indicating that the regulation of PPI is also dependent on a complex
array of structures commonly termed the cortico-striatal-pallidal-pontine (CSPP) circuitry
(see Swerdlow et al., 2001a for review), including the medial prefrontal cortex (mPFC)
(Koch and Bubser, 1994;Lacroix et al., 2000), amygdala (Wan and Swerdlow,
1997;Fendt et al., 2000), NAc (Swerdlow et al.,1986;1990;1994;Wan and Swerdlow,
1996), and hippocampus (Caine et al., 1992;Swerdlow et al., 1995;2001b;Wan et al.,
1996;Bakshi and Geyer, 1998;Klarner et al, 1998;Zhang et al., 1999;2002a;Bast et al.,
2001;Caine et al., 2001). Importantly, the systemic administration of DA or DA agonists
(Mansbach et al, 1988;Swerdlow et al., 1990;Zhang et al., 2000a) and N-Methyl-D-
Aspartate (NMDA) antagonists (Mansbach and Geyer, 1989;Bakshi and Geyer, 1998),
likely acting in areas of the CSPP circuitry, also disrupt PPI in animals (Geyer et al.,
2001). Recently, a complex role of NAc DA and Glu via D2, NMDA, and (±)-a-amino-
3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) receptors in the
modulation of PPI has been described (Mansbach et al., 1988;Swerdlow et al, 1990;Wan
and Swerdlow, 1993;1996;Reijmers et al., 1995;Wan et al., 1995).
Hippocampal dysfunction has been linked to symptoms of schizophrenia
(Silbersweig et al., 1995;Heckers et al., 1998;Harrison, 1999;Dierks et al., 1999;Grace,
2000), and consequently the roles of distinct hippocampal subregions in the modulation
of PPI have been examined extensively. Although permanent lesions of the ventral
hippocampus (vHip) generally do not disrupt PPI (Swerdlow et al., 1995;2000b;Pouzet et
al., 1999 but see also (Caine et al., 2001), temporary inactivation of vHip or dorsal
hippocampal (dHip) with either the GABA A agonist muscimol or the sodium channel
blocker tetrodotoxin disrupts PPI (Zhang et al., 2002b). Debate also exists as to whether
38

infusion of glutamatergic antagonists, such as MK801, into the dHip, but not the vHip,
disrupts PPI (Bakshi and Geyer, 1998;1999;Bast et al., 2000;Zhang et al., 2000b).
Nevertheless, compelling evidence exists for the disruption of PPI following chemical
stimulation of the vHip (Wan et al., 1996;Klarner et al., 1998;Zhang et al., 1999;Bast et
al., 2001;Swerdlow et al., 2001b;Zhang et al., 2002a), but not dHip (Swerdlow et al.,
2001b;Zhang et al., 2002a) with infusions of the ionotropic Glu agonist NMDA. As
these studies suggest, various manipulations of the vHip and dHip can impair
sensorimotor gating behavior, and regional differences clearly exist.
Both the vHip and dHip are well positioned neuroanatomically to interact with the
CSPP circuitry to modulate PPI. The vHip projects densely to the NAc shell, amygdala,
and mPFC (Kelley and Domesick, 1982;Groenewegen et al., 1987;Brog et al.,
1993;Conde et al., 1995) whereas the dHip projects to the NAc core (Swanson and
Cowan, 1977;Whitter, 1986), and also communicates with the vHip via intra-
hippocampal connections (Amaral and Whitter, 1995). At the level of the ventral
striatum, projections from the vHip exert powerful control over DA efflux in the NAc
shell and firing of NAc medium spiny projection neurons (O'Donnell and Grace,
1995;Taepavarapruk et al., 2000;Floresco et al., 2001). More specifically, stimulation of
the vHip with NMDA (Brudzynski and Gibson, 1997;Legault et al., 2000) or brief trains
of higher frequency electrical current (20 Hz, 10 s) (Blaha et al., 1997;Taepavarapruk et
al., 2000) cause a long-lasting increase in NAc DA efflux and an increase in locomotor
activity. Activation of ionotropic Glu receptors (iGluR's) in the NAc underlies the
changes in NAc DA efflux observed following electrical stimulation of the vHip (Blaha
et al., 1997;Taepavarapruk et al., 2000). In contrast, preliminary studies performed in our
39

laboratory demonstrate that lower frequency (2 Hz) stimulation of the vHip for 100 s can
significantly reduce NAc DA efflux for at least 2 hr, an effect that may result from the
activation of intra-NAc group 2 and 3 metabotropic Glu receptors (Taepavarapruk et al.,
1998). The consequences of stimulating the dHip on NAc DA efflux have been poorly
characterized, although stimulation of the previously kindled dHip results in a brief
increase in NAc DA efflux (Strecker and Moneta, 1994).
In the following experiments, the effects of electrical stimulation of the Hip on
PPI were examined. Electrical stimulation was chosen because it has a number of
distinct advantages over chemical stimulation, including precise control over the duration
and intensity of the stimulation. Additionally, electrical stimulation parameters can
approximate the physiological firing activity of the neurons that are stimulated. Given
that intra-vHip NMDA infusions and 20 Hz electrical stimulation of the vHip produce
similar increases in NAc DA efflux (Brudzynski and Gibson, 1997;Legault et al.,
2000;Taepavarapruk et al., 2000), and that vHip NMDA infusions disrupt PPI (Wan et
al., 1996;Klarner et al, 1998;Zhang et al, 1999;2002a;Bast et al., 2001;Swerdlow et al.,
2001b), the first objective of the present study was to assess whether 20 Hz stimulation of
the vHip would induce a reversible disruption of PPI. In a complementary experiment,
we examined the effect of 20 Hz electrical stimulation to the dHip on PPI, hypothesizing
that if dHip NMDA infusions fail to disrupt PPI (Swerdlow et al., 2001b;Zhang et al,
2002a), then electrical stimulation at this frequency should also fail to reduce PPI. The
final behavioral experiment examined the effect of 2 Hz vHip stimulation on PPI.
Additional microdialysis experiments addressed two issues related to the effects of 20 Hz
stimulation of the hippocampus. The first assessed whether unilateral vHip stimulation
40

would result in an increase in DA efflux bilaterally in the NAc. Secondly, as the effects
of 20 Hz dHip stimulation on NAc DA are unknown, a similar microdialysis experiment
assessed the effects of these stimulation parameters on NAc DA efflux. Male Long-
Evans (LE) rats were used in all experiments in an effort to maintain consistency between
the neurochemical data collected previously and the present behavioral experiments.
Methods
Subjects
Male LE rats (Charles River Canada, St. Constant, Quebec, Canada) were pah-
housed in Plexiglas cages and handled daily until surgery. The colony was kept on a
12/12 hour light/dark cycle (lights on at 0700), at a temperature of 22±1°C. Rats were
given food (Purina Rat Chow) and tap water ad libitum. Experiments were conducted in
accordance with the standards of the Canadian Council on Animal Care and the
Committee on Animal Care at the University of British Columbia approved all
procedures.
Surgery
At the time of surgery, subjects weighed 330 to 370 g. Rats were anaesthetized
with ketamine hydrochloride (100 mg/kg, i.p., MTC Pharmaceuticals) and xylazine (10
mg/kg, i.p., Rompun), and placed in a stereotaxic frame. The dorsal surface of the skull
was exposed, and holes were drilled. A bipolar stimulating electrode (Plastics One,
Roanoke, VA) was implanted into either the vHip (AP -5.8 mm from bregma, ML ±5.5
mm from midline, DV -6.0 mm from dura) or dHip (AP -3.0 mm, ML ±1.5 mm, DV -
3.0 mm) of all rats. Guide cannulae were also implanted dorsal to the NAc in those rats
used in the microdialysis experiments (AP +1.7 mm, ML ±1.1 mm, DV -1.0 mm).

Cannulae and the electrode were secured to the skull with four jeweler's screws and
dental acrylic. Wire obdurators were inserted into the cannula to keep them patent.
Animals were allowed at least 7 days to recover from surgery before testing began, and
were regularly handled beginning three days following surgery.
Prepulse Inhibition Testing
Testing was conducted in a single sound-attenuating startle chamber (ambient
noise level 62 dB), containing a transparent Plexiglas tube (8.2 cm in diameter, 20 cm in
length), mounted on a Plexiglas frame (SR-LAB, San Diego Instruments, San Diego,
CA). Noise bursts were presented through a speaker mounted 24 cm above the tube. An
accelerometer below the frame of the apparatus measured whole body startle amplitude,
defined as the average of 100 1 ms accelerometer readings collected beginning at
stimulus onset. Each PPI test session began with a 5 min acclimatization period during
which a 70 dB background noise level was presented, which remained constant for the
entire test session. Following the acclimatization period, six pulse alone trials (120 dB,
40 ms) were presented to achieve a relatively stable level of startle amplitude before
presentation of the prepulse + pulse trials. The data from these pulse alone trials was not
considered in the analysis of PPI. Immediately following the six initial pulse alone trials,
presentation of the trials that were used in the calculation of PPI levels began. A total of
84 trials of five different types were presented. Trials presented were of three types:
pulse alone (12 trials; 120 dB, 40 ms), prepulse + pulse (10 trials X 3 prepulse intensities
- discussed below), or no stimulus (42 trials). Prepulse + pulse trials consisted of the
presentation of a 20 ms prepulse of 73, 76, or 82 dB 80 ms before the presentation of the
pulse. The pulse and prepulse + pulse trials were presented in a pseudorandom order.
42

One no stimulus trial was presented between each pair of pulse and prepulse + pulse
trials. The inter-trial interval varied randomly from 3 to 12 s (average 7.5 s). The total
length of each PPI session was approximately 18 min. Calibration of the apparatus was
performed using a RadioShack Digital Sound Level Meter and adjustments were made as
necessary.
A within-subjects design was used in all PPI experiments. Experiments were
conducted in two successive weeks; all animals were tested four times during these two
weeks. The four PPI sessions were referred to as follows. The 'stim' PPI session
occurred 2 min following electrical stimulation of either the vHip or dHip, 'stim+48' was
the session 48 hr following stimulation, 'control' was the PPI session 2 min following
exposure to the stimulation box without stimulation, and 'control+48' followed the
'control' session by 48 hrs. Animals were randomly assigned to one of two sequences to
control for potential order effects. Rats in sequence one received their PPI sessions in the
following order: stim, stim+48, control, and control+48. Rats in sequence two received
their PPI sessions in the control, control+48, stim, and stim+48 order.
Following recovery from surgery, animals were habituated to the stimulation
environment on the two days immediately preceding their first PPI test. Each rat was
removed from the colony and taken to the stimulation room. This small room was
immediately adjacent to the room used for PPI testing, and contained two transparent
Plexiglas chambers (32 cm X 32 cm X 41 cm) used for stimulation. In the stimulation
room, each rat was connected to a stimulation lead and placed in a Plexiglas chamber for
10 min, following which they were returned to the colony. On the day of their first PPI
test, the animals were treated exactly as on the two previous days except that after 10 min
43

in the stimulation box, electrical stimulation (20 Hz: 200 cathodal, constant current
pulses, 300 uA, 20 Hz, pulse width 0.5 ms; 2 Hz: identical except frequency of pulses
was 2 Hz) was applied to half the rats, while the other half were given no current.
Stimulation was delivered through an isolator (Iso-flex, A.M.P.I, Israel) via a Master-8
stimulator (A.M.P.I.). The experimenter observed the animals for 90 sec following
stimulation, disconnected them, and quickly placed them in the PPI chamber. This
procedure took a total of 2 min. Immediately following the PPI session, the animals were
returned to the colony. All rats were then tested for PPI 48 hours later. For this test, the
rats were removed from the colony and immediately placed in the PPI apparatus.
Following the second PPI session, the animals were handled daily for five days. On the
sixth day following the first PPI test session, all rats were given one habituation session
in the stimulation chamber for 10 min. The following day, the PPI testing procedure
described above was repeated, with control animals receiving electrical stimulation and
previously stimulated animals receiving no current. Forty-eight hours later, all rats
received their last PPI session.
Neurochemical Experiments
The microdialysis procedure commonly employed in our laboratory has been
described in detail elsewhere (Taepavarapruk et al, 2000;Howland et al., 2002). Briefly,
concentric-style microdialysis probes (2 mm of exposed membrane) were constructed in
our laboratory. Probes were attached to gas-tight syringes via a liquid swivel containing
perfusion medium (147 mM NaCl, 3.0 mM KC1,1.3 mM CaCl 2.H 20, 1.0 mM
MgCl 2.6H 20, 0.01 sodium phosphate buffer; pH 7.3-7.4) and flushed for 10 to 20 min
using a syringe pump. Probes were then secured in a copper collar and inserted into the
44

NAc (7.8 mm ventral to dura) and the rats were allowed to move freely in a Plexiglas box
(32 cm x 32 cm x 41 cm high) with access to food and water for 12 to 18 hours before
experimental testing began the following morning. The probes were continuously
perfused at 1 pL/min overnight. Probes were inserted bilaterally for the vHip stimulation
experiment and unilaterally ipsilateral to the electrode for the dHip stimulation
experiments.
Two high-performance liquid chromatography systems with electrochemical
detection (HPLC-ED) consisting of an ESA 582 pump (ESA Inc., Bedford, MA),
Rheodyne Inert manual injector (Rheodyne, Rohnert Park, CA), an Ultrasphere column
(Beckmann, Fullerton, CA.; ODS 5 pm, 15 cm x 4.6 mm), an ESA 5011 analytical cell,
and a Coulochem II EC detector (ESA Inc.) were used to quantify DA levels in all
experiments. The working potentials were: +450 mV (electrode 1), -300 mV (electrode
2), and +450 mV (guard cell). The mobile phase (pH 3.5) consisted of 6 g/L sodium
acetate, 10 mg/L ethylenediaminetetra-acetic acid (EDTA), 150 mg/L octyl sulfate
(adjustable), 35 ml/L glacial acetic acid and 865 mL Milli Q purified water.
Chromatograms were registered on a dual-pen chart recorder (Kipp and Zonen, Bohemia,
NY). All samples were injected immediately after collection and DA peak heights were
measured manually.
Dialysate samples were collected at 10 min intervals throughout the experiment.
Once four baseline samples were collected that did not differ by more than ± 10%, the
rats received 20 Hz electrical stimulation of either the vHip or dHip (same parameters
described for the PPI experiments). Stimulation was timed to ensure that the next
dialysis sample reflected only "stimulation-evoked" changes in DA efflux. The
45

experimenter remained in the testing room following the stimulation to record any
behavioral effects of the stimulation. Nine samples were collected following stimulation,
after which the animals were disconnected and sacrificed.
Histology
Upon completion of each experiment, animals were sacrificed with an overdose of
pentobarbital, and perfused transcardially with 0.9% saline followed by 10%
formaldehyde. Brains were stored in 10% sucrose in 10% formaldehyde for at least 1
week, after which they were sectioned (50 pm) using a cryostat and stained with cresyl
violet. Placements of the electrodes and probes were confirmed under a light microscope
with the assistance of a rat brain atlas (Paxinos and Watson, 1997).
Data Analysis
Data were analysed using repeated measures analyses of variance with the aid of
SPSS (version 10.0). All post-hoc tests were performed separately. Variance is indicated
on all graphs with the standard error of the mean (SEM). Significance levels for all
statistical tests were set at 0.05.
For the PPI experiments, two measures were calculated for each animal. The
startle amplitude represented the mean startle amplitude of the 12 pulse alone trials
presented after the six habituation trials. For calculation of PPI, startle amplitudes were
averaged for each trial type. The percent PPI for each prepulse intensity was calculated
using the formula: [100 - (100 X startle amplitude on prepulse + pulse trials) (startle
amplitude on pulse alone trials)]. Two-way (prepulse intensity, test session as factors)
repeated measures ANOVAs were performed on the data obtained from each stimulation
site. As the prepulse intensity factor did not significantly interact with any of the other
46

factors, percent PPI data was averaged across the three prepulse intensities for each rat,
thereby creating a global PPI score (Wan et al., 1995;Bakshi and Geyer, 1998;1999).
Both startle amplitude and percent PPI were then analyzed using separate one-way
ANOVAs with condition as a repeated measures factor. The repeated measures
assumption of sphericity was met in all cases. Post-hoc analyses were performed using
the Neuman-Keul's test where appropriate.
Following the microdialysis experiments, baseline DA levels for each rat were
calculated by averaging the four baseline samples. In all figures, DA levels are expressed
as a percentage change from baseline (baseline is expressed as 0%). One-way repeated
measures analyses of variance with time as a within-subjects factor were performed on all
data and Dunnett's post-hoc tests were performed where appropriate. The baseline
sample taken immediately before the first stimulation was used as the critical value
during computation of the Dunnett's post-hoc tests.
Results
Immediate Behavioral Effects of Stimulation
During the 2 min that the rats remained in the stimulation apparatus after
stimulation was applied, a variety of behavioral effects were noted. The rats were
generally awake and alert before administration of stimulation. At the onset of
stimulation, behavioral arrest was observed in all animals. Higher frequency (20 Hz)
stimulation of the vHip resulted in 5 to 20 wet dog shakes (WDS) that were restricted to
45 s following initiation of the stimulation in 20 of the 21 animals (rats used in the
dialysis studies are included in this description). Additionally, all rats engaged in
vigorous forward locomotor behavior following cessation of the 20 Hz stimulation, as has
47

een previously quantified (Taepavarapruk et al., 2000). Stimulation of the dHip resulted
in an initial period of WDS activity in 4 of the 28 animals. All rats displayed a period of
increased activity and rearing within 30 sec of the cessation of stimulation. Between 70
and 100 s following the cessation of stimulation, 18 of the 28 rats had a second bout of
WDS activity. One to 20 WDS were displayed during this period. Two Hz stimulation
of the vHip caused an initial period of behavioral arrest followed by behavioral activation
manifested by grooming and some rearing. Wet dog shakes were observed in 3 of 15
animals during application of the 2 Hz stimulation.
Differential Effects of 20 Hz Stimulation of the vHip or dHip on PPI and Startle
Amplitude
Twenty Hz stimulation of the vHip (n=14) resulted in a significant and reversible
disruption of PPI (Fig. 2-1 A). A one-way repeated measures ANOVA performed on the
PPI data for the vHip stimulation group revealed a significant main effect for the test
condition factor (F(3, 39) = 9.32, p< 0.001). Post-hoc analyses revealed that percent PPI
levels were significantly lower in the stim session (20.19 ± 6 %) than in the stim+48
(41.73 ± 4%), control (42.39 ± 4 %), or control+48 (44.47 ± 4 %) sessions. This effect
was replicated with rats tested previously with 2 Hz stimulation (data discussed in section
3.3 below). In contrast, 20 Hz stimulation of the dHip (n=13) failed to induce any
significant changes in percent PPI (Fig. 2-1C; F(3, 36) = 2.01, N.S.). Percent PPI levels
were similar in the stim (39.40 ± 3%), control (46.94 ± 5 %), stim+48 (51.40 ± 4 %), and
control+48 (48.67 ± 5 %) sessions.
48

stim control stim control
+48 +48
Condition
stim control stim: control
+48 +48
Condition
startle
stim control stim control
+48 +48
Condition
dHip - startle
stim control stim control
+48 +48
Condition
Figure 2-1. Effects of 20 Hz stimulation of the hippocampus on PPI and acoustic startle
amplitude. A, Percent PPI scores 2 min (stim) or 48 hr (stim+48) following 20 Hz
stimulation (10 s) of the vHip (n=14), and respective control PPI scores (control and
control+48). The asterisk denotes a significant reduction in PPI in the stim group when
compared to all other groups (p

Presentation of the auditory pulse alone induced robust startle in all rats (Fig. 2-
1B, D). A one-way repeated measure ANOVA revealed that average startle amplitudes
for the 4 PPI test sessions of the vHip 20 Hz stimulation group did not differ significantly
(F(3, 39) = 1.40, N.S.). Thus, stimulation of the vHip did not significantly affect startle
amplitude (average startle amplitude 116.61 ± 20 arbitrary startle units). Although
stimulation of the dHip did not alter percent PPI levels (Fig. 2-1C), a significant main
effect of test session was observed when startle amplitude was examined (Fig. 2-1D, F(3,
36) = 3.37, p < 0.03). Post-hoc analyses revealed that when tested in the stim+48
condition, rats had significantly lower startle amplitudes (74.24 ± 10) than when tested in
the control condition (101.30 ± 13). Although a similar trend was noted between the stim
and stim+48 test sessions, it was not significant.
Effects of 2 Hz Stimulation of the vHip on PPI and Startle Amplitude
Two hertz stimulation of the vHip (n=15) did not significantly alter PPI levels
(Fig. 2-2A, F(3, 42) = 0.34, N.S.). When tested in the stim (46.93 ± 4 %), control (42.35
± 4 %), stim+48 (43.03 ± 4 %), and control+48 (43.52 ± 3 %) conditions, the 2 Hz group
demonstrated similar levels of PPI. In order to verify that 20 Hz stimulation would
disrupt PPI in this group of rats, one week following their last PPI session, nine of the
rats received 20 Hz stimulation of the vHip, and were tested for PPI 2 min and 24 hr later.
Results revealed that 2 min following stimulation, PPI scores were significantly lower
(30.01 ± 6 %) than 24 hr following stimulation (46.96 ± 4 %, F(l, 8) = 5.88, p < 0.05,
data not shown). These data confirmed the short-term disruptive effect of 20 Hz
stimulation of the vHip on PPI previously described.
Stimulation (2 Hz) of the vHip produced an unexpected significant increase in
50

A
vHip-PPI
stim control stim control
+48 +48
Condition
B
300, vHip - startle
stim control stim control:
+48 +48
Condition
Figure 2-2. Effects of 2 Hz stimulation of the vHip on PPI and acoustic startle
amplitude. A, Percent PPI scores resulting from 2 Hz stimulation (100 s) of the vHip
(n=15) either 2 min (stim) or 48 hr (stim+48) prior to testing, and respective control PPI
scores (control and control+48). Control testing sessions were run exactly the same as
'stim' sessions; however, no stimulation was applied to the vHip. B, Amplitude of the
startle response during the same test conditions depicted in panel A. The cross denotes a
significant difference between the stim condition and the stim+48 and control+48 groups
(p

startle amplitude (Fig. 2-2B, F(3, 42) = 9.04, p < 0.001). Post-hoc analyses revealed that
following 2 Hz stimulation, startle amplitudes were significantly higher (213.68 ± 29)
than during the stim+48 (137.04 ± 15) or control+48 (134.71 ± 19) test sessions.
Effects of Stimulation of the vHip and dHip on NAc DA Efflux
As depicted in Fig. 2-3, baseline levels of DA in the NAc were relatively stable in
all groups prior to stimulation. Stimulation of the vHip (20 Hz, n=7) induced a robust,
long-lasting 35.41 ±4% increase in DA efflux in the NAc that was restricted to the
hemisphere ipsilateral to the stimulating electrode. Separate one-way repeated measures
ANOVA's confirmed that the increase in DA efflux in the ipsilateral hemisphere was
significantly greater than baseline for 20 min (F(12, 72) = 4.04, p < 0.001, Dunnett's p <
0.05), whereas no significant changes in DA efflux were observed in the contralateral
hemisphere following vHip stimulation (F(12, 72) = 1.23, N.S.). Stimulation of the dHip
(n=7) failed to evoke an increase in DA efflux in the NAc (F(12, 72) = 1.52, N.S.).
Histology
The locations of representative stimulation electrodes and the active tips of
microdialysis probes are depicted in Fig. 2-4A-C. Ventral hippocampal placements (Fig.
4A) were similar to those previously reported in the CAl/ventral subiculum (Wan et al.,
1996;Blaha et al., 1997;Klarner et al, 1998;Zhang et al., 1999;2002a;Taepavarapruk et
al, 2000;Bast et al., 2001;Swerdlow et al., 2001b). Electrodes aimed at the dHip (Fig.
4B) were similarly positioned to dHip cannulae reported by a number of different groups
(Swerdlow et al., 2001b;Zhang et al., 2002a) in the CA3/dentate gyrus region of the
dHip. The placements of the active tips of the microdialysis probes aimed at the NAc did
52

^ -20 -I 1 1 1 1 1 1 1 1 1 1 1 1
1 2 3 4 5 6 7 8 9 10 11 12 13
Time (X 10 min)
Figure 2-3. Unilateral stimulation (20 Hz, 10 s) of either the vHip (n=7, stim) or dHip
(n=7), and its effect on NAc DA efflux in the ipsilateral (vHip - black diamonds; dHip -
white triangles) or contralateral (vHip - white squares) hemisphere. Data points
represent the mean level of DA obtained over a 10 min sampling period, and error bars
represent SEM. Asterisks denote a significant difference from baseline (p

Figure 2-4. Schematic diagram of the placements of stimulating electrodes and
microdialysis probes in all experiments. A, Representative placements of electrode tips
(black circles) aimed at the vHip. B, Representative placements of electrode tips (black
circles) aimed at the dHip. C, Representative placements of microdialysis probes aimed
at the NAc. The black bars illustrate the position of the probes and are scaled to 2 mm in
length to accurately reflect only the area of the brain from which each probe sampled.
Numbers correspond to the anterior or posterior distance (in mm) of each plate from
bregma. Plates adapted from Paxinos and Watson (Paxinos and Watson, 1997).
54

not substantially differ between the vHip and dHip stimulation experiments and are
depicted on the same panels (Fig. 2-4C).
Discussion
In the present study, the effects of electrical stimulation of the hippocampus on
PPI were site and frequency specific. Unilateral 20 Hz stimulation of the vHip, but not
the dHip, caused a significant reduction in PPI at all prepulse intensities when testing
began 2 min, but not 24 or 48 hr, after stimulation. Two Hz stimulation of the vHip
failed to produce any changes in PPI. Changes in startie amplitude were unpredictable
and difficult to interpret. Higher frequency stimulation of the vHip did not change startle
amplitude significantly, whereas 2 Hz stimulation significantly increased startle
amplitude 2 min following stimulation. When tested in the control condition, rats in the
dHip group showed significantly higher startle amplitudes than when tested in the
stim+48 condition. Experiments performed with microdialysis in freely moving rats
confirmed our previous report that 20 Hz stimulation of the vHip results in a significant
increase in NAc DA efflux (Taepavarapruk et al., 2000;Bast et al., 2001), and showed
further that this increase was restricted to the hemisphere ipsilateral to the stimulating
electrode. Finally, 20 Hz stimulation of the dHip failed to significantly increase NAc DA
efflux.
Potent Disruption of Sensorimotor Gating Induced by 20 Hz Stimulation of the vHip, but
not dHip
Numerous studies have reported that prolonged activation of the vHip, but not
dHip, induced by bilateral infusion of NMDA, disrupts PPI in both Sprauge-Dawley (SD)
and Wistar (W) rat strains (Wan et al., 1996;Klarner et al., 1998;Zhang et al,
55

1999;2002a;Bast et al., 2001;Swerdlow et al, 2001b). The present study demonstrated a
similar disruption of PPI following 20 Hz electrical stimulation of the vHip in the LE
strain. Although hooded rats, such as the LE strain, are less commonly used in PPI
experiments than the SD and W strains, they have been employed in studies of the
genetic determinants (Swerdlow et al., 2001c;2003) and other pharmacological aspects of
PPI (Rasmussen et al., 1997;Faraday et al., 1999;Binder et al., 2001). Recent
experiments suggest that the three strains are differentially sensitive to the disruptive
effects of DA agonists (Swerdlow et al., 2001c;2003), which is interesting given the role
of the vHip in modulating NAc DA efflux discussed below.
The degree (~ 50 %) and duration (< 24 hr) of PPI disruption following the brief
20 Hz electrical stimulation employed in these studies were similar to that observed
following NMDA infusion (Wan et al, 1996;Zhang et al., 1999;2002a;Bast et al.,
2001;Swerdlow et al., 2001b), although some inconsistencies have been reported
(Klarner et al., 1998). By using electrical stimulation of the vHip, we have demonstrated
the brevity (10 s) and specificity (20 Hz vs. 2 Hz) by which aberrant activity in the vHip
results in disrupted PPI. Clearly, the mechanism by which vHip stimulation disrupts PPI
must significantly outlast the duration of stimulation. Analysis of the startle amplitude
data revealed that no significant changes in startle occurred following 20 Hz stimulation
of the vHip. Inspection of the startle amplitude data following NMDA infusion into the
vHip suggests that startle amplitude is typically either unchanged (Wan et al.,
1996;Klarner et al., 1998;Bast et al., 2001) or reduced (Zhang et al.,
1999;2002a;Swerdlow et al., 2001b) following chemical stimulation of the vHip. Given
the inconsistent changes in startie amplitude relative to consistent disruption of PPI
56

following stimulation of the vHip, it is unlikely that the reduction in PPI observed here is
due to changes in startle amplitude.
The role of the hippocampus in the modulation of PPI has also been assessed with
a variety of other manipulations. Permanent lesions of either the vHip or dHip do not
affect PPI (Swerdlow et al., 1995;2000b;Pouzet et al., 1999 but see also Caine et al.,
2001), although lesions of the vHip increase the sensitivity of rats to apomorphine-
induced disruption of PPI (Swerdlow et al., 1995;2000b). Interestingly, temporary
inactivation of either the vHip or dHip reversibly disrupts PPI (Zhang et al., 2002b).
Data regarding modulation of PPI by the dHip are inconsistent and difficult to interpret.
Infusion of the noncompetitive NMDA antagonist MK-801 into the vHip does not affect
PPI (Bakshi and Geyer, 1998), whereas MK-801 infusions into the dHip may (Bakshi and
Geyer, 1998;1999) or may not (Bast et al., 2000;Zhang et al., 2000b) alter PPI
responding. In summary, these previous data demonstrate a subtle role for the vHip in
the modulation of PPI under normal conditions. However, the present 20 Hz stimulation
data, and those of others groups using NMDA (Wan et al., 1996;Zhang et al.,
1999;2002a;Bast et al., 2001;Swerdlow et al., 2001b), indicate that overactivity in the
vHip can cause a potent reduction in PPI. Although the dHip may also modulate PPI
under certain conditions, data from this study and others (Swerdlow et al., 2001b;Zhang
et al., 2002a) clearly indicate that induced overactivity of this structure does not
significantly disrupt PPI.
Frequency Dependence of the vHip Stimulation-induced Disruption of PPI
Two Hz stimulation of the vHip, which causes a significant reduction in NAc DA
efflux that may be mediated by the activation of group 2/3 mGluR's (Taepavarapruk et
57

al., 1998), failed to significantly affect PPI in this study (Fig. 2-2). Available
pharmacological evidence is consistent with the observed lack of effect on PPI following
2 Hz vHip stimulation. For instance, blockade of DA receptors with DA antagonists,
such as haloperidol, does not alter levels of PPI in rats (Mansbach et al., 1988;Depoortere
et al., 1997;Hart et al., 1998;Feifel and Priebe, 1999). Additionally, systemic
administration of the group 2/3 mGluR agonist LY354740 has no effect on PPI levels
(Schreiber et al., 2000). Thus, pharmacological treatments that mimic some of the effects
of 2 Hz vHip stimulation are also without effect on PPI responding.
Unexpectedly, 2 Hz stimulation of the vHip resulted in a significant increase in
startle amplitude immediately following the stimulation when compared to the stim+48
and control+48 conditions. Treatment with group 2/3 mGluR agonists or haloperidol
decreased or had no effect on startle magnitude respectively (Mansbach et al.,
1988;Depoortere et al., 1997;Hart et al., 1998;Feifel and Priebe, 1999;Schreiber et al.,
2000). Given that these drugs were administered systemically, it is possible that their
effects may result from actions in brain areas other than those affected by 2 Hz
stimulation. However, it is interesting that stimulation of the vHip with two different
frequencies had different effects on both startle and PPI. Given the inconsistencies in
startle changes observed following NMDA stimulation (Wan et al., 1996;Zhang et al.,
1999;2002a;Legault et al., 2000;Taepavarapruk et al., 2000;Bast et al., 2001;Swerdlow et
al., 2001b), it will be important to replicate these findings in separate groups and/or
strains of rats to ensure their reliability.
58

Increases in NAc DA Efflux following Hip Stimulation are Site and Hemisphere Specific
Stimulation of the vHip with either NMDA or brief periods of 20 Hz electrical
stimulation produces a long-lasting increase in NAc DA efflux (Legault et al.,
2000;Taepavarapruk et al., 2000). The effect of 20 Hz stimulation is mediated by
iGluR's in the NAc, thereby implicating the direct glutamatergic projection from the
vHip to NAc in this effect (Blaha et al, 1997;Taepavarapruk et al., 2000). The present
neurochemical experiments (Fig. 3) extend our previous findings, as the increase in NAc
DA efflux following 20 Hz electrical stimulation of the vHip was restricted to the
hemisphere ipsilateral to the stimulating electrode. This finding is consistent with the
fact that the vHip projections to forebrain areas such as the NAc are primarily unilateral
(Kelley and Domesick, 1982;Groenewegen et al, 1987;Brog et al., 1993).
In contrast to stimulation of the vHip, 20 Hz stimulation of the dHip failed to
increase NAc DA efflux significantly. Although the dHip sends glutamatergic efferents
to the NAc, it is important to note that the vHip projects primarily to the NAc shell
whereas the dHip projects primarily to the NAc core (Swanson and Cowan, 1977;Kelley
and Domesick, 1982;Whitter, 1986;Groenewegen et al., 1987;Brog et al., 1993). Probe
placements adjacent to both the shell and core subregions of the NAc were employed in
studies in which stimulation of the vHip increased NAc DA efflux (Fig. 2-4C); therefore,
we monitored DA efflux in a similar region of the NAc following stimulation of the
dHip. This raises the possibility that any effect of the dHip stimulation on NAc DA
efflux was restricted to the NAc core. However, unpublished observations failed to
confirm an increase in NAc DA efflux with dialysis probes located in the core of the NAc
(Howland, Taepavararpuk, and Phillips).
59

Microdialysis experiments have examined dynamic changes in NAc DA efflux
during PPI test sessions. Zhang and colleagues (Zhang et al., 2000a) demonstrated that
systemic administration of amphetamine disrupted PPI, whereas treatment with cocaine
had no effect (but also see Martinez et al., 1999). Interestingly, both drugs increased
NAc DA efflux by at least 100 % of baseline during the PPI test sessions. Consequently,
these authors suggest that the behavioral effects of DA agonists on PPI are critically
dependent on their mechanism of action. Other experiments have shown that
presentation of startle pulses alone cause a significant decrease in NAc DA efflux that is
not observed when prepulses of a moderate intensity are presented prior to the startle
pulses (Humby et al., 1996). These data suggest that during PPI, the presentation of the
prepulses prevents perturbation of the mesoaccumbens DA system in response to the
startling stimuli. Together, these data imply that certain drugs, such as amphetamine, can
interfere with the effect of a prepulse on DA efflux in the NAc, thereby disrupting PPI.
Given these data, it is intriguing that two different methods of stimulating the
vHip, both of which increase DA efflux in the NAc, can disrupt PPI. The fact that 20 Hz
stimulation of the dHip or 2 Hz stimulation of the vHip failed to disrupt PPI and did not
increase NAc DA efflux is consistent with the hypothesis that increased NAc DA efflux
following 20 Hz vHip stimulation may be a factor in the disruption of PPI. However,
systemic administration of the D 2 antagonist haloperidol (Wan et al., 1996;Zhang et al.,
1999;Bast et al., 2001) or intra-NAc infusions of the non-NMDA receptor antagonist
CNQX (Wan et al., 1996) fail to block the disruptive effects of intra-vHip NMDA
infusion on PPI responding. Therefore, it is unlikely that the disruptive effects of
60

stimulation of the vHip on PPI are mediated solely by activation of the mesoaccumbens
DA system or non-NMDA receptors in the NAc (Wan et al., 1996;Bast et al., 2001).
Potential Mechanisms Underlying vHip Stimulation-induced Disruption in PPI
Normal DA and Glu transmission appears to be important for PPI (Zhang et al.,
2000a;Geyer et al., 2001). For example, infusions of DA or DA agonists into the NAc
disrupt PPI (Mansbach et al., 1988;Swerdlow et al., 1990;Wan et al., 1995), and infusions
of Glu agonists and antagonists such as AMPA, APV, CNQX, and MK-801 into the NAc
have complex and regionally specific effects (Reijmers et al., 1995;Wan et al., 1995;Wan
and Swerdlow, 1996). Recent physiological experiments raise the possibility that the
effects of vHip stimulation on PPI may involve interactions between multiple receptor
subtypes in the NAc. Stimulation of the vHip shifts NAc neurons to an activated or 'up'
state (O'Donnell and Grace, 1995) and recent experiments from our laboratory indicate
that 20 Hz stimulation (10 s) of the vHip alters the responses of medium spiny neurons in
the NAc shell to afferent input from the vHip and BLA (Floresco et al., 2001). These
effects are dependent on the 20 Hz stimulation-induced increase in NAc DA and a
complex pharmacology involving activation of both D t and NMDA receptors, but not D 2
and non-NMDA receptors (Floresco et al., 2001). Importantly, the potentiating effect of
20 Hz stimulation of the vHip on NAc neurons is maintained for at least 20 min, which
corresponds to the duration of PPI trials in the present study.
Stimulation of the vHip may also disrupt PPI via the widespread connections of
the vHip to a number of areas in the CSPP circuitry critical for normal PPI (Klarner et al.,
1998). Data obtained in the present study do not refute this hypothesis. The vHip is
reciprocally connected to a number of areas other than the NAc, such as the mPFC,
61

amygdala, thalamus, septum, and ventral pallidum (Groenewegen et al, 1987;Amaral and
Whitter, 1995;Conde et al., 1995), all of which are important for the regulation of PPI
(Swerdlow et al., 2001a). Stimulation of the vHip with doses of NMDA that disrupt PPI
cause increased fos-protein expression in a number of limbic and cortical areas such as
the NAc, lateral septum, mPFC, and dorsomedial striatum (Klarner et al., 1998).
Stimulation of the vHip also induces various types of plasticity in the mPFC such as
long-term potentiation and depression (Laroche et al., 2000). Therefore, subtle changes
in multiple areas induced by vHip stimulation may disrupt PPI (Klarner et al., 1998).
Although the dHip projects to the NAc core, it is not directly connected to other areas
implicated in PPI (Swanson and Cowan, 1977;Kelley and Domesick, 1982;Whitter,
1986;Amaral and Whitter, 1995), and therefore aberrant activity generated in the dHip
may not have access to the neural substrates critical to disrupt PPI. This conjecture is
consistent with the failure of 20 Hz stimulation of the dHip to increase NAc DA efflux in
the NAc or to disrupt PPI in the present study.
Conclusion
Our data clearly demonstrate that brief, higher frequency stimulation of vHip, but
not dHip, disrupts PPI and increases DA efflux in the NAc. Given the anatomical and
functional abnormalities frequently observed in the hippocampal formation of patients
with schizophrenia, these data support the assertion that abnormalities in limbic-cortico-
striatal interactions may underlie deficits in sensorimotor gating in schizophrenia.
Identification of the mechanism underlying the disruptive effects of vHip stimulation on
PPI may provide insight into potential targets for pharmacological treatment strategies for
schizophrenia.
62

\
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69

CHAPTER THREE: DELAYED ONSET OF PREPULSE INHIBITION DEFICITS
FOLLOWING KAINIC ACID TREATMENT ON POSTNATAL DAY SEVEN IN
RATS. 2
Introduction
Given the heterogeneity and complexity of schizophrenia, its etiology has been
difficult to define. The neurodevelopmental hypothesis of schizophrenia proposes that
adverse events or genetic abnormalities may disrupt early brain development, and interact
with environmental factors or influence subsequent brain development to cause the
disorder (Weinberger, 1995;Benes et al., 2000;McClure and Weinberger, 2001). Once
the illness is expressed, a diverse array of subde changes are found in temporal and
frontal brain areas such as the hippocampus, striatum, thalamus, and frontal cortex
(Pakkenberg, 1990;Suddath et al., 1990;Bogerts, 1997;Jones, 1997;Heckers etal.,
1998;Harrison, 1999;Gothelf et al., 2000;McClure and Weinberger, 2001;Meyer-
Lindenberg et al., 2002). Neurodegeneration in these areas is rarely observed in the
brains of patients with schizophrenia (but see Stevens, 1994), therefore alterations in
normal patterns of connectivity between affected brain areas may underlie the expression
of some symptoms of schizophrenia (Harrison, 1999;Friston, 1999;Benes, 2000;Penn,
2001).
Accordingly, the consequences of disrupting the normal development of limbic
and cortical areas on behaviors in rodents related to schizophrenia have been examined
(for reviews see Lipska and Weinberger, 2000;Van den et al., 2003). Specific behavioral
abnormalities have been observed in adult, but not prepubescent rats, that received
2
A version of this chapter has been published: Howland JG, Hannesson DK, Phillips AG (2004). Delayed
onset of prepulse inhibition deficits following kainic acid treatment on postnatal day seven in rats.
European Journal of Neuroscience 20:2639-2648.
70

ventral hippocampal (vHip) lesions on postnatal day (PND) 7 (Lipska and Weinberger,
2000). Behavioral changes in prepulse inhibition (PPI) and locomotor responses to
novelty, which resemble certain positive symptoms in schizophrenia, are robust and can
be exacerbated by acute administration of dopamine (DA) agonists immediately before
testing in adulthood (Lipska et al., 1993; 1995;Le Pen and Moreau, 2002). Importantly,
temporary reduction of neural activity within the vHip on PND7, with the sodium
channel blocker tetrodotoxin (TTX) increased locomotor activity in response to novelty,
injection stress, and amphetamine after puberty (Lipska et al., 2002). Similar results have
been observed following neonatal lesions of the amygdala (Daenen et al., 2001; 2002;
2003), but not the medial prefrontal cortex (Lipska et al., 1998;Brake et al., 2000;Van
den et al., 2003 but see Flores et al., 1996). Additionally, systemic administration of the
non-competitive NMDA receptor antagonist dizocilpine (MK-801) on PND7 can reduce
PPI levels and increase locomotor activity in female rats (Harris et al., 2003; but see
Beninger et al., 2002).
The apparent sensitivity of limbic-cortical circuits on PND7 to functional
disruption raises the possibility that increased neural activity induced by the ionotropic
glutamate agonist kainic acid (KA) at this developmental age would result in behavioral
changes in rats similar to those discussed above. Kainic acid has been used extensively
to study limbic-motor seizures and excitotoxicity in adult rats (for reviews see Sperk,
1994;Ben Ari and Cossart, 2000); however, its effects during early development are not
as well characterized. During the first two postnatal weeks, systemic administration of
KA results in tonic-clonic seizures and the selective activation of the hippocampus and
lateral septum without the high levels of cell death normally observed in limbic and
71

cortical areas following administration during or after the 3 r
postnatal week (Nitecka et
al., 1984;Tremblay et al., 1984;Stafstrom et al., 1992;Khalilov et al., 1999;Koh et al.,
1999;Lynch et al., 2000;Silveira et al., 2002). Although few studies have analyzed
behavioral changes in adulthood following a single administration of KA during the first
week of life, there are reports of deficits in conditioned avoidance (de Feo et al., 1986)
and spatial learning using the radial arm maze (Lynch et al., 2000), but not the water
maze (Stafstrom et al., 1993;Koh et al., 1999).
The present study sought to examine whether systemic KA administration on
PND7 would alter the behavior of rats in a pattern similar to that observed in other animal
models of schizophrenia (Lipska et al., 1993;Harris et al., 2003). Kainic acid or saline
was administered to rat pups on PND7 and their PPI levels and locomotor responses to a
novel environment were measured before and after puberty. To assess potential
alterations of the DA system following KA administration, we tested the potential
interactive effects of KA treatment and apomorphine, a direct DA agonist, on PPI and
also with amphetamine-induced locomotion. Subgroups of KA- and saline-treated rats
were also tested on a standard hippocampal-dependent Morris Water Maze task in
adulthood to examine aspects of spatial learning and memory that are dependent on
normal hippocampal function (Morris et al., 1990).
Methods
Subjects
Two independent groups of animals (i.e. Group 1 and Group 2) were tested in this
study. Testing protocols for both groups were similar with small procedural differences
noted in the methods section where appropriate. Pregnant Long-Evans rats were obtained
72

from Charles River (Quebec, Canada) at 13 to 15 days of gestation. They were singly
housed and left undisturbed until giving birth. The colony was maintained on a 12/12
hour light/dark cycle (lights on at 0700), at a temperature of 22+1 °C. All rats were given
food (Purina Rat Chow) and tap water ad libitum. Experiments were conducted in
accordance with the standards of the Canadian Council on Animal Care and were
approved by the Committee on Animal Care at the University of British Columbia.
Kainic Acid Administration
The day of birth of the pups was designated post-natal day (PND) 0. On PND3,
the litters were sexed and culled to include only males (6-9 rats per litter). On PND7, all
but one of the pups were removed from the nest, weighed and individually placed in
small compartments of a cardboard box for KA administration. They were then removed
from the colony and taken to a small heated room. KA (1.5 mg/kg, Tocris) or saline was
injected (i.p.) with a 30-gauge needle (10 ml/kg). Care was taken to ensure that both KA
and saline was administered to members from each litter. Previous reports indicated that
KA administration of 1-2 mg/kg on PND7 causes behavioral seizures in most pups
without high mortality rates (Stafstrom et al., 1993;Lynch et al., 2000). The behavior of
all rats was recorded with an overhead video camera for 180 minutes. Before being
returned to their mothers, the pups were earmarked according to treatment condition.
The litters were then left undisturbed (except for normal cage changing) until weaning on
PND25. Weanling rats were housed in cages of 2 or 3 with members of their litter. All
rats were handled for at least 5 days before behavioral testing.

Prepulse Inhibition
On PND35 and 56, rats were removed individually from the colony and taken
immediately to the PPI apparatus. Testing was conducted in a single sound-attenuating
startle chamber (ambient noise level 64 dB), containing a transparent Plexiglas tube (8.2
cm in diameter, 20 cm in length), mounted on a Plexiglas frame (SR-LAB, San Diego
Instruments, San Diego). Noise bursts were presented through a speaker mounted 24-cm
above the tube. An accelerometer below the frame of the apparatus measured whole
body startle amplitude, defined as the average of 100 1-ms accelerometer readings
collected from stimulus onset. Each PPI test session began with a 5-min acclimatization
period during which a 70-dB background noise level was presented, which remained
constant for the entire test session. Following the acclimatization period, six pulse alone
trials (120 dB, 40 ms) were presented to achieve a relatively stable startle amplitude
before PPI testing. Data from these pulse-alone trials was not considered in the analysis
of PPI. Immediately following the six initial pulse-alone trials, presentation of the trials
that were used in the calculation of PPI levels were initiated. Trials presented were of
three types: pulse alone (12 trials, 120 dB, 40 ms), prepulse + pulse (10 trials X 3
prepulse intensities - discussed below), or no stimulus (42 trials). Prepulse + pulse trials
consisted of the presentation of a 20 ms prepulse of 73, 76, or 82 dB 80 ms before the
presentation of the pulse. The pulse and prepulse + pulse trials were presented in a
pseudorandom order. One no-stimulus trial was presented between each pair of pulse and
prepulse + pulse trials. The inter-trial interval varied randomly from 3 to 12 s (average
7.5 s). Calibration of the apparatus was performed using a RadioShack Digital Sound
Level Meter and adjustments were made as necessary.
74

Between PND98 and 105, a randomly selected subset of the rats from Group 2
were re-tested on PPI following injection with vehicle (0.1% ascorbic acid) and
apomorphine (Sigma; 0.2 mg/kg; s.e.) 72 hr later to assess: 1) the consistency of the PPI
reduction in KA-treated animals and 2) the potential role of dopaminergic mechanisms in
the observed PPI decreases in the KA-treated animals. The PPI sessions were conducted
in a manner identical to that described above, except that immediately before the PPI
session, all rats were weighed and injected with the appropriate volume of vehicle or
drug.
Locomotor Activity
On PND36 and 57, rats were weighed, removed from the colony, and
immediately placed in locomotor boxes. For Group 1, the locomotor boxes were in two
similar, small adjacent rooms (4 boxes in one, 3 in the other) constructed from clear
Plexiglas (32 cm X 32 cm X height 41 cm) and fitted with four pairs of infra-red beams
positioned 10 cm apart and 2.5 cm above a metal grid floor. All sensors were interfaced
to a computer-controlled system (MANX). Motor activity counts were collected in 10-
min bins during testing. Care was taken to ensure that rats from each group were tested
in both rooms and in all boxes. Spontaneous locomotor activity levels in a novel
environment were measured for 60 min. Immediately following the spontaneous
locomotor activity test, all rats were injected with D-amphetamine (1.5 mg/kg, i.p.) and
returned to the locomotor boxes. Locomotor activity was then monitored for an
additional 90 min before the rats were returned to the colony room. For Group 2, testing
procedures were identical, except that a Med Associates System was used to collect the
data. Eight Med Associates Test Chambers (ENV-008; 30.5 cm X 24.1cm X height 21.0
75

cm) were fitted with 4 pairs of infrared photocells 3.5 cm from the floor evenly spaced on
the long walls. The chambers had metal grid floors and two operant levers (which were
retracted during locomotor activity testing). Each chamber was contained within a Med
Associates sound attenuating cubicle with a house light that was illuminated during
testing.
Water Maze Testing
A subgroup of the animals from Group 1 was tested between PND65 and 75 in a
water maze. Testing was performed in a circular, white swimming pool 180 cm in
diameter, 54 cm in height. The escape platform was 34 cm in height and 14 cm in
diameter. The pool was filled with water such that the top of the platform was 3 cm
below the surface of the water. For visible platform trials, an additional piece of wood
and wire mesh was secured to the top of the invisible platform such that it extended 3 cm
out of the water. The water was rendered opaque by the addition of white Tempura
Powder paint. Data were recorded using a computerized tracking system (HVS Image,
Hampton, England). Rats were transported to the pool room in groups and testing was
conducted over 3 consecutive days. Rats were released facing the wall of the pool from
one of the geographic poles in a pseudorandom order. Each rat was allowed 60 s to swim
to the platform, after which they were left on the platform for 10 s. They were then
removed from the water maze, and the next rat was tested. The intertrial interval for each
rat was approximately 7 min. On the first day, rats were trained to swim to the visible
platform over 5 trials to allow habituation to the testing environment and swimming. The
location of the platform was varied for each trial, and was never in the location of the
hidden platform used on day 2. Hidden platform training was performed on day 2. Rats
76

were given 10 trials to learn to find the constant location of the hidden platform. Finally,
on day 3, the rats were tested in a 30-s probe trial without the platform present. The
percent time spent in the quadrant that contained the hidden platform on day 2 was taken
as a measure of spatial memory.
Histology
Following behavioral testing, a subgroup of animals (n=5 from each group) was
randomly selected, sacrificed with an overdose of pentobarbital, and perfused
transcardially with 0.9% saline followed by 10% formalin. Brains were stored in 10%
sucrose/10% formalin for at least 1 week, after which coronal sections (40 u.m)
containing the hippocampus were taken using a vibratome. Every fourth section was
saved and stained with cresyl violet. An experimenter blind to the treatment group
examined each section using a light microscope for gross cell loss in the hippocampus
with particular attention paid to the CA3 region due to its established sensitivity to the
excitotoxic effects of KA in adult rats (Ben Ari and Cossart, 2000).
Data Analysis
For the PPI experiments, two measures were calculated for each animal. The
startle amplitude represented the mean startle amplitude of the 12 pulse-alone trials
presented after the 6 habituation trials. Startle amplitude data were compared with an
independent samples t-test for each age. PPI was calculated by averaging startle
amplitudes for each trial type. The percent PPI for each prepulse intensity was calculated
using the formula: [100 - (100 X startle amplitude on prepulse + pulse trials) -s- (startle
amplitude on pulse alone trials)]. A repeated measures ANOVA was performed on the
data obtained from each age with prepulse intensity as a within subjects factor and
77

treatment on PND7 as a between subjects factor. Results from the apomorphine
experiment were also analyzed with a mixed ANOVA (prepulse intensity and test as
within subjects factors, and treatment at PND7 as a between subjects factor). Locomotor
activity data were compared using repeated measures ANOVA at each age with test type
as a within subjects factor and treatment on PND7 as a between subjects factor. For the
water maze task, trials were blocked into pairs to reduce variance; latencies and path
lengths to find the hidden platform were analyzed using a repeated measures ANOVA
(trial block as a within subjects factor, treatment on PND7 as a between subjects factor).
Additionally, swim speeds were measured for all rats and compared using a repeated
measures ANOVA. After the probe trials, the percent time spent in each quadrant was
calculated by dividing the time spent in that quadrant by the total length of the probe trial
(30 s). A repeated measures ANOVA was performed on the data with quadrant as a
within subjects factor and treatment on PND7 as a between subjects factor. For all
ANOVA's, post-hoc analyses were performed using the Neuman-Keuls test where
appropriate. The significance level for all statistical tests was p < 0.05.
Results
Immediate Behavioral Effects ofPND7 KA Administration
Ten to 20 min following KA administration, the majority of pups began to show
behavioral signs of seizure activity as previously reported (Tremblay et al.,
1984;Stafstrom et al, 1992;Lynch et al., 2000;Silveira et al, 2002). Initially, the
majority of the pups showed evidence of a hunched posture and heavy breathing followed
by the appearance of a continuous, clonic 'scratching' activity of the rear limbs. Many of
the rats developed swimming seizures during which they rolled onto their sides during
78

outs of forelimb and hindlimb clonus, which continued for 30 to 120 min. Some of the
pups developed more severe tonic seizures in the second hour after KA administration,
which lasted up to 60 min. Only data from pups that displayed behavioral signs of
seizure activity for at least 30 minutes were analyzed in these experiments, and no
differences in seizure severity between pups from Group 1 and Group 2 could be readily
quantified. Six pups (11%) died as a result of the KA administration, while 10 (18%)
were not used for the behavioral experiments due to mild, intermittent seizures. None of
the saline-treated animals displayed seizure-related behaviors.
Effects ofPND7 KA Administration on Body Weight
On PND7, pups randomly selected for KA administration weighed 18.13 ± 0.37 g
while those administered saline weighed 17.86 ± 0.46 g. On PND8, the pups were
weighed again to ensure that suckling behavior was not altered between the groups after
treatment on PND7. The KA- and saline-treated rats weighted 20.45 ± 0.45 g and 20.77
± 0.55 g on PND8 respectively. An independent samples t-test revealed that both groups
of animals gained a similar amount of weight during the 24 hr after treatment (t(50) = -
1.28, N.S.). On PND36, the rats treated with KA on PND7 weighed slightly more than
those treated with saline (KA: 181.90 ± 2 g; saline: 175.90 ± 2 g); however, when tested
on PND57, the rats treated with KA weighed significantly more than those treated with
saline (KA: 395.33 ± 5 g; saline: 379.66 ± 5 g) as revealed by a significant age by weight
interaction (F(l, 68) = 3.98, p = 0.05).
79

70-,
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Figure 3-1. Prepulse inhibition (PPI) scores from rats in Group 1 treated on postnatal day
(PND) 7 with saline (white bars, n=17) or kainic acid (KA; black bars, n=20). A, Effects
of.PND 7 KA treatment on PPI at PND35. B, Average starde amplitudes of the rats on
PND35. C, Percent PPI scores for those rats tested at PND56. D, Average startle
amplitudes of the rats on PND56. Asterisks indicate a significant difference between
groups (p

Prepulse Inhibition is Reduced in Post-Pubescent, but not Pre-pubescent Rats, Following
PND7 KA Administration
Group 1: As shown in Fig. 3-1 A, rats administered either KA (n=20) or saline
(n=17) on PND7 had similar levels of PPI on PND35. Percent PPI scores were similar in
response to trials with 3 and 6 dB prepulses, whereas PPI was substantially increased for
trials during which a 12 dB prepulse was presented.' A repeated measures ANOVA
revealed a significant effect of prepulse (F(2, 70) = 78.92, p< 0.001), but no significant
prepulse by treatment interaction (F(2, 70) = 0.26, N.S.) or main effect of treatment (F(l,
35) = 2.04, N.S.). On PND35, rats in the KA-treated group demonstrated slightly lower
starde amplitudes than those in the saline treated group to the pulse alone trials (55.27 ± 5
vs. 67.51 ± 7 arbitrary startle units) groups (Fig. 3-1B). However, this difference was not
significant (t(35) = -1.41, N.S.).
In contrast to PND35, PPI levels obtained from KA- and saline-treated rats in
young adulthood (PND56) were significantly different (Fig. 3-1C). A repeated measures
ANOVA revealed a significant effect of prepulse intensity (F(2, 70) = 33.05, p < 0.001),
as well as a significant prepulse by treatment interaction (F(2, 70) = 5.57, p < 0.01). Rats
in the saline-treated condition showed a typical pattern of increasing percent PPI in
response to prepulses of increasing intensity (24.80 ± 5% for PP3, 41.77 ± 4% for PP6,
and 54.38 ± 4% for PP12 trials). When compared to the saline-treated animals, KA-
treated rats responded with significantly lower percent PPI scores for the PP6 trials
(19.17 ± 6% PPI, p < 0.05), and attenuated PPI in response to PP12 trials (47.15 ± 4%).
As a result, animals in the KA-treated group demonstrated a 25% reduction in average
percent PPI scores when compared with the saline-treated group (KA-treated = 30.18 ±
81

3%; saline-treated = 40.32 ± 4%), which approached significance (F(l, 35) = 3.93, p =
0.055). The average startle amplitude of the KA-treated rats (77.20 ± 12 arbitrary startle
units) was lower than that of the saline-treated group (102.11 ± 18), although this
difference was not significant (t(35) = -1.17, N.S.).
Group 2: As in Group 1, rats in both the KA- (n=19) and saline- (n=14) treated
groups tested on PND35 had similar levels of PPI (Fig. 3-2A). A repeated measures
ANOVA revealed a significant main effect of prepulse intensity (F(2, 62) = 28.62, p

£-•30
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Figure 3-2. Prepulse inhibition (PPI) scores from rats in Group 2 treated on postnatal day
(PND) 7 with saline (white bars, n=14) or kainic acid (black bars, n=19). A, Effects of
postnatal day (PND) 7 KA treatment on PPI at PND35. B, Average startle amplitudes of
the rats on PND35. C, Percent PPI scores for those rats tested at PND56. D, Average
startle amplitudes of the rats on PND56. The asterisk indicates a significant difference
between groups (p

shown). Additionally, a significant main effect of test (F(l, 16) = 16.94, p < 0.005) and a
test by prepulse interaction (F(2,32) = 4.82, p < 0.02) were observed. Inspection of the
data confirmed that apomorphine treatment significantly disrupted PPI in both groups of
rats (Fig. 3-3A) while post-hoc analyses revealed that apomorphine disrupted PPI
significantly more during the 6 dB prepulse trials than during either the 3 or 12 dB trials
(p < 0.05, data not shown).
Importantly, the absence of a significant test by treatment interaction (F(l,16) =
0.09, N.S.) does not support the hypothesis that animals treated with KA on PND7 are
more sensitive to the disruptive effects of low doses of apomorphine on PPI. A separate
analysis of the data obtained following the vehicle injection revealed that those animals
treated with KA (n=10) on PND7 had lower PPI scores (38.36 ± 4%) than those treated
with saline (n=8; 50.00 ± 6%, Fig. 3-3 A). Although this difference was of a similar
magnitude to the differences in PPI reported at PND56 for groups 1 and 2 above, it failed
to reach significance perhaps due to insufficient power (t(16) = -1.65, p = 0.12).
Apomorphine treatment significantly decreased startle amplitude in both groups (Fig. 3-
3B). This is reflected by a significant main effect of test (F(l,16) = 6.15, p < 0.05), but
not group (F(l, 16) = 0.13, N.S.) or a group by test interaction (F(l, 16) = 0.07, N.S.).
Locomotor Activity in Response to Novelty and Amphetamine in Rats Following PND7
KA Administration
Group 1: As depicted in Fig. 3-4A and B, KA- (n=20) and saline- (n=17) treated
rats in this group showed similar levels of spontaneous locomotor activity in a novel
environment (60 min) or following amphetamine administration (1.5 mg/kg, 90 min) on
84

S-V K-V S-A K-A
Treatment
S-V K-V S-A K-A
VTreatment
Figure 3-3. A, Effects of pretreatment with vehicle (0.1% ascorbic acid, A) or
apomorphine (0.2 mg/kg, B) on average percent PPI scores in adulthood. Animals were
administered either saline (n=8, white bars) or kainic acid (n=10, black bars) on postnatal
day 7. B, Average startle amplitudes of those rats depicted in panel A. In both panels,
asterisks indicate a significant difference between groups. The cross indicates a between
group difference that approached significance (p

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Figure 3-4. Locomotor activity levels in response to novelty (A, C) or amphetamine (1.5
mg/kg; B, D) of all rats tested on either postnatal day (PND) 36 (A, B) or PND57 (C, D).
Group labels on the x-axis of each panel reflect the treatment of the rats on PND7 (5 =
saline, K - kainic acid) and their group (1 = group 1,2 = group 2). The asterisk indicates
a significant difference between the denoted groups (p

PND36. A repeated measures ANOVA failed to confirm a significant treatment effect
(F(l, 35) = 0.16, N.S.) or treatment by test interaction (F(l, 35) = 0.12, N.S.). At PND57,
a repeated measures ANOVA did not reveal a main effect of treatment (F(l, 35) = 2.01,
N.S.), but did reveal a significant test by treatment interaction (F(l, 35) = 4.10, p = 0.05).
Post-hoc analyses revealed that although both groups showed similar locomotor
responses in the novelty condition (Fig. 3-4C), rats treated with KA on PND7 had
significantly higher locomotor responses (2640.25 ± 225 beam crosses) following
injection with amphetamine than rats treated with saline (2145.06 ± 143; Fig. 3-4D).
Group 2: Similar to the data presented for Group 1, the locomotor responses of
rats treated with either KA (n=19) or saline (n=14) on PND7 did not differ in either the
spontaneous activity or amphetamine conditions on PND36 (Fig. 3-4A and B). This is
reflected in an insignificant main effect of treatment (F(l, 31) = 1.30, N.S.) and treatment
by test interaction (F(l, 31) = 2.29, N.S.). In contrast to the data obtained from Group 1,
when tested on PND57, rats in Group 2 failed to show significant differences in
locomotor activity in either condition (main effect of treatment: F(l, 31) = 1.70, N.S.;
treatment by test interaction: F(l, 31) = 1.66, N.S.).
Spatial Learning and Memory in the Morris Water Maze is not Altered in Rats Following
PND7 KA Administration
On the first test day, all rats learned to escape by swimming to the visible
platform (data not shown). On the second day of testing, rats in both groups learnt to
swim to the hidden platform (Fig. 3-5A). A repeated measures ANOVA performed on
the latency data revealed a significant main effect of trial block (F(4, 52) = 17.43, p <
0.001), but not a significant effect of group (F(l, 13) = 0.279, N.S.) or a significant block
87

E
F
50
40
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20
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1 • ' / .2 . . 3 ;
Quadrant
Figure 3-5. A, Average latencies (s) to locate the hidden platform in the water maze of
rats treated with saline (white diamonds, n=7) or kainic acid (black squares, n=8) on
postnatal day 7. Trial blocks represent the average search time of 2 trials for each rat.
The asterisk indicates a significant difference between trial blocks (p

y group interaction (F(4, 52) = 0.487, N.S.). Post-hoc analyses revealed that both the
KA- (n=8) and saline-treated (n=7) rats were significantly slower at finding the platform
during block 1 (i.e. trials 1 and 2) than the subsequent 4 blocks (average search time -
block 1: 38.66 ± 6 s; blocks 2 to 5: 14.38 ± 3 s; p < 0.05). Path lengths were significantly
reduced across trial blocks in both groups (F(4, 52) = 14.33, p < 0.01; data not shown);
however, neither total path length (KA-treated = 4,029.10 ± 483 cm, saline-treated =
4597.04 ± 609 cm; F(l, 13) = 1.56, N.S.) nor swim speed (KA-treated = 22.33 ± 1 cm/s,
saline-treated = 25.14 ± 1 cm/s; F(l, 13) = 3.21, N.S.) differed significandy between the
groups. When retention of the platform location was tested 24 hr later using a 30 s probe
trial, both groups spent most of their time searching in the quadrant which had contained
the platform the previous day (Fig. 3-5B). A repeated measures ANOVA revealed a
significant main effect of quadrant (F(3, 39) = 20.88, p < 0.001), but no significant group
by quadrant interaction (F(3, 39) = 0.581, N.S.). Post-hoc analyses revealed that both
groups spent significandy more time in quadrant 2 (41.65 ± 5 %), which contained the
platform, than in either quadrants 3 (19.22 ± 4 %) or 4 (8.24 ± 3 %). Inspection of the
data presented in Fig. 3-5B reveals that the animals also spent more time in quadrant 2
than quadrant 1 (30.89 ± 3 %), although this difference failed to reach significance.
Histology
In agreement with previous studies (Nitecka et al., 1984;Stafstrom et al.,
1992;Koh et al., 1999;Lynch et al., 2000), histological examination of the hippocampi of
rats which had received either KA or saline on PND7 revealed no signs of gross cell loss
or hippocampal damage (Fig. 3-6).
89

Figure 3-6. A representative cresyl violet stained section of the dorsal hippocampus of a
KA-treated (A) and saline-treated rat (B).
90

Discussion
The present study demonstrates that treatment with KA on PND7 results in the
delayed emergence of behavioral changes in adulthood not seen before puberty.
Following treatment with KA on PND7, rats in two independent groups showed disrupted
PPI on PND56, but not PND35. When re-tested 6 weeks later, a subgroup of the KA-
treated animals continued to exhibit relatively lower PPI than saline-treated controls.
Additionally, administration of a low dose of apomorphine (0.2 mg/kg) disrupted PPI in
both KA- and saline-treated rats; however, KA-treated rats failed to show enhanced
sensitivity to the disruptive effects of apomorphine. When the locomotor responses of
these animals in response to a novel environment and amphetamine challenge were
analyzed, an inconsistent pattern emerged. KA- and saline-treated rats had similar
locomotor responses to both novelty and amphetamine on PND36, as well as novelty on
PND57. Following amphetamine administration (1.5 mg/kg) on PND57, locomotor
activity was significantly increased in the KA-treated rats from Group 1, but not from
Group 2. Furthermore, neither spatial learning nor memory were affected by the
administration of KA on PND7 as assessed in the Morris water-maze, confirming
previous studies (Stafstrom et al., 1993;Koh et al., 1999).
Effects of Neonatal KA Administration on PPI
In two separate groups of rats, we observed that KA treatment on PND7
significantly disrupted PPI in postpubescence, but not before puberty. It is important to
note that PPI has been reliably measured in rat pups as young as 16 days of age (Martinez
et al., 2000;Swerdlow et al., 2000), and that the rats tested in the present experiments on
PND35 showed consistent levels of PPI with relatively low variance. Post-pubescent rats
91

treated with KA on PND7 had mean PPI levels that were 75 to 80% of those shown by
saline-treated animals. This effect was observed consistently in two groups of animals
tested independendy (Fig. 3-1C and 3-2C) and was evident without changes in startle
amplitude (Fig. 3-1D and 3-2D). Previous studies have reported the effects of neonatal
manipulations on PPI in adult rats. Data presented by Lipska and colleagues (Lipska et
al, 1995; Fig. 4) from rats that had sustained permanent lesions of the vHip on PND7,
indicate PPI levels that were approximately 70% of their control littermates, although a
significant difference between groups was noted only at the lowest prepulse intensity (4
dB above baseline). Other studies report slightiy larger effects over a wider range of
prepulse intensities following neonatal lesions of the vHip (Le Pen and Moreau,
2002;Daenen et al., 2003) and the amygdala (Daenen et al., 2003) or treatment with MK-
801 on PND7 (Harris et al., 2003). Given that these treatments result in relatively subtle
disruptions in PPI under baseline testing conditions, it is perhaps not surprising that KA
treatment on PND7, which does not cause gross brain damage (Nitecka et al.,
1984;Stafstrom et al., 1992;Koh et al., 1999;Lynch et al, 2000; present data), causes
relatively small disruptions in PPI.
Rats with neonatal vHip lesions also show heightened sensitivity to the disruptive
effects of apomorphine (0.1 mg/kg), thereby supporting the hypothesis that mesolimbic
DA transmission is altered in the lesioned rats (Lipska et al., 1995). In the present study,
KA treatment on PND7 failed to enhance the disruptive effects of apomorphine treatment
on PPI. However, administration of 0.2 mg/kg of apomorphine immediately before
testing disrupted PPI in both the KA- and saline-treated animals (Fig. 3-3A). This result
is surprising because Long-Evans rats are reported to be less sensitive than Sprague
92

Dawley rats (the strain used by Lipska et al., 1995) to the disruptive effects of
apomorphine on PPI. Indeed, it has been demonstrated that Long-Evans fats fail to show
disrupted PPI following administration of doses of apomorphine as high as 0.5 mg/kg
(Swerdlow et al., 2001). Although no clear explanation exists for these discrepant data,
complex interactions between rat strain and PPI have been studied extensively (Varty and
Geyer, 1998;Ellenbroek and Cools, 2000;Swerdlow et al., 2000). In this context, further
examination of the effect of early KA-treatment's potentially interactive effects with
apomorphine may be profitable in other rat strains.
Using C57BL6 mice, Yee and colleagues (Yee et al., 2004a; 2004b) elegandy
demonstrated that pretreatment with dopamine agonists such as apomorphine or non­
competitive A/-methyl-D-aspartate antagonists, such as dizocilpine and phencyclidine,
disrupts PPI, a finding that has previously been well documented (Geyer et al., 2001).
However, when prepulse-elicited reactivity was examined, a dissociation between the two
classes of drugs was observed whereby apomorphine increases and dizocilpine and
phencyclidine decrease prepulse-elicited reactivity (Yee et al., 2004a; 2004b).
Incorporation of a prepulse alone condition in future experiments may allow for parallels
to be drawn between these pharmacological studies and KA treatment. For example, if
KA administration on PND7 decreased prepulse-elicited startle, it could be argued that
KA administration has effects similar to dizocilpine or phencyclidine pretreatment, but
not apomorphine pretreatment.
Effects of Neonatal KA on Locomotor Activity
Increased locomotor activity in response to treatments that activate the
mesolimbic DA system is a cardinal feature of many animal models of schizophrenia
93

(Lipska et al., 1993;Hall, 1998;Weiss et al., 2000;Lipska et al., 2002;Harris et al, 2003).
Neonatal KA treatment in the present study failed to increase locomotor behavior
consistently following exposure to a novel environment or an amphetamine injection,
although Group 1 did show a significant increase in locomotor activity following
treatment with amphetamine (Fig. 3-4D). It is unclear why KA-treated rats in Group 2
failed to show elevated locomotor responses following amphetamine treatment on
PND57 given that both groups displayed similar seizure-related behaviors following KA
administration on PND7 and similar deficits in PPI when tested at PND56. These results,
considered in conjunction with the failure of apomorphine to affect PPI, question the role
of postsynaptic alterations in the mesolimbic DA system in the observed effects of KA
treatment on PPI. Furthermore, the absence of consistent changes in locomotor behavior
in early adulthood following KA-induced aberrant activity in developing cortico-limbic
circuits on PND7, underscores important differences between these data and the
decreased activity induced by the TTX administration into the vHip (Lipska et al., 2002)
and systemic MK-801 administration (Harris et al., 2003).
Potential Mechanisms Underlying the Observed PPI Changes
TTX infusions into the vHip on PND7 potentiate locomotor activity in early
adulthood (Lipska et al., 2002) and systemic administration of the NMDA antagonist
MK-801 on PND7 may also increase locomotor activity and disrupt PPI (Harris et al.,
2003, but see Beninger et al., 2002). Lipska and colleagues failed to detect any gross
morphological changes in the adult brains of animals injected with TTX on PND7
(Lipska et al., 2002). In contrast, administration of MK-801 on PND7 increases
apoptosis in limbic and cortical brain areas such as the hippocampus and thalamus
94

(Harris et al., 2003), and this effect may underlie the observed behavioral changes in
adult animals. Taken together, these data suggest that neural circuits subserving
locomotor behavior and PPI are particularly sensitive to alterations in neural activity
during the first postnatal week.
All rats included in this study displayed generalized tonic-clonic seizures for 1 to
2 hr following KA treatment on PND7, therefore it is reasonable to assume that aberrant
activity would have occurred in neural circuits that regulate PPI and locomotor activity in
adulthood (i.e. the hippocampus and septum; (Tremblay et al., 1984;Khalilov et al.,
1999;Silveira et al., 2002). This raises the question of whether the behavioral effects
observed in this study are the result of the activation of glutamate receptors and/or the
consequence of seizures early in postnatal development. Obviously, these possibilities
cannot be resolved based on the present experiments. However, it is well known that
ionotropic glutamate receptor activity is critical for the normal formation of cortico-
limbic circuits (Ben Ari et al., 1997;Feldman and Knudsen, 1998;Luthi et al., 2001).
Although most of this research has focused on NMDA and AMPA receptor mediated
events, some recent studies suggest that kainate receptors are dynamically regulated
during critical periods of experience-dependent plasticity in thalamocortical synapses
(Kidd and Isaac, 1999;Kidd et al., 2002). As a result, it is conceivable that
hyperstimulation of kainate receptors could result in developmental alterations of the
neural circuitry known to regulate PPI. Given that KA was administered systemically in
the present study and genes encoding kainate receptor subunits are widely expressed in
the brain during early postnatal development (Bahn et al., 1994), KA administration
95

could have subtly altered a number of brain regions, thereby producing the observed
behavioral effects.
The dose of KA (1.5 mg/kg) used in the present study induced seizures in the
neonatal rats, therefore the effects of seizures, and not ionotropic glutamate receptor
hyperstimulation per se, may also underlie the behavioral effects observed. Seizures
induced by systemic administration of KA on PND7 increase glucose metabolism
primarily in the dorsal hippocampus and lateral septum (Tremblay et al., 1984) and
transiendy increase c-fos expression in the hippocampus (Silveira et al., 2002).
Significant changes in the morphology of the hippocampus observed routinely following
KA administration post-weaning are not evident after acute systemic treatment with KA
during postnatal weeks 1 and 2 (Nitecka et al., 1984;Stafstrom et al., 1992;Koh et al.,
1999;Lynch et al., 2000).
More subtle alterations in the excitability of limbic circuits have been noted
following administration of KA on PND7 including a reduction in long-term potentiation
in the dentate gyrus, slower kindling rates, and increased paired pulse inhibition in the
perforant path in adult rats, accompanied by impairments of spatial learning as assessed
on a radial arm maze (Lynch et al., 2000). Exposure to KA on PND15 may also
exacerbate cognitive impairment and cell loss in the hippocampus induced by seizures in
adulthood (Koh et al., 1999). Recently, i.c.v. administration of KA (10-50 nmol) on
PND7 has been demonstrated to cause a relatively subtle loss of hippocampal neurons,
particularly in the CA3 and CA1 subregions of the hippocampus (Montgomery et al.,
1999;Dong et al., 2003a). Although some cell loss is observed immediately following
KA administration on PND7, greater cell loss occurs in adulthood (Humphrey et al.,
96

2002). Interestingly, dynamic alterations in neurogenesis are correlated with this cell
loss; when more hippocampal cells are lost, the rate of neurogenesis in the dentate gyrus
is increased (Dong et al., 2003b). These results obtained with i.e.v. administration
suggest that hippocampal circuits may be particularly vulnerable to alterations caused by
KA during the end of the first postnatal week. It is important to note that the histological
methods used in the present study preclude a detailed analysis of subtle changes in
hippocampal circuits such as those discussed above following i.e.v. administration.
However, given the findings of Lynch and colleagues (2000) and those using i.e.v.
administration of KA, the changes in PPI observed in the present study may be attributed
to alterations in the development of the hippocampus, particularly the dentate gyrus.
This hypothesis receives further support from studies in adult rats that confirm a
role for the hippocampus and dentate gyrus in the neural circuitry that regulates PPI. For
example, stimulation or deactivation of the hippocampus disrupts PPI (for review see
Bast and Feldon, 2003), and infusion of the cholinergic agonist carbachol into the dentate
gyrus disrupts PPI (Caine et al., 1991; 1992), an effect that is not reversed by
pretreatment with the D2 antagonist spiperone (Caine et al., 1991). Additionally, reduced
synaptophysin immunoreactivity (Varty et al., 1999) and alterations in specific
interneurons (Greene et al., 2001) are observed in the dentate gyri of socially-isolated rats
that exhibit disrupted PPI.
Conclusion
Attempts to model the etiologies of psychiatric disorders such as schizophrenia in
animals are challenging. All current models have drawbacks, mainly attributable to the
specific brain manipulations performed (Lipska and Weinberger, 2000;Penn, 2001 ;Van
97

den Buuse et al., 2003). The data reported here demonstrate that a single period of
intense cortico-limbic activity induced by the systemic administration of KA on PND7
can cause a selective disruption of PPI that is only observed in adult rats. This provides
further support for the hypothesis that in rats, the end of the first postnatal week is a
critical period for the development of cortico-limbic circuits that mediate behaviors such
as PPI and locomotor activity (Lipska et al., 1993; 1995; Wood et al., 1997;Daenen et al.,
2002; 2003;Harris et al., 2003). However, the present apomorphine-PPI experiment and
locomotor activity experiments do not support the notion that acute administration of KA
on PND7 results in the expression of an array of behavioral changes consistent with
symptoms of schizophrenia.
98

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CHAPTER FOUR: BEHAVIORAL CONVULSIONS INDUCED BY EARLY
POSTNATAL ADMINISTRATION OF THE NR2B ANTAGONIST R025-6981
FAIL TO AFFECT SENSORIMOTOR GATING OR LOCOMOTOR BEHAVIOR
IN PRE-AND POST-PUBESCENT RATS. 3
Introduction
Schizophrenia is a complex disorder resulting from multiple etiological risk
factors. Adverse events during early development have been proposed to contribute to
the etiology of schizophrenia (Weinberger, 1987). Supporting evidence includes
increased risk of developing schizophrenia following perinatal events such as maternal
infection and obstetrical complications (for review see Lewis and Levitt, 2002).
Additionally, subtle signs of cognitive and behavioral impairment are present during
childhood and early adolescence in those who go on to develop schizophrenia in
adulthood (Lewis and Levitt, 2002). These observations, among others, led to the
proposal that the primary pathology of schizophrenia occurs during early development
and its effects somehow remain largely silent until adulthood (Weinberger, 1987;Lewis
and Levitt, 2002). The effects of disturbing the early neural development of rats on
behavioral measures relevant to schizophrenia are being examined to test this hypothesis.
During early postnatal life, glutamate neurotransmission, particularly via NMDA
receptors, is critical for the formation of neural circuits due to its established role in
processes such as synaptogenesis (Rabacchi et al., 1992;Fox et al., 1996;Luthi et al.,
2001), cell birth (Gould et al., 1994), and cell survival (Vallano, 1998;Ikonomidou et al,
1999). Abnormalities of the glutamate system, with an emphasis On NMDA
hypofunction, have been linked to patients with schizophrenia (Jentsch and Roth,
3
A version of this Chapter will be submitted for publication: Howland JG, Choi FY, Phillips AG (2005)
Behavioral convulsions induced by early postnatal administration of the NR2B antagonist Ro25-
6981 fail to affect sensorimotor gating or locomotor behavior in pre- and post-pubescent rats.
105

1999;01ney et al., 1999;Goff and Coyle, 2001;Coyle and Tsai, 2004). Accordingly,
previous studies have administered non-competitive NMDA antagonists such as
phencyclidine, ketamine, and MK-801 to neonatal rats and demonstrated alterations in
several behavioral paradigms including prepulse inhibition (PPI; (Wang et al.,
2001;Harris et al., 2003), locomotor activity (Facchinetti et al, 1993;Wang et al.,
2001;Harris et al., 2003;Fredriksson et al., 2004), stereotypy (Semba et al., 2001), set-
shifting (Stefani and Moghaddam, 2005), and spatial memory (Gorter and de Bruin,
1992;Wang et al., 2001;Fredriksson et al., 2004;Stefani and Moghaddam, 2005).
Importandy, blocking NMDA receptor function at the end of the first postnatal week
causes extensive apoptosis throughout forebrain areas implicated in schizophrenia such as
the hippocampus, prefrontal cortex, striatum, and thalamus (Ikonomidou et al.,
1999;Wang et al., 2001;Harris et al., 2003;Fredriksson et al, 2004) thereby providing a
potential mechanism that may underlie the behavioral changes observed in adulthood.
Recently, an explosion in knowledge surrounding the composition of NMDA
receptors and their subunit composition has emerged (Cull-Candy et al, 2001). This
provides a unique opportunity to examine the potential role of various subclasses of the
receptors in disorders such as schizophrenia. NMDA receptors are heterodimers
composed of a variety of subunits (Fig. 4-1) and over the course of development, the
expression levels of some subunits undergo dynamic change. At birth, NR2B subunits
are expressed at high levels, whereas expression of NR2A subunit levels is low. Over the
first two to three weeks of development, this pattern reverses, and during late adolescence
and into adulthood, NMDA receptors containing NR2A subunits predominate (Monyer et
al, 1994;Guilarte and McGlothan, 1998;Ritter et al., 2002;Zhang et al., 2002). As the
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Figure 4-1. Cartoon illustrating an NMDA receptor within the cell membrane. NMDA
receptors are assemblies of various subunits including NR1, NR2A to NR2D, and NR3A
and NR3B. Functional receptors contain two NR1 subunits and some combination of two
additional subunits. As is depicted, the binding site for non-competitive NMDA
antagonists such as MK-801 and phencyclidine is located within the pore of the receptor.
Thus, the receptor must be open in order for these antagonists to bind. In the present
experiments, the NR2B-specific antagonists Ro25-6981 and ifenprodil were used, which
have been reported to bind in the region of the polyamine site. Interestingly, these drugs
only bind to receptors termed diheteromers that contain two NR1 subunits and two NR2B
subunits. In this manner, their high degree of specificity is achieved.
107

subunit composition of NMDA receptors determine their precise kinetics, the unique
subunit composition of NMDA receptors during the neonatal period is likely to be an
important factor in normal development (Cull-Candy et al., 2001).
Interestingly, numerous lines of evidence suggest that subunit-specific changes of
the NMDA receptor may exist in schizophrenia: (1) significant increases in the
expression of NR2B subunits have been noted in the hippocampus (Gao et al., 2000),
thalamus (Clinton and Meador-Woodruff, 2004), and temporal cortex (Grimwood et al.,
1999) of schizophrenic patients, (2) increased expression of NR2D in the prefrontal
cortex (Akbarian et al., 1996) and decreases in NR1 expression in the hippocampus (Gao
et al., 2000) have been demonstrated, (3) novel polymorphisms and variants in the
promoter regions of the NR2A (Itokawa et al, 2003) and NR2B genes (Miyatake et al.,
2002; Qin et al., 2005) may also exist in schizophrenia, (4) behavioral and neurochemical
abnormalities consistent with the disorder are observed in mice with genetically altered
NR1 (Mohn et al., 1999;Duncan et al., 2004) and NR2A (Miyamoto et al., 2001)
subunits. Therefore, in an effort to further understand the effects of transiently disrupting
glutamate receptors containing specifically NR2B subunits, we administered the subunit-
selective NMDA antagonist [(+/-)-(R*,S*)-alpha-(4-hydroxyphenyl)-beta-methyl-4-
(phenylmethyl)-l-piperidine propanol] (Ro25-6981) to neonatal rats on postnatal days
(PND) 6 and 7.
Ro25-6981 is a high affinity non-competitive antagonist of receptors containing
the NR2B subunit (Fischer et al., 1997). As NR2B-containing NMDA receptors are
overexpressed in the neonatal period, administration of Ro25-6981 provides a unique
opportunity to assess the importance of these specific receptors in the maturation of the
108

neural circuits that mediate behaviors with potential relevance to schizophrenia.
Importantly, Ro25-6981 has low affinities for a-adrenergic and serotoninergic receptors,
a problem recognized for other NR2B antagonists such as ifenprodil (Mutel et al., 1998).
Following neonatal treatment, rats were tested for PPI and locomotor responses to a novel
environment and amphetamine challenge both before and after puberty. These behavioral
tests are extensively used to model the information processing deficits and striatal
dopamine elevations observed in patients with schizophrenia (Lipska and Weinberger,
2000;Braff et al., 2001 ;van den et al., 2003;Bast and Feldon, 2003). Finally, the
disruptive effect of the dopamine agonist apomorphine on PPI was also assessed in a sub­
group of adult rats.
Unexpectedly, the dose of the Ro25-6981 used (15 mg/kg) caused behavioral
convulsions when administered on PND 6. As is detailed in the methods section, this
dose has been used extensively in adult rats without reports of convulsive activity, and
was chosen based on "affinity estimates for NR2B-containing receptors. Due to this
observation, we also performed a series of parametric studies to further examine the
convulsive effects of a number of NMDA antagonists during early development.
Additionally, previous work performed in our laboratory demonstrated that seizures
induced by kainic acid on PND 7 resulted in the delayed expression of PPI deficits in rats
(Howland et al., 2004). From this standpoint, the long-term behavioral effects of
convulsions following Ro25-6981 were of considerable interest.
109

Methods
Experiment 1 - Incidence and Characteristics of Behavioral Convulsions in Postnatal
Rats Following Antagonism of NMDA Receptors Containing the NR2B Subunit
Subjects
Long-Evans or Sprague-Dawley rat pups were used in all experiments. Pregnant
Long-Evans rats were obtained from Charles River (Quebec, Canada) at 13 to 15 days of
gestation. They were singly housed and left undisturbed until giving birth. The colony
was maintained on a 12/12 hour light/dark cycle (lights on at 0700), at a temperature of
22±1°C. All rats were given food (Purina Rat Chow) and tap water ad libitum. Sprague-
Dawley rat pups were obtained from the University of British Columbia Animal Care
Center. The pups were delivered to our animal colony on the desired postnatal day
without their mother. All experiments were initiated within 60 min of the arrival of the
rat pups in the laboratory. Experiments were conducted in accordance with the standards
of the Canadian Council on Animal Care and were approved by the Committee on
Animal Care at the University of British Columbia.
Experimental Procedures
The day of birth of the pups was designated PND 0. Rat pups of the appropriate
age (PND 6, 9, 12,15) were weighed and separated into individual compartments of a
cardboard box for drug administration. The pups were removed from the colony and
taken to a room where the behavioral effects of the administered drugs were videotaped
and scored by an observer. The subunit-selective NMDA-NR2B antagonists Ro25-6981
(7.5, 15, and 30 mg/kg) and ifenprodil (15 and 30 mg/kg) were injected (10 ml/kg; i.p.)
and the behavior of the animals was videotaped for 60 minutes. Reports indicate that 15
110

mg/kg of Ro25-6981 is in the dose range necessary to block most NR2B-containing
receptors in the adult rodent brain (Murray et al., 2000;Lee and Rajakumar, 2003), and
doses of 20 to 30 mg/kg have been used in vivo (Chaperon et al., 2003;Higgins et al.,
2005). Similar data exists for ifenprodil (Murray et al., 2000). Following the
experiment, the pups were sacrificed with carbon dioxide. All drugs were obtained from
Sigma.
Experiment 2 - Long-term Behavioral Effects of Convulsions Resulting from Antagonism
of NMDA Receptors Containing the NR2B Subunit
Subjects
Two independent groups of Long-Evans rats (i.e. Group 1 and Group 2) were
tested in this study. The animals were obtained, housed and cared for in a manner
identical to that described in Experiment 1. Testing protocols for both groups were
similar with small procedural differences noted in the methods section where appropriate.
Ro25-6981 Administration
On PND3, the litters were sexed and culled to include only males (4-9 rats per
litter). At 4:00 p.m. on the afternoon of PND 6 and 9:00 a.m. and 4:00 p.m. of PND 7, all
pups were removed from the nest, weighed and placed individually in small
compartments of a cardboard box. They were then removed from the colony and taken to
a small heated room. Ro25-6981 (15 mg/kg) or saline was injected (i.p.) with a 30-gauge
needle (10 ml/kg). Care was taken to ensure that both treatments were administered to
members of each litter. The behavior of all rats was recorded with an overhead video
camera for 60 min. Before being returned to their mothers, the pups were earmarked
according to treatment condition. The litters were then left undisturbed (except for
111

normal cage changing) until weaning on PND 25. Weanling rats were housed in cages of
2 or 3 with members of their litter and these groups remained together for the duration of
the experiment. All rats were handled before behavioral testing.
Prepulse Inhibition
On PND 35 and 56, rats were removed individually from the colony and taken
immediately to the PPI apparatus. Testing was conducted in a single sound-attenuating
startle chamber (ambient noise level 64 dB), containing a transparent Plexiglas tube (8.2
cm in diameter, 20 cm in length), mounted on a Plexiglas frame (SR-LAB, San Diego
Instruments, San Diego). Noise bursts were presented through a speaker mounted 24-cm
above the tube. An accelerometer below the frame of the apparatus measured whole
body startle amplitude, defined as the average of 100 1-ms accelerometer readings
collected from stimulus onset. Each PPI test session began with a 5-min acclimatization
period during which a 70-dB background noise level was presented, which remained
constant for the entire test session. Following the acclimatization period, six pulse alone
trials (120 dB, 40 ms) were presented to achieve a relatively stable startle amplitude
before PPI testing. Data from these pulse-alone trials was not considered in the analysis
of PPI. Immediately following the six initial pulse-alone trials, presentation of the trials
that were used in the calculation of PPI levels were initiated. Trials presented were of
four types: pulse alone (12 trials, 120 dB, 40 ms), prepulse + pulse (12 trials X 3 prepulse
intensities - discussed below), prepulse alone (12 trials X 3 prepulse intensities) or no
stimulus (12 trials). Prepulse + pulse trials consisted of the presentation of a 20 ms
prepulse of 73, 76, or 82 dB 80 ms before the presentation of the pulse. Prepulse alone
trials consisted only of a 20 ms prepulse (73, 76, 82 dB). All trials were presented in a
112

pseudorandom order. After the trials used for calculation of PPI were presented, 6
additional pulse-alone trials were presented. These, along with the first 6 pulse-alone
trials, were used to calculate the level of habituation over the testing period. As no
significant differences were observed between the groups in habituation and or for the
prepulse-alone trials, these data are not presented. The inter-trial interval varied
randomly from 3 to 12 s (average 7.5 s). Calibration of the apparatus was performed
using a RadioShack Digital Sound Level Meter and adjustments were made as necessary.
Spontaneous Locomotor Activity
On PND 36, rats were weighed, removed from the colony, and immediately
placed in 1 of 8 Med Associates Test Chambers (ENV-008; 30.5 cm X 24.1cm X height
21.0 cm) to measure spontaneous locomotor activity for 60 min. The chambers were
fitted with 4 pairs of infrared photocells 3.5 cm from the floor evenly spaced on the long
walls and had metal grid floors and two operant levers (which were retracted during
locomotor activity testing). Each chamber was contained within a Med Associates sound
attenuating cubicle with a house light illuminated during testing.
Dopaminergic Challenges of PPI and Locomotor Activity
For those rats in Group 1, 7 to 14 days after PPI testing in early adulthood, the
responses of the animals to administration of dopamine agonists were measured in both
the PPI and locomotor activity tests. Some rats were tested for PPI first followed by
locomotor activity while others were tested in the reverse order. A minimum of 7 days
separated locomotor activity and PPI testing. The PPI sessions were conducted in a
manner identical to that described above, except that immediately before the PPI session,
all rats were weighed and injected with the appropriate volume of vehicle (ascorbic acid,
113

0.1%) or drug (apomorphine, Sigma, 0.2 mg/kg; s.e.). Each rat was tested twice (i.e.
following either vehicle or apomorphine injection), and the PPI tests were at least 5 days
apart. For locomotor activity testing, the rats were weighed, removed from the colony,
and placed in the locomotor activity boxes for 60 min (as described above for PND 36).
All rats were then immediately injected with D-amphetamine (1.5 mg/kg, i.p.) and
returned to the locomotor boxes. Locomotor activity was monitored for an additional 90
min before the rats were returned to the colony room.
For rats in Group 2, PPI responses following apomorphine challenge were not
measured. However, locomotor responses to D-amphetamine were examined at both
PND 36 and PND 57. Rats were weighed and placed in the locomotor boxes as
previously described, however, at both ages, they were injected with D-amphetamine (1.5
mg/kg) immediately following the spontaneous locomotor activity test. Locomotor
activity following D-amphetamine was measured for 90 min in all cases.
Data Analysis
For the PPI experiments, two measures were calculated for each animal. The
startle amplitude represented the mean startle amplitude of the 12 pulse-alone trials
presented after the 6 habituation trials. Startle amplitude data were compared using an
independent samples t-test for each age. PPI was calculated by averaging startle
amplitudes for each trial type. The percent PPI for each prepulse intensity was calculated
using the formula: [100 - (100 X startle amplitude on prepulse + pulse trials) (startle
amplitude on pulse alone trials)]. A repeated measures ANOVA was performed on the
data obtained from each age with prepulse intensity as a within-subjects factor and
treatment on PND 6 and 7 as a between-subjects factor. Results from the apomorphine
114

experiment were also analyzed using a repeated measures ANOVA (prepulse intensity
and test as within-subjects factors, and treatment at PND 7 as a between-subjects factor).
Locomotor activity data were compared using repeated measures ANOVA at each age
with test type as a within-subjects factor and treatment on PND 7 as a between-subjects
factor. For all ANOVA's, post-hoc analyses were performed using the Neuman-Keuls
test where appropriate. The significance level for all statistical tests was p < 0.05.
Results
Experiment 1 - Behavioral Effects of Ro25-6981 Administration in the Early Postnatal
Period
Approximately 5 min following injection with Ro25-6981, the majority of PND 6
pups displayed behavioral convulsions. The convulsions were similar those typically
described following administration of kainic acid (Howland et al., 2004) or bicuculline
(Lai et al., 2002) in neonatal rats. Generally, they consisted of loss of posture,
vocalizations, and rapid bursts of motor activity (especially with the hind legs). The
majority of the animals showed bouts of bilateral forelimb and hindlimb clonus and/or
tonus on their backs, in what has been called a 'swimming' posture by other authors
(Stafstrom et al., 1993). In most cases, the behavioral convulsions subsided within 30 to
60 min following injection. Interestingly, following their initial bout of convulsions,
some animals would have another period of convulsive activity if they were handled by
the experimenter. Those pups injected repeatedly with Ro25-6981 (see Experiment 2)
showed no evidence of tolerance to the convulsive effects of the drug.
As depicted in Table 4-1, the convulsive effects of Ro25-6981 were age and dose
dependent. Whereas all pups treated with 15 mg/kg of the drug on PND 6 had
115

convulsions, only 25% of PND 9 pups, and 0% of PND 12 or 15 (using either 15 or 30
mg/kg) pups displayed evidence of convulsions. However, pups injected with Ro25-
6981 were more active than saline-injected controls. Additionally, none of the rats
treated with 7.5 mg/kg of Ro25-6981 displayed behavioral convulsions. In an effort to
rule out the possibility that the convulsions observed in the PND 6 pups were caused by
an unknown property of Ro25-6981 unrelated to NR2B antagonism, ifenprodil was also
administered to some animals on PND 6. Similarly to the results obtained with Ro25-
6981, almost all pups (89%) treated with ifenprodil had behavioral convulsions.
Experiment 2 - Effects of Neonatal Ro25-6981 Administration on Body Weight
As described in detail for experiment 1, administration of Ro25-6981 (15 mg/kg;
Group 1: n=25; Group 2: n=16) resulted in behavioral convulsions for 30 to 60 minutes.
Some individual differences in the severity of the convulsions were noted; although these
did not correlate with performance on the behavioral tests performed. As a result, data
from all rats is included.
All rats were weighed on PND 6, twice on PND 7, and once on PND 9, 36 and 57.
Repeated measures ANOVA's performed on these data indicate that administration of
Ro25-6981 to the pups had no significant effect on body weight at any age (Group 1: F(l,
41)=0.90, N.S.-; Group 2: F(l, 30)=0.49, N.S.). However, comparing the average weights
of rats in Group 1 and Group 2 revealed that the animals in Group 1 were significandy
lighter than those in Group 2 (F(l, 73)=48.97, p < 0.001). On PND 6 and 7, rats in Group
1 weighed approximately 70% of those in Group 2 (PND 6: Group 1 mean = 12.16 g;
Group 2 = 17.00 g). During puberty and early adulthood, the difference in weights
116

Drug Postnatal Dose (i.p., Total Number Total Number
Day mg/kg) Injected Exhibiting
Convulsions
Ro25-6981 6 7.5 8 0
Ro25-6981 6 15 8 8
Ro25-6981 9 15 4 1
Ro25-6981 12 15 5 0(A)
Ro25-6981 15 15 4 0(A)
Ro25-6981 15 30 3 0(A)
Ifenprodil 6 15 4 4
Ifenprodil 6 30 5 4
Table 4-1. The incidence of behavioral convulsions in rats administered NR2B-selective
NMDA antagonists at various postnatal ages. i.p. = intraperitoneal.
117

narrowed to a 10 to 15% difference (PND 36: Group 1 = 159.77 g; Group 2 = 173.58 g;
PND 57: Group 1 = 339.26 g; Group 2 = 398.96 g).
Neonatal Ro25-6981 Administration Does not Alter PPI Responding Either before
Puberty or in Early Adulthood
Before Puberty (PND 35): As is shown in Figure 4-2A and 4-3A, comparable
levels of PPI was elicited in both saline- and Ro25-6981-treated rats for Groups 1 (saline:
n=19, average PPI: 27.86±2%; Ro25-6981: n=25, average PPI: 26.53±3%) and 2 (saline
n=16, average PPI: 32.24±3%; Ro25-6981: n=16, average PPI: 26.38±4%). Repeated
measures ANOVA's performed separately for each Group revealed a significant effect of
prepulse (Group 1: F(2, 84)=96.62, p < 0.001; Group 2: F(2, 60)=49.92, p < 0.001), but
no significant treatment effects (Group 1: F(l, 43)=0.13, N.S.; Group 2: F(l, 30)=1.45,
N.S.) or treatment by prepulse interactions (Group 1: F(2, 84)=0.08, N.S.; Group 2: F(2,
60)=0.14, N.S.). Figures 4-2B and 4-3B depict the average startie amplitudes for all
animals tested in Groups 1 and 2. Independent samples t-tests revealed no significant
differences between saline- and Ro25-6981-treated rats in either Group (Group 1:
t(42)=1.35, N.S.; Group 2: t(30)=0.03, N.S.).
Early Adulthood (PND 56): Similar to before puberty, all groups tested
demonstrated robust PPI in early adulthood (Fig. 4-2C and 4-3C), and no significant
differences were observed between animals treated with either saline (average PPI, Group
1: 30.60±3%; Group 2: 31.74±3%) or Ro25-6981 (average PPI, Group 1: 28.33±3%;
Group 2: 37.00±5%). Repeated measures ANOVA's confirmed these observations with
no significant treatment (Group 1: F(l, 42)=0.29, N.S.; Group 2: F(l, 30)=0.68, N.S.) or
treatment by prepulse interactions (Group 1: F(2, 84)=0.63, N.S.; Group 2: F(2, 60)=0.69,
118

Q_
Q.
:

pp3 pp6 pp12 average
Prepulse Intensity :
pp3 pp6 pp12
Prepulse Intensity average
B
160i
D
160!
1120
I 80]
I 40]
saline Ro
Group
saline Ro
Group
Figure 4-3. Prepulse inhibition (PPI) scores from rats in Group 2 treated on postnatal day
(PND) 6 and 7 with saline (white bars, n=16) or Ro25-6981 (Ro, 15mg/kg; black bars,
n=16). A, Effects of postnatal day (PND) 7 KA treatment on PPI at PND 35. B, Average
starde amplitudes of the rats on PND 35. C, Percent PPI scores for those rats tested at
PND 56. D, Average startle amplitudes of the rats on PND 56.
120

N.S.)- As expected, significant main effects for prepulse intensity were found for both
groups (Group 1: F(2, 84)=70.90, p < 0.001; Group 2: F(2, 60)=32.71, p < 0.001).
Additionally, startle amplitudes did not differ as a result of treatment in either Group 1
(Fig. 4-2D; t(42)=l .05, N.S.) or Group 2 (Fig. 4-3D; t(30)=0.73, N.S.)
Apomorphine Challenge in Adulthood has Similar Effects on PPI in Neonatally Saline- or
Ro25-6981 -treated Rats
To examine the potential effects of neonatal Ro25-6981 treatment on the
dopaminergic regulation of PPI, 16 saline-treated and 18 Ro25-6981 -treated rats from
Group 1 were retested for PPI following injection with either vehicle (0.1% ascorbic acid
in saline) or apomorphine (0.2 mg/kg). Figure 4-4A summarizes the results of these tests.
Following vehicle injection, PPI levels in both saline- and Ro25-6981-treated rats were
similar to those observed when PPI was tested on PND 56. Apomorphine (0.2 mg/kg)
significantly disrupted PPI in both the saline- and Ro25-6981-treated rats, as reflected by
a significant main effect of test (vehicle versus apomorphine; F(l, 32)=31.18, p < 0.01)
and a significant test by prepulse interaction (F(2, 64)=10.75, p < 0.001, post-hoc, p <
0.05). Additional analyses revealed that significant effects only existed for trials with 6
and 12 dB prepulses in both groups (p < 0.05). As shown in Fig. 4-4B, startle amplitude
was also significantly reduced by apomorphine administration in both groups (F(l,
32)=8.59, p < 0.01).
Neonatal Treatment with Ro25-6981 and Amphetamine-Induced Locomotor Activity in
Adult Rats
Group 1: Spontaneous locomotor activity in response to a novel environment was
assessed for 60 min in the saline- and Ro25-6981-treated rats. Both before puberty (Fig.
121

GL
D-:
a>
70-
60-
50-
40-
30-
20 \
lO-
CI
A
asal+veh
•Ro+veh
•sal+apol
oRo+apo
Condition
average
sal+ Ro+ sal+ Ro+
veh veh apo apo
Group
Figure 4-4. A, Effects of pretreatment with vehicle (0.1% ascorbic acid) or apomorphine
(0.2 mg/kg) on percent PPI scores in adulthood. Animals were administered either saline
(n=16) or Ro25-6981 (15 mg/kg; n=18) on postnatal day 6 and 7. B, Average starde
amplitudes of those rats depicted in panel A. In both panels, asterisks indicate a
significant difference between groups.
122

Figure 4-5. Locomotor activity levels (photobeam breaks) in response to novelty (A, B
left side) or amphetamine (1.5 mg/kg; B right side) of rats tested on either postnatal day
(PND) 36 (A) or PND 57 (B) in Group 1. Rats were treated with either saline (white
bars) or Ro25-6981 (black bars, 15 mg/kg) on PND 6 and 7. The asterisk denotes a
significant difference between amphetamine-treated groups (p

Figure 4-6. Locomotor activity levels (photobeam breaks) in response to novelty or
amphetamine (1.5 mg/kg) of rats tested on either postnatal day (PND) 36 (A) or PND 57
(B) in Group 2. Rats were treated with either saline (white bars, n = 12) or Ro25-6981
(black bars, 15 mg/kg, n = 12) on PND 6 and 7. Note the difference in scale for the units
of activity levels (y-axis) for each panel.
124

4-5A) and in early adulthood (Fig. 4-5B), exploration levels were similar in both groups
(before puberty: t(42)=0.45, N.S.; early adulthood: see ANOVA below). Interestingly,
administration of the dopamine agonist D-amphetamine (1.5 mg/kg) further increased
locomotor activity by approximately 28% in rats neonatally treated with Ro25-6981
(2193.75 ± 143 beam breaks) than saline (1712.67 ± 133 beam breaks) during the 90 min
test (Fig. 4-5B). This effect was confirmed by a significant treatment by test interaction
(F(l, 22)=7.83, p < 0.05), and post-hoc analyses (p < 0.05).
Group 2: Spontaneous and amphetamine-induced locomotor activity was compared
between treatment groups both before puberty (Fig. 4-6A) and in early adulthood (Fig. 4-
6B) for those rats in Group 2. Unexpectedly, the groups did not significandy differ on
either test at either age. Statistical analyses revealed insignificant main effects of
treatment (PND 36: F(l, 22)= 0.27, N.S.; PND 57: 0.18, N.S.) and treatment by test
interactions (PND 36: F(l, 22)=0.12, N.S.; PND 57: F(l, 22)=0.18, N.S.).
Discussion
The present experiments assessed the acute and delayed behavioral effects of the
administration of Ro25-6981, an antagonist of NR2B-containing NMDA receptors, in
rats. During PND 6 or 7, administration of Ro25-6981 resulted in behavioral convulsions
that typically lasted 30 to 60 minutes. The convulsions were dose-dependent and could
be elicited with a second NR2B antagonist, ifenprodil. Parametric experiments
demonstrated that convulsions could not be reliably elicited in rats older than PND 7.
The delayed behavioral effects of administering Ro25-6981 on PND 6 and 7 were
inconsistent. Ro25-6981-treated rats in Group 1 performed similarly to saline-treated
animals on PPI and spontaneous locomotion tests both before and after puberty.
125

However, the Ro25-6981 -treated animals were significantly more active when treated
with amphetamine in early adulthood, but failed to show increased sensitivity to the
disruptive effects of apomorphine on PPI. In contrast, Ro25-6981-treated rats tested in
Group 2 failed to show significant differences from saline-treated animals in any of the
behavioral tests conducted.
Antagonism of NR2B-containing NMDA Receptors Induces Behavioral Convulsions
The convulsive effects of Ro25-6981 were unexpected in the present experiments;
however, they appear to be reliable as they were dose-dependently elicited in two strains
of rats by both Ro25-6981 and ifenprodil. Interestingly, some groups (Fischer et al.,
1997;Mutel et al., 1998;Mallon et al., 2005) have described agonist-like properties of
Ro25-6981 when the drug is applied in conjunction with low concentrations of NMDA.
In Xenopus oocytes, Ro25-6981 (1 uM) profoundly inhibits the current elicited by 100
u.M of NMDA, but surprisingly potentiates the current elicited by 1 uM of NMDA
(Fischer et al., 1997). In another study using hippocampal slices from pubescent rats,
Ro25-6981 (3 uM) potentiated the effects of NMDA (4 to 10 uM) on CA1 field
potentials and paired-pulse interactions (Mallon et al., 2005). As also reported by Fisher
et al. (1997), these effects were more significant with lower concentrations of NMDA
(Mallon et al., 2005). Interestingly, the NR2A specific antagonist NVP-AAM077
(Auberson et al., 2002) effectively blocked the effects of Ro25-6981 in the hippocampal
slice suggesting that the NR2B subunit may reduce NMDA receptor activity by exerting
tonic inhibitory effects on receptors containing the NR2A subunit (Mallon et al., 2005).
A number of issues make integrating the present data with other reports in the
literature challenging. Most importantly, the subunit composition of NMDA receptors
126

dynamically changes over development; therefore, care must be taken when comparing
data sets gathered from animals of different ages (for example, comparing the present
data to those of Mallon et al., 2005). During the first postnatal week, NR2A subunits are
expressed at very low levels in the rodent brain, whereas in adulthood NR2A subunits
predominate (Monyer et al., 1994;Guilarte and McGlothan, 1998;Cull-Candy et al.,
2001;Ritter et al., 2002;Zhang et al., 2002). Given these expression differences, it is
unlikely that the observations made by Mallon and colleagues (2005) apply during the
early neonatal period since very few NR2A subunits are expressed at that age.
Additionally, numerous reports indicate that in vivo Ro25-6981 antagonizes NR2B
containing NMDA receptors at doses within the range used in the present study (Lee and
Rajakumar, 2003;Chaperon et al., 2003;De Vry and Jentzsch, 2003;Boyce-Rustay and
Holmes, 2005). Therefore, the most likely explanation of the present data is that the
behavioral convulsions observed resulted from the antagonist action of Ro25-6981 on
NR2B-containing NMDA receptors.
Although convulsions resulting from NMDA receptor antagonism are
counterintuitive, they are not without precedence in the literature. For example, systemic
administration of the non-competitive NMDA antagonist MK801 (0.1, 0.5, 1.0 mg/kg) to
neonatal rat pups induces behavioral and electrographic seizures and exacerbates seizures
caused by kainic acid (Stafstrom et al., 1997). Given that MK-801 would primarily act
on NMDA receptors containing NR2B subunits at this developmental age, these results
support our assertion that the neonatal brain may be prone to convulsions following the
blockade of receptors containing NR2B subunits.
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Further experiments are necessary to understand the mechanism underlying the
convulsive effects of neonatal NMDA receptor antagonism. Seizures are more common
neonatally than later in development, an effect that may be related to differences in the
net ratio of excitation and inhibition early in development (Ben Ari et al., 1997;Holmes et
al, 2002). As previously described, intricate developmental regulation of NMDA
receptor expression exists during the first few weeks of life. The expression patterns and
function of other receptors, including GABA A and AMPA receptors, are also
developmentally regulated. For instance, GABA A receptors are expressed early in the
embryonic period and provide much of the excitatory drive in the developing brain until
the end of the first postnatal week when NMDA and AMPA currents begin to
predominate (Ben Ari et al., 1997;Holmes et al., 2002;Khazipov et al., 2004). The
excitatory action of GABA is primarily due to delayed expression of the K +
/Cf co-
transporter KCC2, resulting in increased intracellular Cf concentrations. Thus, when
GABA A channels open under these conditions, Cl" ions exit the neuron resulting in
depolarization (Holmes et al., 2002). However, under some conditions, GABA A
activation may also increase the resting membrane conductance sufficiently that the net
effect of opening these channels is inhibitory (Leinekugel et al., 1999;Khalilov et al.,
1999a;Holmes et al., 2002). As resting membrane conductance at this age is also
critically dependent on glutamate receptor conductances (Khalilov et al., 1999a), one
possible explanation of our observations is that antagonism of NMDA receptors with
Ro25-6981 may 'tip the balance' in favor of excitation, thereby triggering a convulsion.
128

The Long-Term Behavioral Effects of Ro25-6981 on PPI and Locomotor Activity
The levels of PPI and locomotor activity observed before and after puberty in the
present study are similar to previous experiments from our group (Howland et al., 2004).
Accordingly, percent PPI increased with higher prepulse intensities (Fig. 4-2 and 4-3).
Average PPI levels were also lower in prepubescence than in early adulthood.
Interestingly, a relatively low dose of apomorphine (0.2 mg/kg) disrupted PPI in the
present study (Fig. 4-4A). This effect is consistent with previous results using the Long-
Evans strain in our laboratory (Howland et al., 2004), but not others (Swerdlow et al.,
2001).
Although the significantly potentiated locomotor activity following amphetamine
administration in the Ro25-6981-treated rats of Group 1 (Fig. 4-5B) is intriguing, the
failure to replicate this effect in Group 2 (Fig. 4-6B) questions the reliability of these
data. However, an interaction between Ro25-6981 administration and the factors that led
to significantly reduced body weight in Group 1 may explain these behavioral changes.
When the present body weights are compared to previous studies from our laboratory
(Howland et al., 2004) in which Long-Evans pups were reared under identical conditions
to those described herein, it is clear that the pups tested in Group 1 had abnormally low
body weights. The cause of these differences cannot be confirmed, although some
renovations to our facility occurred during the period which the pregnant females arrived
and gave birth to the pups in Group 1. Thus, the mothers of the rats tested in Group 1
may have been abnormally stressed during the pre- and postnatal period. Indeed, stress
during these developmental periods has been shown to regulate dopamine-dependent
behaviors such as locomotor activity in adult rats (Kofman, 2002;Brake et al., 2004).
129

The present experiments fail to provide convincing evidence that neonatal
treatment with Ro25-6981 results in specific behavioral changes related to schizophrenia.
However, these data stand in contrast to reports of significant behavioral effects of
neonatal NMDA antagonism in adulthood using non-competitive antagonists. For
example, neonatal treatment with phencyclidine (PND 7, 9, and 11) reduces PPI and
significantly increases locomotor activity in response to a phencyclidine challenge, both
of which were reversed following antipsychotic treatment (Wang et al., 2001). Increased
spontaneous locomotor activity is also observed following ketamine administration to
mice on PND 10 (Fredriksson et al., 2004). Finally, neonatal administration of MK-801
alters PPI (Harris et al., 2003), locomotor activity (Harris et al., 2003), and set-shifting
(Stefani and Moghaddam, 2005), although the PPI and locomotor effects were not
independently replicated (Beninger et al., 2002) and were only found in female animals
(Harris et al, 2003).
A number of differences between non-competitive NMDA antagonists and Ro25-
6981 may underlie the different behavioral effects observed following their
administration. For example, non-competitive NMDA antagonists have actions at
dopamine D 2 and serotonin 5-HT 2 receptors, in addition to their effects of NMDA
receptors (Kapur and Seeman, 2002). Importantly, the neonatal administration of non­
competitive NMDA antagonists also causes significant apoptosis in areas such as the
hippocampus, thalamus, cortex, and striatum 24 to 48 hours after treatment (Wang et al.,
2001 ;Lai et al., 2002;Beninger et al., 2002;Harris et al., 2003-/Fredriksson et al., 2004).
Given that cell loss or disarray in these areas is reported in schizophrenia (Pakkenberg,
1990;Jones, 1997;Harrison, 1999;Gothelf et al., 2000), these findings strengthen the
130

validity of neonatal non-competitive NMDA antagonist administration as a model of
schizophrenia, and may explain their behavioral effects. It is not known whether Ro25-
6981 administration causes similar neuropathological effects, although the potential
exists given that various competitive and non-competitive NMDA antagonists cause
apoptosis in the developing brain (Dconomidou et al., 1999).
Conclusion
The convulsions caused by Ro25-6981 on PND 6 and 7 failed to affect PPI or
locomotor responding in the present experiments. In contrast, febrile seizures early in
life are associated with increased risk for schizophrenia (Vestergaard et al., 2005) and
psychosis (Kanemoto et al., 2001). Additionally, kainic acid-induced seizures on PND 7
in rats result in the delayed emergence of subtle PPI disruptions in early adulthood
(Howland et al., 2004) and have short and long-term effects on temporal lobe
electrophysiology, especially within the hippocampus (Tremblay et al., 1984;Stafstrom et
al., 1992;Khalilov et al., 1999b;Lynch et al., 2000;Silveira et al., 2002). As the
electrographic characteristics of the convulsions induced by Ro25-6981 are presently
unknown, experiments designed to address this issue may enable more specific
hypotheses to be generated regarding the brain areas important for long-term behavioral
changes following neonatal seizures and their relationship to schizophrenia.
131

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CHAPTER FIVE: GENERAL DISCUSSION
The experiments contained within the present dissertation were designed to
address two main objectives. First, Chapter Two detailed a number of experiments
conducted to test the effects of electrical stimulation of discrete sub-regions of the
hippocampus on PPI. Results of these experiments suggest brief periods of higher
frequency activity that result in increased locomotor activity and ventral striatal
dopamine efflux reversibly disrupt PPI when applied to the ventral, but not dorsal,
hippocampus. The experiments described in Chapters Three and Four were designed to
further the hypothesis that a period at the end of the first postnatal week is particularly
sensitive for the development of the circuits mediating PPI and locomotor activity
(Lipska and Weinberger, 2000). The experiments summarized in Chapter Three support
this hypothesis by demonstrating that a single exposure to the kainate receptor agonist
kainic acid on postnatal day seven disrupted PPI in early adulthood, but not before
puberty. In Chapter Four, the effects of administration of the NR2B-subunit selective
NMDA receptor antagonist Ro25-6981 on postnatal day six and seven were also
assessed. Serendipitously, Ro25-6981 was observed to induce convulsions at this age;
however, no consistent effects of this treatment on PPI or locomotor activity were noted
either before puberty or in early adolescence. Although the implications of these results
have been discussed at some length in the proceeding chapters, the present chapter will
serve to further integrate these data with contemporary strategies of modeling
schizophrenia in rodents.
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The Role of the Hippocampus in the Regulation of Prepulse Inhibition and Locomotor
Activity
As detailed in the discussion section of Chapter Two, hippocampal regulation of
PPI is complex and incompletely understood. However, a great deal of evidence using
both NMDA (Wan et al., 1996;Klarner et al., 1998;Zhang et al, 1999;Swerdlow et al.,
2001b;Zhang et al., 2002) and higher frequency electrical (Howland et al., 2004b)
stimulation protocols, indicates that over-activity of the ventral, but not dorsal,
hippocampus disrupts PPI. Numerous studies have also assessed the role of hippocampal
activity in locomotor behavior patterns of adult rats. Similar to the effects described for
PPI, stimulation of the ventral hippocampus with either NMDA (Mogenson and Nielsen,
1984;Bardgett and Henry, 1999;Bast et al., 2001;Zhang et al., 2002) or higher frequency
electrical current (Taepavarapruk et al., 2000) increases locomotion, whereas comparable
stimulation of the dorsal hippocampus has no effect (Zhang et al., 2002). Here it is
important to note that the dorsal hippocampus can influence locomotor activity under
some conditions as dorsal hippocampal infusions of carbachol (Flicker and Geyer,
1982a;Mogenson and Nielsen, 1984), picrotoxin (Flicker and Geyer, 1982b) or other
agents (Bast and Feldon, 2003), can alter the behavior. Thus, although the regulation of
PPI and locomotor activity by the dorsal and ventral hippocampus is complex, both
behaviors are clearly more sensitive to stimulation of the ventral than the dorsal region
(Zhang et al., 2002;Bast and Feldon, 2003;Howland et al., 2004b). Previous research
supports functional differentiation within the hippocampus, especially in regards to
spatial learning and memory (supported by the dorsal hippocampus) and fear-related
behavior (ventral hippocampus) (Moser and Moser, 1998; Kjelstrup et al., 2002).
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When the roles of dopamine and glutamate in mediating the effects of either
NMDA or electrical stimulation of the ventral hippocampus on PPI or locomotor activity
are considered, an interesting dissociation has emerged (Bast and Feldon, 2003). As was
previously described for PPI, neither dopamine antagonists (systemically administered)
nor glutamate antagonists (infused into the nucleus accumbens) effectively block the
disruption of PPI induced by infusion of NMDA into the ventral hippocampus (Wan et
al., 1996;Bast et al., 2001). In contrast, both dopamine and glutamate antagonists block
the increases in locomotion observed following stimulation of the ventral hippocampus
(Bardgett and Henry, 1999;Taepavarapruk et al., 2000;Bast et al., 2001). Additionally,
locomotor activity in a novel environment increases dopamine efflux in the nucleus
accumbens, an effect that can be reversed by inactivation of the ventral hippocampus or
administration of a glutamate receptor antagonist into the ventral tegmental area (Legault
and Wise, 2001). Thus, these findings indicate that dopamine and glutamate transmission
is critical for the ventral hippocampal modulation of locomotor activity, but not for PPI.
Although the circuitry underlying the role of dopamine and glutamate in the
ventral hippocampal stimulation-elicited increases in locomotor behavior is still being
investigated, evidence suggests that dopamine and glutamate receptors in the nucleus
accumbens (Taepavarapruk et al., 2000) and ventral tegmental area (Legault and Wise,
2001) play an important role, although other brain regions may also be involved.
Stimulation of the ventral hippocampus with NMDA increases dopamine efflux in the
medial prefrontal cortex (Peleg-Raibstein et al., 2005), and co-administration of
dopamine antagonists with NMDA into the ventral hippocampus blocks the expected
increase in locomotor behavior observed when NMDA is administered alone (Gimenez-
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Llort et al., 2002). Thus, strong parallels exist between the neural circuitry and
pharmacology underlying the regulation of locomotor activity by the ventral
hippocampus and that commonly described for schizophrenia.
On the other hand, the mechanisms underlying the disruption of PPI following
stimulation of the ventral hippocampus are less clear. As neither dopamine nor glutamate
antagonists reverse the PPI disruptions following ventral hippocampal stimulation (Wan
et al., 1996;Bast et al., 2001), direct activation of glutamatergic afferents from the ventral
hippocampus to the nucleus accumbens is unlikely to underlie these effects. A recent
study provides further support for this notion by demonstrating that lesions of the fornix,
a tract containing the ventral hippocampal efferents to the nucleus accumbens, fail to
block the disruption of PPI following stimulation of the ventral hippocampus with
NMDA (Swerdlow et al., 2004). Therefore, the modulation of PPI by the ventral
hippocampus likely involves either (1) interactions with brain areas other than those
directly implicated in locomotor control (i.e., areas other than the nucleus accumbens) or
(2) poly-synaptic pathways to the nucleus accumbens that do not include projections via
the fornix.
One potential circuit that may underlie the effects of stimulation of the ventral
hippocampus includes the ventral hippocampal projections to the perirhinal and
entorhinal cortex (Swerdlow et al., 2004). These areas have been implicated in the
modulation of PPI (Swerdlow et al., 2001b;Goto et al, 2002) and are connected, via non-
fornical routes, to structures such as the nucleus accumbens (Totterdell and Meredith,
1997), basolateral amygdala (Pitkanen et al., 2000), and medial prefrontal cortex (Insausti
et al., 1997) that are also involved in the modulation of PPI (Swerdlow et al., 2001a).
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Importantly, given that the mechanisms through which stimulation of the ventral
hippocampus disrupts PPI are currendy unknown, it has been suggested that further
understanding of this phenomenon may aid in the development of novel therapeutic
strategies for schizophrenia (Bast and Feldon, 2003).
The Utility of Adult Animal Models of Schizophrenia
As was discussed in Chapter One, animal models of schizophrenia created by
manipulations performed during adulthood generally have poor construct validity given
numerous lines of evidence that support a developmental component to the etiology of
the disorder. However, adult models of schizophrenia can still contribute to a better
understanding of the disorder in a number of different ways. For example, novel insights
into the neural substrates underlying the behavioral symptoms of schizophrenia can be
gained by performing experiments using adult animals. Support for this assertion is
provided by data presented in Chapter Two that suggest increased ventral hippocampal
activity in adulthood may underlie some symptoms of schizophrenia. Interestingly,
results from brain imaging studies confirm the importance of these preclinical findings by
demonstrating that basal levels of hippocampal activity are increased in schizophrenia
(Heckers et al., 1998;Benes, 2000;Heckers, 2001).
Additionally, the present results (Howland et al., 2004b), and those of other
researchers (Zhang et al., 2002), suggest that the ventral hippocampus may be more
involved in the regulation of sensorimotor processes such as PPI and locomotor activity
than the dorsal hippocampus. As a result, examining alterations in specific sub-regions
of the hippocampus in patients with schizophrenia may reveal previously unappreciated
characteristics of the neurobiology of the disorder (Goldman and Mitchell, 2004). In
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humans, the anterior portion of the hippocampus corresponds to the rodent ventral
hippocampus, and some studies indicate that this region may be preferentially altered in
schizophrenia (Csernansky et al., 1998;Szeszko et al., 2003;Narr et al., 2004; but see also
Narr et al., 2001;Velakoulis et al., 2001).
Furthermore, experiments with adult rats enable basic knowledge of the neural
mechanisms underlying performance of behavioral tasks to be generated quickly. This
knowledge can then be used to assess the potential mechanisms underlying behavioral
deficits following developmental manipulations. This type of strategy was used to
demonstrate that the PPI disruptions observed in socially isolated rats are dependent on
dopamine levels in the nucleus accumbens (Powell et al., 2003) and extensively by
Lipska and colleagues regarding various aspects of the altered neural circuitry and
pharmacology underlying changes in the neonatal ventral hippocampal lesion model
(Lipska et al., 1998;Lipska and Weinberger, 2000). Thus, combining information gained
from both adult and developmental approaches will likely enable a more rapid and
thorough understanding of the disorder (Floresco et al., 2005).
Developmental Models of Schizophrenia - Effects of Early Postnatal Glutamate
Manipulations
Clearly, the results gained from both the neonatal kainic acid and Ro25-6981
manipulations are not as strong as was desired at the outset of the experiments. However,
a number of interesting observations have been gained from these data, many of which
should be verified in future experiments. The most important result was the subtle and
reliable decrease in PPI observed following kainic acid administration in Chapter Three.
Consistent with the ventral hippocampal lesion model, the PPI deficits were observed
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only after the animals had reached early adulthood. However, these deficits were not
augmented by apomorphine administration or accompanied consistently by changes in
locomotor activity, as is the case with the PPI deficits following neonatal ventral
hippocampal lesions (Lipska et al., 1993;Lipska et al., 1995).
As was discussed in the introduction, some first degree relatives of patients with
schizophrenia exhibit lower PPI responses than matched controls (Cadenhead et al.,
2000). Thus, disrupted PPI may be considered a candidate marker for a 'trait' (or
underlying predisposition) to develop schizophrenia. Accordingly, the kainic acid model,
while not reproducing the full-blown expression of schizophrenia, may be useful as a
method of producing an underlying predisposition for the disorder in rodents. Further
support for this assertion is gained from studies suggesting that the hippocampus is also
altered in first degree relatives of schizophrenic patients (Seidman et al., 2002) and in
adult rats by neonatal kainic acid administration (Lynch et al., 2000).
Assessing the interaction of neonatal kainic acid administration with additional
manipulations may support the notion that schizophrenia results from multiple 'hits' over
the course of development (McCarley et al., 1999;Bayer et al., 1999;Lewis and Levitt,
2002;Wong and Van Tol, 2003;Ellenbroek, 2003). To date, such strategies have been
rarely used in an attempt to model the disorder although in one report, rats with neonatal
ventral hippocampal lesions treated repeatedly with PCP in early adulthood displayed
enhanced locomotor responses when compared to lesioned rats that had not been treated
with PCP (Hori et al., 2000). Support for this experimental design is provided by
observations indicating that neonatal kainic acid administration renders rats more
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sensitive to morphological damage and behavioral impairments following kainic acid
administration in adulthood (Koh et al., 1999).
It is disappointing that the long-term behavioral experiments performed with
Ro25-6981 failed to yield positive results. However, investigating novel mechanisms of
brain development in the context of schizophrenia will improve understanding of the
disorder. The rational for the use of Ro25-6981 was conceived from detailed knowledge
surrounding the dynamic changes of specific NMDA subunits during early development,
and similar experiments developed to test mechanistically-driven hypotheses will likely
be an important component of preclinical schizophrenia research in the future.
Further consideration of the null results of the experiments assessing the
behavioral effects of neonatal administration of the NR2B antagonist Ro25-6981 can be
focused in two main directions: (1) why the experiments performed fail to yield
significant results and (2) what conclusions may be drawn from these experiments with
respect to the existing literature regarding early developmental animal models of
schizophrenia.
Although the experiments using Ro25-6981 were carefully designed, the effects
of this drug have not been extensively studied, especially in regard to the developing
mammalian brain. In contrast, the acute effects of kainic acid administration are well
known during development, and fit logically with some conceptions of the etiology of
schizophrenia (Howland et al., 2004a). For example, kainic acid administration early in
the neonatal period alters the activity of the hippocampus (Stafstrom et al., 1992;Khalilov
et al., 1999;Lynch et al., 2000;Silveira et al., 2002) and additional limbic and cortical
areas including the entorhinal cortex (Khalilov et al., 1999) implicated in schizophrenia.
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Additionally, rats treated neonatally with kainic acid exhibit subtle physiological
alterations in the hippocampus during adulthood, which may underlie some of the
behavioral alterations observed in these animals (Lynch et al., 2000). To my knowledge,
the present experiments are the first to assess the effects of Ro25-6981 administration in
neonatal rats. In retrospect, it may have been profitable to more carefully assess the acute
effects of Ro25-6981 administration on the neonatal brain before the long-term
behavioral experiments described herein were performed.
Two main avenues will likely be especially profitable for understanding both the
acute and long-term effects of neonatal Ro25-6981 administration on brain and behavior.
Initially, determining which brain areas are activated with either convulsive or sub-
convulsive doses of the drug will enable the development of a more detailed hypothesis-
driven approach regarding the potential long-term behavioral effects of the convulsions
induced by Ro25-6981. I expect that the activity of structures such as the hippocampus,
cortex, and striatum will be altered by administration of Ro25-6981 due to high levels of
NR2B expression in those areas neonatally (Monyer et al., 1994;Loftis and Janowsky,
2003), and the well documented role of these areas in seizures (Ben Ari and Cossart,
2000). However, high levels of NR2B-containing NMDA receptors are also expressed in
other areas including the thalamus and cerebellum at this developmental age (Loftis and
Janowsky, 2003). Interestingly, connections between these areas and the frontal cortex
have been implicated in schizophrenia (Andreasen et al., 1999;Konarski et al., 2005).
Thus, altered activity patterns in distributed neural circuits may underlie the convulsions
produced by Ro25-6981.
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Secondly, an assessment of the effects of Ro25-6981 administration on cell
survival in the developing brain is essential. As detailed in Chapter Four, a number of
competitive and non-competitive NMDA receptor antagonists induce massive cell death
in the developing brain, a factor which may be important in the long-term behavioral
effects of their administration (Ikonomidou et al., 1999;Wang et al, 2001;Harris et al.,
2003;Fredriksson et al., 2004). The influence of Ro25-6981 on cell survival in the
developing brain is currently unknown, however the high proportion of NR2J3-containing
NMDA receptors early in development supports the hypothesis that treatment with Ro25-
6981 may cause apoptosis during this developmental age.
Although a detailed understanding of the effects of Ro25-6981 administration is
lacking, the results of the behavioral experiments conducted prior to and after puberty are
still surprising as the induction of three strong convulsions during the end of the postnatal
week failed to alter PPI or locomotor responding. One factor that could be especially
important in the different behavioral effects observed between the convulsions elicited by
kainic acid and Ro25-6981 is that convulsions elicited by kainic acid are significantly
longer in duration than those elicited with Ro25-6981. Longer convulsions may disturb
developing neural networks to a significantly greater extent than shorter ones (Jensen and
Baram, 2000;Holmes, 2004). Previous studies using other convulsive agents such as
flourothyl, which have a short duration of action (10 to 15 minutes), support this
assertion. These experiments indicate that a series of 25 to 50 convulsions during the
first week of life must be elicited for long-term alterations in behavior to be observed
(Holmes et al., 1998;Huang et al., 1999). Further support for this hypothesis is gained
from data suggesting that a history of prolonged febrile seizures is a significant risk factor
147

for psychosis in adulthood epileptics (Kanemoto et al., 2001) and that prolonged febrile
seizures result in long-term changes in hippocampal excitability in rodents (Chen et al.,
1999;Jensen and Baram, 2000). Therefore, it is tempting to speculate that repeated
convulsions induced by Ro25-6981 during the first postnatal week would be related to
alterations in PPI, locomotor activity, and cognitive deficits in adult rats.
Developmental Models of Schizophrenia - The Time Course of Symptom Emergence
Although developmental animal models of schizophrenia are likely more
ecologically valid than adult models, they are much more difficult to establish. One of
the cardinal features of the majority of patients with schizophrenia is the delayed onset of
many symptoms of the disorder (Marcotte et al., 2001;Wong and Van Tol, 2003),
however, this feature of the disorder remains elusive in animal models (Marcotte et al.,
2001). Research examining recovery of function after brain injury generally supports the
assertion that behavioral impairment is less severe if the injury is suffered early in life - a
phenomenon commonly termed the Kennard principle (Kolb et al., 2000;Marcotte et al.,
2001). However, it is important to note that the pattern of recovery from injuries
sustained during early brain development show some exceptions to the Kennard
principle. For example, lesions of the frontal cortex induced at specific developmental
stages (i.e. late during the embryologic period or after the first postnatal week) result in
significant recovery over time, while other periods exist (i.e. the first postnatal week)
where recovery from similar injuries is significantly poorer than expected (Kolb and
Whishaw, 1989;Kolb et al., 2000).
In the case of schizophrenia, the relation between adverse events early in
development and subsequent manifestation of symptoms suggest a delayed response in
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which behavioral deficits emerge after a period of relative normality. The neonatal
ventral hippocampal lesion model provides important proof that alterations in the neural
substrates implicated in schizophrenia during certain developmental stages may result in
the delayed onset of various behavioral and electrophysiological alterations related to the
disorder (Lipska et al., 1993;Lipska et al., 1995;Wood et al., 1997;Lipska and
Weinberger, 2000;Lipska et al, 2002;Goto and O'Donnell, 2002). Additional research
suggests that manipulations other than ventral hippocampal lesions such as immune
activation (Zuckerman et al., 2003), administration of the anti-mitotic agent arabinoside
(Elmer et al., 2004), or kainic acid (Howland et al., 2004a) result in the delayed onset of
behavioral abnormalities associated with schizophrenia (unfortunately, unlike the design
employed in this thesis, the behavioral effects of many early neonatal treatments
developed have not been tested before adulthood). Taken together, these data suggest
that certain critical periods exist in the neonatal brain for the normal development of the
neural circuits mediating behaviors commonly ascribed to schizophrenia in animals
(Lipska and Weinberger, 2000;Marcotte et al., 2001;Howland et al., 2004a). Future work
related to the mechanisms underlying these changes will likely provide novel insights
into the etiology of the disorder.
Criteria for Establishing Validity in Behavioral Models of Schizophrenia
The present experiments relied heavily on PPI and locomotor behavior in
assessing the experimental manipulations performed in modeling schizophrenia.
Although these behaviors have been used extensively by researchers in the field, they
have a number of shortcomings. Importantly, PPI is disrupted in a number of
neurological and psychiatric disorders in addition to schizophrenia including
149

Huntington's disease, Tourette's syndrome, obsessive-compulsive disorder, and
pathological gambling (Braff et al., 2001). Given the complexity of the neural circuitry
mediating PPI this is not particularly surprising; however, it is problematic in that
disrupted PPI may not be characteristic of schizophrenia per se, but rather of alterations
in the distributed circuitry mediating it (Swerdlow et al., 2001a).
As previously discussed, increased locomotor activity is often used as an indirect
measure of ventral striatal dopamine activity, especially when combined with the
administration of dopamine agonists (Kelly et al., 1975;Castall et al., 1977;Porrino et al.,
1984;Lipska and Weinberger, 2000;Marcotte et al., 2001). Unfortunately, altered
locomotor behavior is a very general phenomenon and can be influenced by a number of
factors including anxiety levels, alterations in sensory processing, and memory
disruptions (Bast and Feldon, 2003). Although some parallels between increased
locomotor activity and certain symptoms of schizophrenia have been suggested (Bast and
Feldon, 2003), these assertions are clearly speculative (Marcotte et al., 2001).
As a result of these shortcomings, testing PPI and locomotor activity levels should
be viewed as an important 'first-pass' in the assessment of potential animal models of
schizophrenia. Future experiments with an array of behaviors relevant to schizophrenia
will serve to strengthen the validity of putative models of schizophrenia (Lipska and
Weinberger, 2000). Recent interest has been generated around preclinical correlates of
the negative and cognitive symptoms of schizophrenia as they have traditionally been
ignored in much of the literature (Ellenbroek and Cools, 2000;Floresco et al., 2005).
Tests assessing social interaction, reward sensitivity, and executive functions such as
cognitive set shifting and working memory are likely to be especially useful in further
150

understanding the more complicated and treatment-resistant symptoms of the disorder
(Ellenbroek and Cools, 2000;Robbins, 2004;Floresco et al., 2005). It is worth pointing
out, however, that many individuals with schizophrenia do not exhibit all symptoms of
the disorder (Wong and Van Tol, 2003). Therefore, reliance on strategies that
successfully model many aspects of the disorder in animals may be counterproductive in
some instances.
Interestingly, some manipulations, such as neonatal ventral hippocampal lesions,
produce changes in many behaviors resembling schizophrenia (Lipska and Weinberger,
2000), while others, such as certain neonatal NMDA antagonist treatment regimes,
produce selective deficits on certain behavioral tasks relevant to one class of symptoms
(e.g. cognitive symptoms) (Stefani and Moghaddam, 2005). The present data do not
indicate how animals treated with either kainic acid or Ro25-6981 may respond if further
testing was conducted. However, in some studies, rats treated neonatally with kainic acid
display deficits in learning and memory (Lynch et al., 2000), although null findings have
also been reported (Stafstrom et al., 1993). Therefore, in the future, it may be profitable
to greatly extend the test battery beyond the tests presently employed.
Finally, testing the efficacy of antipsychotic drugs at reversing the behavioral
disturbances observed following neonatal kainic acid administration could greatly aid in
assessing its validity as a model of schizophrenia. As has been noted throughout the
present dissertation, both typical and atypical antipsychotics are effective at ameliorating
the behavioral disturbances, including PPI deficits, in numerous animal models of
schizophrenia (Lipska and Weinberger, 2000;Marcotte et al., 2001;Bast et al., 2001 ;Le
Pen and Moreau, 2002;Bast and Feldon, 2003;Van den et al., 2003;Le Pen et al., 2003).
151

Conclusion
In the introduction, three main goals for developing animal models of
schizophrenia were introduced: (1) advancing the understanding of the symptoms of the
disorder, (2) furthering the understanding of schizophrenia's etiology, and (3) aiding in
the development of pharmaceutical therapies (Lipska and Weinberger, 2000). The
present dissertation explored two general strategies for developing behaviorally-oriented
animal models of schizophrenia. Although the results were somewhat mixed, the
experiments were successful at providing novel insights into the symptoms and etiology
of the disorder. In general, they support the assertion that short periods of altered activity
in the limbic system, and hippocampus in particular, at different points during
development may underlie the expression of some of the most basic symptoms of
schizophrenia. These data also suggest that the nature and anatomical location of these
alterations critically determines their long-term functional effects.
152

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