APPROACHES TO MODELING SCHIZOPHRENIA FN THE RAT by ...
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<strong>APPROACHES</strong> <strong>TO</strong> <strong>MODELING</strong> <strong>SCHIZOPHRENIA</strong> <strong>FN</strong> <strong>THE</strong> <strong>RAT</strong><br />
<strong>by</strong><br />
JOHN GEORGE HOWLAND<br />
B A. (Hons.), University of Saskatchewan, 1999<br />
M.A., The University of British Columbia, 2001<br />
A <strong>THE</strong>SIS SUBMITTED IN PARTIAL FULFILMENT OF<br />
<strong>THE</strong> REQUIREMENTS FOR <strong>THE</strong> DEGREE OF<br />
DOC<strong>TO</strong>R OF PHILOSOPHY<br />
in<br />
<strong>THE</strong> FACULTY OF GRADUATE STUDIES<br />
(Psychology)<br />
<strong>THE</strong> UNIVERSITY OF BRITISH COLUMBIA<br />
October 2005<br />
© John George Howland, 2005
ABSTRACT<br />
Schizophrenia is a complicated and variable disorder that is notoriously difficult<br />
to study. Converging lines of evidence support the hypothesis that schizophrenia is<br />
characterized <strong>by</strong> a diverse array of distributed changes in limbic and cortical areas of the<br />
brain involving abnormalities in dopamine and glutamate transmission. Furthermore,<br />
genetic and behavioral studies indicate that abnormalities in normal development<br />
contribute to the etiology of the disorder. The present dissertation used two general<br />
strategies in an attempt to model some of the basic characteristics of the disorder. In<br />
Chapter Two, experiments were conducted that demonstrate short periods of higher<br />
frequency stimulation applied to the ventral, but not dorsal, hippocampus in adult rats<br />
reversibly reduce prepulse inhibition, a pre-attentive processing mechanism that is<br />
disrupted in schizophrenic patients. In Chapters Three and Four, the behavioral effects of<br />
reversible pharmacological manipulation of glutamate receptors early in development on<br />
prepulse inhibition and locomotor activity were assessed both before and after puberty.<br />
Additional experiments tested the putative role of dopaminergic abnormalities following<br />
these manipulations. Results from Chapter Three demonstrate that administration of a<br />
convulsive dose of the glutamate receptor agonist kainic acid to neonatal rats on postnatal<br />
day seven results in the delayed emergence of PPI deficits in early adulthood. Levels of<br />
locomotor activity were not reliably altered in a novel environment or following<br />
amphetamine administration. The experiments conducted in Chapter Four were designed<br />
to assess alterations in prepulse inhibition and locomotor activity following<br />
administration of the NR2B-subunit selective NMDA antagonist Ro25-6981.<br />
Unexpectedly, Ro25-6981 administration resulted in behavioral convulsions when
administered during the first postnatal week; however, no consistent behavioral<br />
abnormalities were revealed in rats treated with the drug. Although the present results<br />
are somewhat mixed, the experiments were successful at providing novel insights into the<br />
symptoms and etiology of schizophrenia. In general, they support the assertion that short<br />
periods of altered activity in the limbic system, and hippocampus in particular, at<br />
different points during development may underlie the expression of some of the most<br />
basic symptoms of schizophrenia. These data also suggest that the nature and anatomical<br />
location of these alterations critically determines their long-term functional effects.<br />
iii
TABLE OF CONTENTS<br />
ABSTRACT ii<br />
TABLE OF CONTENTS iv<br />
LIST OF TABLES viii<br />
LIST OF FIGURES ix<br />
ACKNOWLEDGMENTS xi<br />
CO-AUTHORSHD? STATEMENT xii<br />
CHAPTER ONE: GENERAL INTRODUCTION 1<br />
Schizophrenia 2<br />
Brief Overview of the Disorder 2<br />
Etiological Theories 7<br />
Animal Models 8<br />
Genetic Models 11<br />
Adult Pharmacological Models 12<br />
Adult Lesion Models 14<br />
Developmental Animal Models 16<br />
Maternal Separation and Social Isolation Models 16<br />
Neonatal Lesion Models 18<br />
Neonatal Pharmacological Models 22<br />
References 24<br />
CHAPTER TWO: ELECTRICAL STIMULATION OF <strong>THE</strong> HIPPOCAMPUS<br />
DISRUPTS PREPULSE INHIBITION IN <strong>RAT</strong>S: FREQUENCY AND SITE<br />
DEPENDENT<br />
EFFECTS 37<br />
Introduction 37<br />
Methods 41<br />
Subjects 41<br />
Surgery 41<br />
Prepulse Inhibition Testing 42<br />
Neurochemical Experiments 44<br />
Histology 46<br />
Data Analysis 46<br />
Results 47<br />
Immediate Behavioral Effects of Stimulation 47<br />
Differential Effects of 20 Hz Stimulation of the vHip or dHip on PPI and Startle<br />
Amplitude : 48<br />
iv
Effects of 2 Hz Stimulation of the vHip on PPI and Startle Amplitude 50<br />
Effects of Stimulation of the vHip and dHip on NAc DA Efflux 52<br />
Histology 52<br />
Discussion 55<br />
Potent Disruption of Sensorimotor Gating Induced <strong>by</strong> 20 Hz Stimulation of the vHip,<br />
but not dHip 55<br />
Frequency Dependence of the vHip Stimulation-induced Disruption of PPI 57<br />
Increases in NAc DA Efflux following Hip Stimulation are Site and Hemisphere<br />
Specific 59<br />
Potential Mechanisms Underlying vHip Stimulation-induced Disruption in PPI 61<br />
Conclusion 62<br />
References 63<br />
CHAPTER THREE: DELAYED ONSET OF PREPULSE INHIBITION DEFICITS<br />
FOLLOWING KAINIC ACID TREATMENT ON POSTNATAL DAY SEVEN IN<br />
<strong>RAT</strong>S 70<br />
Introduction 70<br />
Methods 72<br />
Subjects 72<br />
Kainic Acid Administration 73<br />
Prepulse Inhibition 74<br />
Locomotor Activity 75<br />
Water Maze Testing 76<br />
Histology ". 77<br />
Data Analysis 77<br />
Results ., 78<br />
Immediate Behavioral Effects ofPND7 KA Administration 78<br />
Effects of PND7 KA Administration on Body Weight 79<br />
Prepulse Inhibition is Reduced in Post-Pubescent, but not Pre-pubescent Rats,<br />
Following PND7 KA Administration 81<br />
Locomotor Activity in Response to Novelty and Amphetamine in Rats Following PND7<br />
KA Administration 84<br />
Spatial Learning and Memory in the Morris Water Maze is not Altered in Rats<br />
Following PND7 KA Administration 87<br />
Histology 89<br />
Discussion 91<br />
Effects of Neonatal KA Administration on PPI 91<br />
Effects of Neonatal KA on Locomotor Activity 93<br />
Potential Mechanisms Underlying the Observed PPI Changes 94<br />
Conclusion 97<br />
References 99<br />
CHAPTER FOUR: BEHAVIORAL CONVULSIONS INDUCED BY EARLY<br />
POSTNATAL ADMINIST<strong>RAT</strong>ION OF <strong>THE</strong> NR2B ANTAGONIST R025-6981<br />
FAIL <strong>TO</strong> AFFECT SENSORIMO<strong>TO</strong>R GATING OR LOCOMO<strong>TO</strong>R
BEHAVIOR IN PRE- AND POST-PUBESCENT<br />
<strong>RAT</strong>S..... : 105<br />
Introduction 105<br />
Methods 110<br />
Experiment 1 - Incidence and Characteristics of Behavioral Convulsions in Postnatal<br />
Rats Following Antagonism of NMDA Receptors Containing the NR2B Subunit.... 110<br />
Subjects 110<br />
Experimental Procedures 110<br />
Experiment 2 - Long-term Behavioral Effects of Convulsions Resulting from Antagonism<br />
of NMDA Receptors Containing the NR2B Subunit 1111<br />
Subjects Ill<br />
Ro25-6981 Administration Ill<br />
Prepulse Inhibition 112<br />
Spontaneous Locomotor Activity 113<br />
Dopaminergic Challenges of PPI and Locomotor Activity 113<br />
Data Analysis 114<br />
Results 115<br />
Experiment 1 - Behavioral Effects ofRo25-6981 Administration in the Early Postnatal<br />
Period 115<br />
Experiment 2 - Effects of Neonatal Ro25-6981 Administration on Body Weight 116<br />
Neonatal Ro25-6981 Administration Does not Alter PPI Responding Either before<br />
Puberty or in Early Adulthood 118<br />
Apomorphine Challenge in Adulthood has Similar Effects on PPI in Neonatally<br />
Saline- or Ro25-6981 -treated Rats 121<br />
Neonatal Treatment with Ro25-6981 and Amphetamine-Induced Locomotor Activity in<br />
Adult Rats 121<br />
Discussion 125<br />
Antagonism of NR2B-containing NMDA Receptors Induces Behavioral Convulsions<br />
126<br />
The Long-Term Behavioral Effects of Ro25-6981 on PPI and Locomotor Activity.. 129<br />
Conclusion 131<br />
References 132<br />
CHAPTER FIVE: GENERAL DISCUSSION 138<br />
The Role of the Hippocampus in the Regulation of Prepulse Inhibition and Locomotor<br />
Activity 139<br />
The Utility of Adult Animal Models of Schizophrenia 142<br />
Developmental Models of Schizophrenia - Effects of Early Postnatal Glutamate<br />
Manipulations 143<br />
Developmental Models of Schizophrenia - The Time Course of Symptom Emergence<br />
148<br />
Criteria for Establishing Validity in Behavioral Models of Schizophrenia 149<br />
Conclusion 152<br />
References 153<br />
vi
LIST OF TABLES<br />
Table 4-1. The incidence of behavioral convulsions in rats administered<br />
NR2B-selective NMDA antagonists at various postnatal ages 117<br />
vii
LIST OF FIGURES<br />
Figure 2-1. Effects of 20 Hz stimulation of the hippocampus on PPI and<br />
acoustic startle amplitude 49<br />
Figure 2-2. Effects of 2 Hz stimulation of the vHip on PPI and acoustic<br />
startle amplitude. 51<br />
Figure 2-3. Unilateral stimulation (20 Hz, 10 s) of either the vHip or dHip,<br />
and its effect on NAc DA efflux in the ipsilateral or contralateral hemisphere 53<br />
Figure 2-4. Schematic diagram of the placements of stimulating electrodes and<br />
microdialysis probes in all experiments 54<br />
Figure 3-1. Prepulse inhibition scores from rats in Group 1 treated on<br />
postnatal day 7 with saline or kainic acid 80<br />
Figure 3-2. Prepulse inhibition scores from rats in Group 2 treated on<br />
postnatal day 7 with saline or kainic acid 83<br />
Figure 3-3. Effects of pretreatment with vehicle or apomorphine on<br />
average percent PPI scores in adulthood 85<br />
Figure 3-4. Locomotor activity levels in response to novelty or amphetamine<br />
of all rats tested on either postnatal day 36 or 57 86<br />
Figure 3-5. Average latencies to locate the hidden platform in the water<br />
maze of rats treated with saline or kainic acid on postnatal day 7 88<br />
Figure 3-6. A representative cresyl violet stained section of the dorsal<br />
hippocampus of a KA-treated and saline-treated rat 90<br />
Figure 4-1. Cartoon illustrating an NMDA receptor within the cell membrane 107<br />
Figure 4-2. Prepulse inhibition scores of rats in Group 1 treated on<br />
postnatal day 6 and 7 with saline or Ro25-6981 119<br />
Figure 4-3. Prepulse inhibition scores from rats in Group 2 treated on<br />
postnatal day 6 and 7 with saline or Ro25-6981 120<br />
Figure 4-4. Effects of pretreatment with vehicle or apomorphine on<br />
percent PPI scores in adulthood 122<br />
Figure 4-5. Locomotor activity levels in response to novelty or<br />
amphetamine of rats tested on either postnatal day 36 or 57 in Group 1 123<br />
viii
Figure 4-6. Locomotor activity levels in response to novelty or amphetamine of rats<br />
tested on either postnatal day 36 or 57 in Group 2. 124
ACKNOWLEDGMENTS<br />
The work contained within this dissertation would not have been completed without the<br />
help of many people. Those who directly assisted me with these projects are co-authors<br />
on my publications. Many of those who indirectiy assisted me in the lab are listed<br />
alphabetically below. I would like to especially acknowledge the wisdom and expert<br />
advise of Drs. Tony Phillips, Stanley B. Floresco, and Darren 'Twinkle-Toes' Hannesson<br />
throughout my graduate career and Dr. Karen Brebner for providing many helpful<br />
comments on the General Introduction and Discussion Sections of my dissertation.<br />
Finally, I would like to acknowledge NSERC, CIHR, and the Michael Smith Foundation<br />
for Health Research for funding.<br />
Thank you:<br />
Soyon Ahn, Steven Barnes, Alasdair Barr, Karen Brebner, Deanna Chavez, Christina<br />
Cheng, Michael Corcoran, Carine Dias, Brennan Eadie, Rachel Genn, Rosie Gilham,<br />
Natalia Gorelova, Lucy Greggorios-Pippas, Rebecca Harrison, Fred Lepiane, Sarah C.<br />
Lidstone, Brandi Ormerod, Julie Pongrac, Kitty So, Aline Stephan, Christina Thorpe,<br />
Giada Vacca, and liana Winrob.<br />
To my friends and family who may not have helped directly with my academic life, but<br />
in many other ways, don't worry, your thanks is coming (and you're likely not reading<br />
this anyway). USB UBC.
CO-AUTHORSHIP STATEMENT<br />
I am the first author on all manuscripts presented in this thesis. I was directly<br />
involved in all stages of the research including, but not limited to, conception of the<br />
ideas, designing the experiments, conducting the studies, analyzing the data, and writing<br />
the manuscripts. Research assistants and other students helped in gathering some of the<br />
data presented in this dissertation and they are included as authors on the manuscripts<br />
with which they were involved. Dr. Phillips was also involved in all aspects of the<br />
research included in this dissertation. He also acted as my PhD supervisor and is<br />
included as an author on all manuscripts.
CHAPTER ONE: GENERAL INTRODUCTION<br />
The present dissertation summarizes several novel approaches to the development<br />
of rodent models of schizophrenia that are reliable and valid. As will become clear in the<br />
introduction, schizophrenia is an incredibly complicated and variable disorder (or group<br />
of disorders). Therefore, it is difficult to put forth a unitary set of criteria <strong>by</strong> which to<br />
verify the success of putative models of schizophrenia. The following pages detail a<br />
number of strategies aimed at reproducing some characteristics of the disorder. As the<br />
experiments of Chapter Two demonstrate, acute changes in hippocampal activity in<br />
adulthood reversibly alter behavior in a manner consistent with schizophrenia.<br />
Additionally, Chapters Three and Four show that under some circumstances, transient<br />
disruption of normal activity patterns in the developing brain is sufficient to subtiy<br />
change adult behavior in a manner consistent with schizophrenia. Finally, the<br />
implications of these studies and a number of directions for future research are discussed<br />
in Chapter Five.<br />
This dissertation is presented in a manuscript-based format, rather than the<br />
traditional thesis format. Chapters Two through Four are stand-alone manuscripts,<br />
containing their own Introduction and Reference sections, while Chapter One and Five<br />
are the General Introduction and Discussion sections of the entire dissertation. While this<br />
format has numerous practical advantages, it has two clear limitations. First, there is<br />
inevitably a certain amount of repetition between chapters, and second, individual<br />
chapters do not necessarily flow into the next. In an effort to address the second problem,<br />
I will include a brief statement of the objectives of each chapter in the General<br />
Introduction, and summarize the findings from all chapters in the General Discussion.<br />
1
Schizophrenia<br />
Brief Overview of the Disorder<br />
Schizophrenia is a debilitating mental illness with a lifetime prevalence of 1.4-4.6<br />
per 1000 people worldwide (Jablensky, 2000). Symptoms usually appear in late<br />
adolescence or early adulthood, although cases with earlier and later onset certainly exist<br />
(Wong and Van Tol, 2003). For many patients, schizophrenia has a chronic course with<br />
unpredictable relapses that require repeated hospitalizations. Although antipsychotic<br />
drugs effectively reduce symptom severity in many patients, their side effects are severe,<br />
and the drugs often fail to improve many of the negative and cognitive symptoms of the<br />
disorder (see below). It is certainly worth noting that the complexities and<br />
inconsistencies of the disorder are far from understood. As a result, the behavioral<br />
symptoms and neuropathological characteristics of the disorder described below have<br />
been simplified. Within the following chapters, pertinent details related to relevant<br />
aspects of schizophrenia are described where necessary.<br />
As the pharmacology underlying antipsychotics drug action has been used to<br />
support hypotheses related to the neural bases of the symptoms of schizophrenia, a brief<br />
review of their main classes is required. Typical antipsychotic drugs, which antagonize<br />
dopamine receptors, were discovered in the 1950's and revolutionized treatment of<br />
schizophrenia (Wong and Van Tol, 2003). During the 1980's and 1990's, psychiatrists<br />
began prescribing numerous atypical antipsychotic drugs to patients who did not respond<br />
to typical antipsychotics (either due to symptom profiles, side effects, or unknown<br />
reasons). Atypical antipsychotics have unique pharmacological effects, including<br />
2
affinities for serotonin, adrenergic, dopamine, histamine, and muscarinic receptors, which<br />
may underlie their effectiveness in some patients (Wong and Van Tol, 2003).<br />
The behavioral symptoms of schizophrenia are varied and involve numerous<br />
advanced functions of the human brain. Generally, symptoms can be divided into three<br />
main categories: the classic psychotic or 'positive' symptoms, deficit or 'negative'<br />
symptoms, and cognitive impairment (Wong and Van Tol, 2003). Positive symptoms are<br />
those typically associated with the illness - disturbances such as hallucinations,<br />
delusions, and thought disorder. Importantly, the appearance of positive symptoms in<br />
late adolescence or early adulthood generally leads to the diagnosis of schizophrenia. A<br />
number of researchers have argued that altered activity of the dopamine system is the<br />
likely cause of positive symptoms, a hypothesis largely based on the effectiveness of<br />
antipsychotic drugs, which are dopamine D2 receptor antagonists, in reducing positive<br />
symptoms and dopamine agonists at inducing or aggravating positive symptoms (Snyder,<br />
1973;Kapur and Mamo, 2003).<br />
Whereas positive symptoms may be thought of as the presence of abnormal<br />
behaviors, negative symptoms may be regarded as a reduction or loss in a number of<br />
normally occurring behaviors. Symptoms such as social withdrawal, apathy, reduced<br />
expression of affect, and the ability to experience pleasure are common examples of<br />
negative symptoms. The causes and treatment of negative symptoms are more poorly<br />
understood than the positive symptoms, although some atypical antipsychotic drugs, with<br />
actions at receptor subtypes other than the D2 receptor, may be more effective than<br />
typical antipsychotics at improving these symptoms (Meltzer et al., 1994;Wong and Van<br />
Tol, 2003).<br />
3
Finally, impairments in cognitive domains such as pre-attentive processing,<br />
executive function, attention, learning, memory, and general intellectual function are an<br />
important component of schizophrenia (Elvevag and Goldberg, 2000;Sharma and<br />
Antonova, 2003;Lewis, 2004). Intense interest has focused recently on these symptoms<br />
because of studies showing that the cognitive symptoms are the most enduring and<br />
consistently debilitating of the disorder (Elvevag and Goldberg, 2000). Additionally,<br />
impairments in cognition may precede the development of the positive symptoms of the<br />
disorder, there<strong>by</strong> providing a potential tool with which to identify those individuals at<br />
risk for expressing full-blown psychosis in early adulthood (Walker, 1994;Walker et al.,<br />
1994;Jones et al., 1994;Lewis, 2004).<br />
In addition to the behavioral changes observed in those suffering from the<br />
disorder, many alterations in the normal structure and function of the brain have been<br />
reported. For example, an overwhelming array of neuroanatomical changes is reported to<br />
exist in schizophrenia, many of which occur in the frontal and temporal lobes (McCarley<br />
et al, 1999;Wright et al., 2000). Ventricular enlargement (Stevens, 1997;Harrison, 1999)<br />
and reductions in temporal (Lawrie and Abukmeil, 1998;Nelson et al., 1998) or frontal<br />
lobe volumes (Wright et al., 1999) are observed in the brains of schizophrenic patients.<br />
Reduced volume of the hippocampus (Suddath et al., 1990;Noga et al., 1996;Bogerts,<br />
1997;Heckers, 2001), reduced cell numbers in the hippocampus (Gothelf et al.,<br />
2000;Heckers, 2001), nucleus accumbens (NAc; Pakkenberg, 1990), and thalamus<br />
(Jones, 1997;Clinton and Meador-Woodruff, 2004), and altered neuronal size and density<br />
in certain frontal cortical areas (Harrison, 1999) have been observed in patients with<br />
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<br />
prefrontal activation (McClure and Weinberger, 2001;Meyer-Lindenberg et al., 2002)<br />
during the performance of a variety of cognitive tasks are also characteristic of<br />
schizophrenic patients (Wong and Van Tol, 2003). Taken together, these<br />
neuroanatomical changes may be considered an enduring trait for schizophrenia in<br />
adulthood. Interestingly, relatives of patients with schizophrenia may also have reduced<br />
cortical volumes (Cannon et al., 1993), altered hippocampi (Seidman et al., 2002) and<br />
enlarged ventricles (Honer et al., 1994;Lawrie et al., 1999), there<strong>by</strong> supporting the<br />
assertion that an underlying genetic abnormality may predispose individuals to develop<br />
the disorder, although this underlying 'trait' for schizophrenia may have to interact with<br />
other environmental factors to result in the expression of a schizophrenic 'state' in early<br />
adulthood. Such conceptions of the etiology of schizophrenia are termed 'two-hit'<br />
hypotheses <strong>by</strong> some authors (McCarley et al., 1999;Bayer et al., 1999;Wong and Van<br />
Tol, 2003;Ellenbroek, 2003).<br />
The neurochemical changes present in schizophrenia are also complex and<br />
incompletely understood. The affinity of typical antipsychotics for the D2 receptor and<br />
psychotomimetic agents such as amphetamine and phencyclidine for the dopamine<br />
transporter and the N-methyl-D-aspartate (NMDA) receptor, respectively, are suggestive<br />
of the importance of dopamine and glutamate neurotransmission in schizophrenia<br />
(Angrist et al., 1974;Jentsch and Roth, 1999). More direct evidence of abnormal<br />
dopamine-glutamate interactions is gained from studies suggesting that alterations in<br />
glutamate and glutamate receptor levels may be present in schizophrenia (for review, see<br />
Goff and Coyle, 2001) and that schizophrenic patients show elevated synaptic levels of<br />
5
dopamine (Abi-Dargham et al., 2000) and potentiated increases in striatal dopamine<br />
release in response to amphetamine administration during episodes of acute psychosis<br />
(Breier et al, 1997;Abi-Dargham et al., 1998;Laruelle et al., 1999;Laruelle et al., 2003)<br />
and periods of relative remission (McGowan et al., 2004). Current theories of cortico-<br />
striatal-limbic function and schizophrenia have begun to delineate mechanisms <strong>by</strong> which<br />
these neurochemical changes and developmental abnormalities may manifest themselves<br />
as the symptoms of schizophrenia. One such example is the tonic-phasic model of<br />
ventral striatal dopamine release proposed <strong>by</strong> Grace (Grace, 1991). The tonic-phasic<br />
model posits that in schizophrenia, reduced activity from cortical glutamatergic afferents<br />
to the ventral striatum reduces basal (or tonic) levels of dopamine in the ventral striatum.<br />
Tonic levels of dopamine are thought to regulate the responsivity of the ventral striatum<br />
to short-lasting (or phasic) changes in dopamine efflux in response to environmental<br />
stimuli. As a result, patients with schizophrenia are predicted to exhibit increased<br />
dopaminergic responsivity to salient environmental stimuli, which may be a partial<br />
explanation of the positive symptoms (Grace, 1991). This model, along with others like<br />
it (Carlsson et al., 1999), illustrates the importance of dopamine-glutamate interactions in<br />
the neurobiology of schizophrenia.<br />
Additionally, the therapeutic efficacy of atypical antipsychotic drugs (Emsley and<br />
Oosthuizen, 2003; A wad and Voruganti, 2004) and a variety of additional<br />
neuropathological findings (Costa et al., 2004;Lewis et al., 2005) underlie the importance<br />
of other neurotransmitter systems in schizophrenia. In particular, alterations in the<br />
GABA and serotonin systems may exist in schizophrenia (Costa et al., 2004;Roth et al.,<br />
2004), although they will not be the focus of the present review.<br />
6
Etiological Theories<br />
Currently, the cause(s) of schizophrenia remain poorly defined. Twin and gene<br />
linkage studies provide convincing evidence that a genetic predisposition to develop<br />
schizophrenia exists, although environmental factors must also contribute as concordance<br />
rates between monozygotic twins are approximately 50 percent, whereas for dizygotic<br />
twins and siblings, the risk is approximately 10 percent (Kennedy et al., 2003;Owen et<br />
al., 2004). One particularly prominent etiological theory of the last 15 to 20 years is the<br />
neurodevelopmental theory (Weinberger, 1987;Lewis and Levitt, 2002;Church et al.,<br />
2002;Eastwood, 2004;Rapoport et al., 2005). In its original form, the theory suggests that<br />
genetic abnormalities may alter early brain development, or make the brain more<br />
susceptible to disruption. This 'compromised brain' then interacts with environmental<br />
factors or later occurring events of brain development and ultimately results in the<br />
expression of the symptoms typically observed in schizophrenic patients (Benes,<br />
2000;Benes et al., 2000;McClure and Weinberger, 2001;Lewis and Levitt, 2002).<br />
The neurodevelopmental theory is supported <strong>by</strong> evidence suggesting that people<br />
who develop schizophrenia have an increased incidence of adverse gestational and<br />
perinatal events, cognitive and behavioral abnormalities before diagnosis, congenital<br />
physical abnormalities, and alterations in brain morphology (for reviews see McClure and<br />
Weinberger, 2001;Lewis and Levitt, 2002). Further support is gained from evidence<br />
suggesting that the neuroanatomical changes observed in schizophrenia are present from<br />
the first episode diagnosis and their severity usually does not increase over time<br />
(Weinberger, 1987), although recent data challenges this notion (Weinberger,<br />
1996;Rapoport et al., 1999;Church et al., 2002;Rapoport et al., 2005). Additionally, signs<br />
7
of active neurodegeneration, such as gliosis, are generally absent from the brains of<br />
schizophrenic patients (Roberts et al., 1986;McClure and Weinberger, 2001), although<br />
such observations do not exclude the possibility that active neurodegenerative processes<br />
such as increased rates of apoptosis occur in patients with schizophrenia (Church et al.,<br />
2002).<br />
Animal Models<br />
Animal models have been used extensively in medicine to simplify complex and<br />
poorly understood pathologies. In psychiatry, and specifically in regards to<br />
schizophrenia, the development of reliable and valid animal models has proven difficult.<br />
Obviously, many symptoms of psychiatric disorders are inherently human, and thus can<br />
never be fully modeled in animals. As a result, attempts at modeling disorders such as<br />
schizophrenia in animals have been met with skepticism. Recently, a number of authors<br />
(Geyer and Markou, 1995;Lipska and Weinberger, 2000;Robbins, 2004) have advanced<br />
the notion that valuable and unique insights can be gained from modeling psychiatric<br />
disorders in animals, including rodents. Paramount to this undertaking is a realistic<br />
notion of the goal of the animal model, and the criteria which will be used to verify<br />
whether or not this goal has been achieved.<br />
With respect to schizophrenia, animal models have been developed for at least<br />
three main goals: furthering our understanding of its etiology, advancing our<br />
understanding of the symptoms (both behavioral and biological), and finally, aiding in the<br />
development and understanding of pharmaceutical therapies (Lipska and Weinberger,<br />
2000;Robbins, 2004). The evaluation criteria (or dependent measures) used to validate<br />
models of schizophrenia are quite varied, mostly due to the inherent difficulties of<br />
8
measuring the disorder in animals. Generally, models are designed to replicate the<br />
abnormal neurochemistry/neuropathology or behavioral changes described for<br />
schizophrenia. Within the present thesis, two behavioral tests - prepulse inhibition and<br />
locomotor behavior - are used extensively to validate the experimental manipulations<br />
used to model schizophrenia.<br />
Prepulse inhibition (PPI) of the acoustic startle response is an operational measure<br />
of sensorimotor gating, and is a normal pre-attentive behavioral response in which a weak<br />
sensory event (or prepulse) inhibits, or gates, the motor response to a starding stimulus<br />
(or pulse) (Fig. 1-1; Swerdlow et al., 2000a). PPI is disrupted in patients with<br />
schizophrenia or schizotypal personality disorder (Braff et al., 2001;Hamm et al.,<br />
2001 ;Ludewig et al., 2003) and in the first degree relatives of patients with schizophrenia<br />
(Cadenhead et al., 2000). These data suggest that PPI deficits are not a result of acute<br />
psychosis (Braff et al., 2001; but see also Meincke et al., 2004). Typical antipsychotic<br />
drugs do not ameliorate PPI deficits in schizophrenic patients (Mackeprang et al.,<br />
2002;Duncan et al., 2003a;Duncan et al., 2003b), although some beneficial effects of<br />
atypical antipsychotic drugs have been reported (Braff et al., 2001;Leumann et al.,<br />
2002;Kumari et al., 2002). More sophisticated longitudinal designs are necessary to<br />
further address the efficacy of antipsychotic drugs in reversing the PPI deficits observed<br />
in patients with schizophrenia (Kumari and Sharma, 2002). Importantly, PPI deficits are<br />
correlated with cognitive and behavioral symptoms such as distractibility, social<br />
perception, and thought disorder (Perry and Braff, 1994;Karper et al., 1996;Braff et al.,<br />
1999;Perry et al., 1999;Swerdlow et al., 2000a;Meincke et al., 2004;Wynn et al., 2005).<br />
Deficits in information processing capabilities, such as those measured <strong>by</strong> PPI, are core<br />
9
cognitive symptoms of schizophrenia and may relate to deficits in stimulus filtering<br />
commonly proposed to exist in the disorder (Swerdlow et al., 2000a).<br />
As PPI is easily and reliably measured in animals, it has been extensively used in<br />
cross-species work as valid behavioral test with relevance to schizophrenia (Swerdlow et<br />
al., 2000a). The use of PPI as an "index" of schizophrenia in animals is validated <strong>by</strong><br />
pharmacological experiments demonstrating that DA agonists such as apomorphine and<br />
NMDA antagonists such as phencyclidine alter PPI responding in animals and healthy<br />
humans, and antipsychotic drugs ameliorate these deficits in animals (Braff et al.,<br />
2001;Geyer et al., 2001). Extensive experimentation has been conducted to understand<br />
the neural substrates underlying the dopaminergic and glutamatergic regulation of PPI<br />
(Swerdlow et al., 2001). These experiments reveal a distributed circuitry in cortical,<br />
limbic, striatal, and pallidal areas important in the regulation of PPI. The pertinent details<br />
of this literature are reviewed in Chapter Two, and therefore will not be elaborated upon<br />
here.<br />
Locomotor responses to a variety of arousing stimuli such as novel environments<br />
or stimulant drugs are commonly measured in rodents as a measure of mesolimbic<br />
dopamine activity (Kelly et al., 1975;Castall et al., 1977;Porrino et al., 1984). More<br />
vigorous locomotor behavior in response to these treatments correlates positively with<br />
elevated ventral striatal dopamine levels (Kelly et al., 1975;Castall et al., 1977;Hooks et<br />
al., 1992). As previously reviewed, ventral striatal dopamine levels are higher in patients<br />
with schizophrenia than controls (Laruelle et al., 2003). As a result, locomotor activity<br />
levels are often used as an indirect measure of ventral striatal dopamine levels in animal<br />
models of schizophrenia (Lipska and Weinberger, 2000;Bast and Feldon, 2003;Boksa,<br />
10
2004). Additionally, some authors have argued that increased locomotor activity may be<br />
homologous to some cognitive abnormalities in schizophrenia patients during acute<br />
episodes (Bast and Feldon, 2003), although these accounts are debatable (Marcotte et al.,<br />
2001).<br />
In the present thesis, PPI and locomotor activity levels were used as the main<br />
endpoints to assess whether the novel experimental manipulations employed would<br />
produce effects resembling aspects of schizophrenia. These tests were selected for the<br />
following reasons: (1) Both tests have been used frequendy to assess previous preclinical<br />
models of schizophrenia. Thus, comparisons could easily be rnade between existing<br />
models and those developed within the present work. (2) These tests can be reliably<br />
conducted before and after puberty, enabling the developmental course of the behavioral<br />
effects of the neonatal manipulations performed in Chapters Three and Four to be<br />
assessed. (3) Pharmacological challenges are easily conducted in conjunction with these<br />
tests there<strong>by</strong> allowing the neurochemical mechanisms of observed behavioral changes to<br />
be evaluated.<br />
Given the multi-dimensional etiology of schizophrenia, researchers have<br />
advanced numerous strategies to model the disorder in animals, each with its own<br />
strengths and limitations. A brief review of the models pertinent to the present discussion<br />
of PPI and locomotor activity will now be presented.<br />
Genetic Models<br />
As previously discussed, the etiology of schizophrenia is at least partly dependent<br />
on complex genetic risk factors, likely involving a number of different genes. Therefore,<br />
there have been numerous attempts to model the disorder with genetic strategies.<br />
11
Knockout and knockdown strategies typically target genes for neurotransmitter systems<br />
implicated in schizophrenia. One well-known example includes dopamine transporter<br />
knockout mice, which show hyperlocomotion and deficits in PPI that are reversed with<br />
antipsychotics (Gainetdinov et al., 2001). Similar results have been gained with the NR-1<br />
knockdown mouse, which has reduced NMDA receptor function (Mohn et al.,<br />
1999;Duncan et al, 2004).<br />
Genetic models targeting genes involved in neurodevelopment and implicated in<br />
schizophrenia have also been developed. Reduced expression of reelin, a protein<br />
important for normal cell positioning in the brain, has been described in schizophrenia<br />
(Impagnatiello et al., 1998). Heterozygous reeler mice, which exhibit a down regulation<br />
of reelin similar to levels observed in schizophrenic patients, also have a variety of<br />
behavioral abnormalities similar to those in schizophrenia (Costa et al., 2002). These<br />
genetic strategies are useful in understanding the impact of genetic alterations on<br />
behavior, neurochemistry, and histology at a systems level, although these single gene<br />
approaches only model part of the complex etiology of schizophrenia. Clearly, the future<br />
challenge for genetic strategies lies in modeling the complex multi-gene abnormalities<br />
that will likely be implicated in schizophrenia (Wong and Van Tol, 2003). Additionally,<br />
combining genetic approaches with environmental factors in a 'two-hit' approach may be<br />
especially valuable in the future (Ellenbroek, 2003).<br />
Adult Pharmacological Models<br />
These models generally rely on producing acute or long-lasting behavioral<br />
changes relevant to schizophrenia <strong>by</strong> administering psychoactive drugs to animals.<br />
Dopamine agonists such as amphetamine and NMDA antagonists such as MK-801,<br />
12
phencyclidine or ketamine are most commonly used. Acute administration of these drugs<br />
induces deficits in PPI and increased locomotor activity (for review see Marcotte et al.,<br />
2001). Not surprisingly, the changes induced <strong>by</strong> dopamine agonists are reversed <strong>by</strong><br />
typical antipsychotics (Marcotte et al., 2001). Additionally, sensitization with<br />
amphetamine, but not phencyclidine, in rats induces long-lasting deficits in PPI, which<br />
may more faithfully model the stable PPI deficits in schizophrenia (Tenn et al., 2005).<br />
Interestingly, administration of NMDA antagonists may more accurately model<br />
schizophrenia <strong>by</strong> inducing a broader range of symptoms, including cognitive deficits and<br />
negative symptoms (Jentsch and Roth, 1999). The effects of NMDA antagonism are also<br />
reversed <strong>by</strong> atypical antipsychotics, there<strong>by</strong> improving the potential of the model to<br />
identify novel antipsychotic compounds.<br />
The data gained from adult pharmacological models is limited for a number of<br />
reasons. First, the effects are typically induced in normally developed adult animals. A<br />
wide range of evidence indicates that people who develop schizophrenia have<br />
abnormalities before adulthood. Importantly, combining neonatal perturbations of brain<br />
development with pharmacological challenges has provided valuable data, and will be<br />
discussed in the next section. Secondly, the changes in the activity of neurotransmitters<br />
such as dopamine are also more regionally specific than can be modeled with systemic<br />
administration of drugs (Kilts, 2001). Nevertheless, some treatment paradigms have<br />
proven to model more faithfully the neurochemical and behavioral changes in<br />
schizophrenia. For example, chronic exposure to phencyclidine in non-human primates<br />
results in cognitive deficits and reduced dopamine turn-over in the prefrontal cortex, both<br />
of which persist after drug treatment in discontinued (Jentsch et al., 1997). These effects<br />
13
can also be reversed following treatment with the atypical antipsychotic clozapine<br />
(Jentsch et al, 1997). Finally, some researchers argue that the development of novel<br />
antipsychotic drugs is precluded <strong>by</strong> dopamine agonist models because the therapeutic<br />
drugs that have emerged from these models block dopamine receptors, there<strong>by</strong> leading to<br />
a circular pattern of discovery (Lipska and Weinberger, 2000).<br />
Adult Lesion Models<br />
Adult lesion models focus on the effects of lesioning limbic and cortical areas<br />
commonly implicated in both subcortical dopamine regulation and schizophrenia, such as<br />
the prefrontal cortex, hippocampus, and thalamus (Lipska and Weinberger,<br />
2000;Marcotte et al., 2001). Obviously, the construct validity of adult lesion models is<br />
compromised <strong>by</strong> their adult initiation and large degree of damage that occurs (at least<br />
compared to that observed in schizophrenic patients); however, such models do provide<br />
unique insights into the neural substrates that may underlie many of the behavioral and<br />
neurochemical abnormalities in schizophrenia.<br />
Adult lesions of the medial prefrontal cortex in rodents result in hyper-<br />
responsiveness to stress (Jaskiw et al., 1990b), increases in spontaneous locomotor<br />
activity (Braun et al., 1993), increased activity in response to amphetamine (Jaskiw et al.,<br />
1990a), and deficits in PPI after challenge with the dopamine agonist apomorphine<br />
(Swerdlow et al, 1995). Similarly, lesions of the ventral, but not dorsal, hippocampus<br />
result in increased spontaneous activity and hyperactivity and PPI deficits following<br />
administration of dopamine agonists (Lipska et al., 1991 ;Lipska et al., 1992;Swerdlow et<br />
al., 1995;Mittleman et al., 1998). These data enable a detailed understanding of the<br />
neural substrates mediating PPI and locomotor activity to be developed, and will be<br />
14
eturned to in greater detail in Chapter Two. Importantly, they can be effectively<br />
integrated with neuroanatomical and neurochemical data, leading to new hypotheses<br />
regarding the mechanisms underlying the behavioral effects of the lesions. For example,<br />
the connectivity of the ventral, but not dorsal, hippocampus with areas such as the<br />
prefrontal cortex, amygdala, nucleus accumbens, and ventral tegmental area of the<br />
midbrain may be linked causally to the behavioral effects observed following its removal<br />
(Bast and Feldon, 2003).<br />
OBJECTIVE ONE: Numerous well-documented shortcomings of permanent lesions<br />
exist, such as compensatory alterations in brain areas other than the lesioned area<br />
(Schoenfeld and Hamilton, 1977;Stein, 1979;Bast and Feldon, 2003). Additionally,<br />
animals may develop strategies that aid in coping with the permanent loss of function<br />
normally carried out <strong>by</strong> the lesioned area (Lomber, 1999;Bast and Feldon, 2003).<br />
Specifically in the context of PPI and locomotor behavior, differences between<br />
permanent and temporary manipulations of the hippocampus have been described<br />
(Swerdlow et al., 1995;Pouzet et al., 1999;Swerdlow et al., 2000b;Zhang et al., 2002).<br />
Therefore, in Chapter Two, I examine the effect of brief stimulation of discrete sub-<br />
regions of the hippocampus on PPI. Given the important role of the hippocampus in<br />
schizophrenia, these experiments may provide further insights into the pathophysiology<br />
of the disorder.<br />
15
Developmental Animal Models<br />
As previously discussed, the neurodevelopmental hypothesis of schizophrenia has<br />
received considerable attention. Therefore, it is not surprising that numerous strategies<br />
have been developed to model schizophrenia using manipulations at distinct periods from<br />
embryological development until the time of weaning. As the present experiments focus<br />
on manipulations in the early neonatal period, most of the studies detailed below will<br />
focus on this time point. Numerous excellent reviews of experiments examining the<br />
effects of prenatal stress, obstetric complications, prenatal infection, and other<br />
developmental manipulations are available (Van den Buuse et al., 2003;Boksa, 2004).<br />
Before discussing developmental models, it is important to note that rodents are<br />
born at later stage of development than humans. Thus, the first week of postnatal life in<br />
rodents is typically described as corresponding to the third trimester of human fetal brain<br />
development (Lipska and Weinberger, 2000). Additionally, the surge of gonadal<br />
hormones that coincide with the onset of puberty occurs during the 5 th<br />
and 6 th<br />
postnatal<br />
weeks of the rodent life span. Thus, assessments of prepubertal function are generally<br />
conducted at approximately postnatal day 35 and assessments of adult function are<br />
performed after postnatal day 56.<br />
Maternal Separation and Social Isolation Models<br />
These two types of models involve removal of developing rats from their normal<br />
sources of social interaction, whether it be from their mothers early in development<br />
(maternal separation) or from their peers immediately following weaning (social<br />
isolation). Stress likely plays a major role in producing the behavioral changes observed<br />
in both models (Hall, 1998;Lipska and Weinberger, 2000;Weiss and Feldon, 2001 ;Van<br />
16
den et al., 2003). In one example of a maternal separation paradigm, a 24 hour separation<br />
period on postnatal day three, six, or nine is sufficient to produce deficits in PPI and<br />
increased sensitivity to dopamine agonists that occur only after puberty (Ellenbroek et al.,<br />
1998;Husum et al., 2002). Interestingly, these deficits can be reversed with typical or<br />
atypical antipsychotics (Ellenbroek et al., 1998). Strain differences are also observed,<br />
with effects present in Wistar rats, but not the Lewis or Fisher 344 strains (Ellenbroek<br />
and Cools, 2000). These data provide important support for the assertion that early life<br />
experiences interact with genetic background to alter adult patterns of behavior with<br />
relevance to schizophrenia (Lipska and Weinberger, 2000;Ellenbroek and Cools, 2000).<br />
The social isolation model involves rearing weanling rat pups individually<br />
without access to their peers, there<strong>by</strong> depriving them of social contact and considerable<br />
sensory stimulation (Hall, 1998;Weiss and Feldon, 2001 ;Van den et al., 2003). This<br />
model is distinct from maternal separation discussed above in that early development of<br />
the animals is not disturbed. Considerable research has been conducted using the social<br />
isolation model showing fairly consistent reductions in PPI, alterations in spontaneous<br />
locomotor activity, and locomotor responses to dopamine agonists and NMDA<br />
antagonists (Hall, 1998;Lipska and Weinberger, 2000;Weiss and Feldon, 2001).<br />
Parametric studies of the duration of rearing reveal that the pups must be in isolation for<br />
greater than three weeks starting at weaning (postnatal day 21 to 28) for behavioral<br />
alterations to be observed (Bakshi and Geyer, 1999;Varty et al., 1999). The behavioral<br />
effects are also sensitive to the strain of rats used (Weiss and Feldon, 2001), and are<br />
usually reversed with either typical or atypical antipsychotic drug treatment (Varty and<br />
Higgins, 1995;Bakshi et al., 1998). Thus, the social isolation model is an important<br />
17
nonpharmacological tool that can be used to identify novel antipsychotic compounds<br />
(Weiss and Feldon, 2001).<br />
With respect to both the maternal separation and isolation rearing paradigms,<br />
reliability is a concern. For example, some groups have failed to observe PPI deficits in<br />
maternally separated rats using similar, but not identical, methods (Lehmann et al.,<br />
2000;Weiss and Feldon, 2001). Factors such as the temperature at which the pups are<br />
maintained and the exact conditions under which they are separated (i.e. separated and<br />
kept in a group versus kept individually) may help explain the conflicting results (Weiss<br />
and Feldon, 2001). With regard to isolation rearing, the robustness of the behavioral<br />
effects has been questioned, as some reports indicate that the model is quite sensitive to<br />
handling or other testing procedures (Weiss and Feldon, 2001). However, a long-term<br />
evaluation of the reliability of PPI deficits in the social isolation model indicate that PPI<br />
deficits occur in approximately 85 percent of groups tested against matched controls<br />
(Cilia et al., 2005).<br />
Neonatal Lesion Models<br />
A great deal of work in the last ten to fifteen years has examined the utility of<br />
performing neonatal lesions of cortical and limbic areas in modeling schizophrenia<br />
(Lipska and Weinberger, 2000). This procedure has a number of distinct advantages over<br />
those discussed above: (1) The normal maturation of specific brain areas is altered and<br />
this simplifies the search for mechanisms underlying observed changes in the model. (2)<br />
The consequences of the lesion can be examined over the course of development there<strong>by</strong><br />
allowing the timing of any changes to be compared to the normal course of the disease.<br />
(3) Manipulating the brain at an early age is consistent with the neurodevelopmental<br />
18
theory of schizophrenia. (4) Further manipulations can be performed later in development<br />
to test the 'two-hit' hypothesis of schizophrenia.<br />
Most prominent among these models is the ventral hippocampal lesion model<br />
initially described <strong>by</strong> Lipska and colleagues (Lipska et al., 1993). In this model, rats are<br />
subjected to bilateral lesions of the ventral hippocampus with the excitotoxin ibotenic<br />
acid on postnatal day seven. The animals are then allowed to develop normally and can<br />
be tested before or after puberty on an array of behavioral tests relevant to schizophrenia.<br />
Numerous reports indicate that rats with neonatal ventral hippocampal lesions display<br />
PPI deficits and increased spontaneous locomotor activity (Lipska et al., 1993;Lipska et<br />
al., 1995). The PPI deficits are potentiated <strong>by</strong> a low dose of apomorphine (Lipska et al.,<br />
1995) and reversed <strong>by</strong> atypical antipsychotic drugs, but not <strong>by</strong> haloperidol (Le Pen and<br />
Moreau, 2002). Locomotor activity is also increased following stress (Lipska et al.,<br />
1993), dopamine agonist administration (Lipska et al., 1993), or treatment with NMDA<br />
antagonists (Al Amin et al., 2000;A1 Amin et al., 2001). The alterations in locomotor<br />
behavior can be normalized with haloperidol, but not atypical antipsychotic drugs (Al<br />
Amin et al., 2000). Treatment with ionotropic glutamate receptor antagonists (Al Amin<br />
et al., 2000) and increasing glycine availability (Le Pen et al., 2003) also reverses some<br />
of the behavioral impairments, there<strong>by</strong> indicating a role of glutamate transmission in the<br />
underlying effects of neonatal ventral hippocampal lesions.<br />
Consistent with the typical course of schizophrenia, the PPI deficits and<br />
locomotor activity changes are not expressed until after the rats reach puberty and persist<br />
at least until mid-adulthood (Lipska and Weinberger, 1993;Lipska et al., 1995;Lipska and<br />
Weinberger, 2000;A1 Amin et al., 2001). Interestingly, lesioned rats show reduced social<br />
19
interaction before and after puberty, a test which may have some relevance to the<br />
abnormalities observed in schizophrenic patients before their first episode (Sams-Dodd et<br />
al., 1997). Parametric experiments also suggest that the delayed onset of behavioral<br />
changes in the animals only occurs if the lesions are created during the first postnatal<br />
week of life (Wood et al., 1997), which corresponds to the third trimester in human<br />
development (Lipska and Weinberger, 2000). Finally, in an important paper, Lipska and<br />
Weinberger (Lipska and Weinberger, 1995) demonstrated that the effects of the neonatal<br />
lesion depend on the genetic background of the animals. Rats highly responsive to stress<br />
(the Fischer 344 strain) showed exaggerated effects of the lesion, whereas rats hypo-<br />
responsive to stress (the Lewis strain) showed reduced lesion effects. Thus, this model<br />
supports the theory that schizophrenia may develop from an interaction between genetic<br />
predisposition to the disorder and adverse environmental events (Lipska and Weinberger,<br />
1995;Benes, 2000;Benes et al., 2000;McClure and Weinberger, 2001;Ellenbroek, 2003).<br />
The results gained from the ventral hippocampal model have led others to<br />
examine the effects of neonatally lesioning other areas such as the medial prefrontal<br />
cortex (Flores et al., 1996;Schneider and Koch, 2005), amygdala (Hanlon and Sutherland,<br />
2000;Daenen et al., 2001;Daenen et al., 2002a;Daenen et al, 2002b;Daenen et al.,<br />
2003a;Daenen et al., 2003b) and thalamus (Lipska et al., 2003) on a similar range of<br />
behaviors. Many of these experiments have yielded results similar to, or less consistent<br />
than, those already discussed. As a result, they will not be elaborated on further.<br />
While it is clear that many characteristics of schizophrenia have been modeled<br />
with great success using neonatal ventral hippocampal lesions, the severity and<br />
permanent nature of the hippocampal lesion limits the construct validity of this model for<br />
20
schizophrenia (Lipska and Weinberger, 2000). As a result, Lipska and colleagues (2002)<br />
demonstrated that reversible temporary inactivation of the ventral hippocampus with<br />
tetrodotoxin on postnatal day seven is sufficient to induce significant changes in<br />
locomotor behavior in adulthood, but not before puberty. This manipulation produces<br />
limited gross morphological damage to the ventral hippocampus, and is short-lasting (24-<br />
48 hr) (Lipska et al., 2002). These data clearly highlight the fragility of the hippocampal-<br />
cortical circuit during this developmental period. Additional data demonstrate that a<br />
single infusion of the NMDA antagonist (±)-2-amino-5-phosphonopentanoic acid (APV)<br />
into the rat medial prefrontal cortex on postnatal day seven results in exaggerated<br />
amphetamine-induced locomotor activity in adulthood (Lafleur et al., 2001). These data<br />
support the hypothesis that an abnormality in activity-dependent plasticity involving<br />
glutamatergic hippocampal-prefrontal interactions may underlie some of the symptoms<br />
observed following neonatal ventral hippocampal lesions (Lipska et al., 2002).<br />
Additionally, these data support the hypothesis that the first postnatal week is a critical<br />
period for the development of cortico-limbic circuits mediating behaviors such as<br />
locomotor activity and PPI.<br />
Although the neonatal ventral hippocampal lesion model provides unique insight<br />
into the consequences of damaging a particular area early in development, it is unlikely<br />
that schizophrenia results from damage to a single brain area. Thus, the efficacy of<br />
altering the activity of the brain pharmacologically during the first postnatal week has<br />
also been tested in an effort to model schizophrenia. These experiments are discussed in<br />
the following section.<br />
21
Neonatal Pharmacological Models<br />
As previously discussed, alterations of the glutamatergic system are likely to be<br />
involved in the expression of the symptoms of schizophrenia. A number of lines of<br />
evidence support the hypothesis that the glutamatergic system may also be involved in<br />
the development of the disorder. For example, adverse events during early development<br />
such as febrile seizures, fetal alcohol exposure, and hypoxia/ischemia involve the<br />
glutamatergic system (Morimoto et al., 1995;01ney et al., 1999;Zornberg et al.,<br />
. 2000;Olney, 2004) and are risk factors for psychosis and/or schizophrenia (Cannon,<br />
1997;Famy et al., 1998;01ney et al., 1999;Kanemoto et al., 2001;Vestergaard et al.,<br />
2005). The normal maturation and development of limbic and cortical areas relevant to<br />
schizophrenia is also dependent on normal glutamatergic function. For example,<br />
processes such as cell birth, survival and synaptogenesis are critically dependent on<br />
glutamate transmission, particularly at NMDA receptors (Rabacchi et al., 1992;Gould et<br />
al., 1994;Fox et al., 1996;Vallano, 1998;Ikonomidou et al., 1999;Luthi et al., 2001).<br />
Given the established role of glutamate neurotransmission in neonatal<br />
development during periods implicated in schizophrenia, a number of studies have<br />
examined the behavioral effects of manipulating glutamate transmission<br />
pharmacologically early in life. Most of these experiments have tested the effects of<br />
systemically administered NMDA receptor antagonists such as MK-801, ketamine or<br />
phencyclidine during the neonatal period. A number of groups have demonstrated<br />
deficits in PPI and alterations in locomotor behavior following treatment with these drugs<br />
(Facchinetti et al., 1993;Wang et al., 2001;Ffarris et al., 2003;Fredriksson et al., 2004).<br />
As will be elaborated further in Chapter Four of the present thesis, the long-lasting<br />
22
morphological effects of administering non-competitive NMDA antagonists early in the<br />
neonatal period are also relevant to schizophrenia (Wang et al., 2001 ;Harris et al., 2003).<br />
OBJECTIVE TWO: Given that the end of the first postnatal week is a sensitive period for<br />
the development of neural circuits mediating PPI and locomotor activity (Wood et al.,<br />
1997;Wang et al., 2001;Lipska et al., 2002;Harris et al, 2003), the second objective of<br />
this thesis was to extend these findings in two novel directions. In Chapter Three, a<br />
series of experiments examined the long-term behavioral effects of administering the<br />
glutamate receptor agonist kainic acid to postnatal day seven rats. Chapter Four<br />
summarizes an independent set of experiments designed to test the effects of<br />
administration of the highly specific NMDA receptor antagonist [(+/-)-(R*,S*)-alpha-(4-<br />
hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-l-piperidine propanol] (Ro25-6981)<br />
during the same time period. These pharmacological manipulations were designed to<br />
subtly alter the normal development of the cortico-limbic circuitry involved in<br />
schizophrenia - kainic acid <strong>by</strong> selectively activating the hippocampus and Ro25-6981 <strong>by</strong><br />
blocking NR2B subunit-containing NMDA receptors, which are highly expressed during<br />
the first postnatal week in rodents.<br />
23
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36
CHAPTER TWO: ELECTRICAL STIMULATION OF <strong>THE</strong> HIPPOCAMPUS<br />
DISRUPTS PREPULSE INHIBITION IN <strong>RAT</strong>S: FREQUENCY AND SITE<br />
DEPENDENT EFFECTS. 1<br />
Introduction<br />
Prepulse inhibition (PPI) is the normal reduction in an acoustic startle response<br />
produced when a brief, quieter stimulus or prepulse is presented 30-500 ms prior to<br />
presentation of the starde-evoking stimulus (Koch, 1999;Swerdlow et al., 2000a). PPI<br />
provides a measure of sensorimotor gating, the inhibitory process <strong>by</strong> which responses to<br />
sensory stimuli are suppressed, there<strong>by</strong> facilitating appropriate responding in complex,<br />
sensory rich environments (Swerdlow, 1996). Interest in the neural mechanisms<br />
underlying PPI has increased because it is often disrupted in humans diagnosed with a<br />
variety of psychiatric disorders including schizophrenia (Weike et al., 2000;Braff et al.,<br />
2001). As similar stimuli elicit PPI in rodents and humans, interventions in rodents that<br />
disrupt PPI may reveal aspects of neuronal dysfunction that underlie disrupted<br />
sensorimotor gating in schizophrenic patients (Swerdlow, 1996;Weike et al., 2000).<br />
Similarities exist in the neuroanatomical and neurochemical substrates related to<br />
both schizophrenia and PPI. A large body of research suggests that schizophrenic<br />
patients show abnormalities in cortico-limbic-striatal circuitry, in areas such as the frontal<br />
cortex, nucleus accumbens (NAc), and hippocampus (Pakkenberg, 1990;Bogerts,<br />
1997;Harrison, 1999;Wright et al., 1999) likely involving alterations in dopamine (DA)<br />
and glutamate (Glu) transmission (Breier et al., 1997;Carlsson et al., 1999;Laruelle et al.,<br />
1999;Goff and Coyle, 2001;Meyer-Lindenberg et al., 2002). Consistent with these<br />
1<br />
A version of the chapter has been published: Howland JG, Mackenzie EM, Yim TT, Taepavarapruk P,<br />
Phillips AG (2004). Electrical stimulation of the hippocampus disrupts prepulse inhibition in rats:<br />
frequency and site dependent effects. Behavioural Brain Research 152:187-197.<br />
37
findings is evidence indicating that the regulation of PPI is also dependent on a complex<br />
array of structures commonly termed the cortico-striatal-pallidal-pontine (CSPP) circuitry<br />
(see Swerdlow et al., 2001a for review), including the medial prefrontal cortex (mPFC)<br />
(Koch and Bubser, 1994;Lacroix et al., 2000), amygdala (Wan and Swerdlow,<br />
1997;Fendt et al., 2000), NAc (Swerdlow et al.,1986;1990;1994;Wan and Swerdlow,<br />
1996), and hippocampus (Caine et al., 1992;Swerdlow et al., 1995;2001b;Wan et al.,<br />
1996;Bakshi and Geyer, 1998;Klarner et al, 1998;Zhang et al., 1999;2002a;Bast et al.,<br />
2001;Caine et al., 2001). Importantly, the systemic administration of DA or DA agonists<br />
(Mansbach et al, 1988;Swerdlow et al., 1990;Zhang et al., 2000a) and N-Methyl-D-<br />
Aspartate (NMDA) antagonists (Mansbach and Geyer, 1989;Bakshi and Geyer, 1998),<br />
likely acting in areas of the CSPP circuitry, also disrupt PPI in animals (Geyer et al.,<br />
2001). Recently, a complex role of NAc DA and Glu via D2, NMDA, and (±)-a-amino-<br />
3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) receptors in the<br />
modulation of PPI has been described (Mansbach et al., 1988;Swerdlow et al, 1990;Wan<br />
and Swerdlow, 1993;1996;Reijmers et al., 1995;Wan et al., 1995).<br />
Hippocampal dysfunction has been linked to symptoms of schizophrenia<br />
(Silbersweig et al., 1995;Heckers et al., 1998;Harrison, 1999;Dierks et al., 1999;Grace,<br />
2000), and consequently the roles of distinct hippocampal subregions in the modulation<br />
of PPI have been examined extensively. Although permanent lesions of the ventral<br />
hippocampus (vHip) generally do not disrupt PPI (Swerdlow et al., 1995;2000b;Pouzet et<br />
al., 1999 but see also (Caine et al., 2001), temporary inactivation of vHip or dorsal<br />
hippocampal (dHip) with either the GABA A agonist muscimol or the sodium channel<br />
blocker tetrodotoxin disrupts PPI (Zhang et al., 2002b). Debate also exists as to whether<br />
38
infusion of glutamatergic antagonists, such as MK801, into the dHip, but not the vHip,<br />
disrupts PPI (Bakshi and Geyer, 1998;1999;Bast et al., 2000;Zhang et al., 2000b).<br />
Nevertheless, compelling evidence exists for the disruption of PPI following chemical<br />
stimulation of the vHip (Wan et al., 1996;Klarner et al., 1998;Zhang et al., 1999;Bast et<br />
al., 2001;Swerdlow et al., 2001b;Zhang et al., 2002a), but not dHip (Swerdlow et al.,<br />
2001b;Zhang et al., 2002a) with infusions of the ionotropic Glu agonist NMDA. As<br />
these studies suggest, various manipulations of the vHip and dHip can impair<br />
sensorimotor gating behavior, and regional differences clearly exist.<br />
Both the vHip and dHip are well positioned neuroanatomically to interact with the<br />
CSPP circuitry to modulate PPI. The vHip projects densely to the NAc shell, amygdala,<br />
and mPFC (Kelley and Domesick, 1982;Groenewegen et al., 1987;Brog et al.,<br />
1993;Conde et al., 1995) whereas the dHip projects to the NAc core (Swanson and<br />
Cowan, 1977;Whitter, 1986), and also communicates with the vHip via intra-<br />
hippocampal connections (Amaral and Whitter, 1995). At the level of the ventral<br />
striatum, projections from the vHip exert powerful control over DA efflux in the NAc<br />
shell and firing of NAc medium spiny projection neurons (O'Donnell and Grace,<br />
1995;Taepavarapruk et al., 2000;Floresco et al., 2001). More specifically, stimulation of<br />
the vHip with NMDA (Brudzynski and Gibson, 1997;Legault et al., 2000) or brief trains<br />
of higher frequency electrical current (20 Hz, 10 s) (Blaha et al., 1997;Taepavarapruk et<br />
al., 2000) cause a long-lasting increase in NAc DA efflux and an increase in locomotor<br />
activity. Activation of ionotropic Glu receptors (iGluR's) in the NAc underlies the<br />
changes in NAc DA efflux observed following electrical stimulation of the vHip (Blaha<br />
et al., 1997;Taepavarapruk et al., 2000). In contrast, preliminary studies performed in our<br />
39
laboratory demonstrate that lower frequency (2 Hz) stimulation of the vHip for 100 s can<br />
significantly reduce NAc DA efflux for at least 2 hr, an effect that may result from the<br />
activation of intra-NAc group 2 and 3 metabotropic Glu receptors (Taepavarapruk et al.,<br />
1998). The consequences of stimulating the dHip on NAc DA efflux have been poorly<br />
characterized, although stimulation of the previously kindled dHip results in a brief<br />
increase in NAc DA efflux (Strecker and Moneta, 1994).<br />
In the following experiments, the effects of electrical stimulation of the Hip on<br />
PPI were examined. Electrical stimulation was chosen because it has a number of<br />
distinct advantages over chemical stimulation, including precise control over the duration<br />
and intensity of the stimulation. Additionally, electrical stimulation parameters can<br />
approximate the physiological firing activity of the neurons that are stimulated. Given<br />
that intra-vHip NMDA infusions and 20 Hz electrical stimulation of the vHip produce<br />
similar increases in NAc DA efflux (Brudzynski and Gibson, 1997;Legault et al.,<br />
2000;Taepavarapruk et al., 2000), and that vHip NMDA infusions disrupt PPI (Wan et<br />
al., 1996;Klarner et al, 1998;Zhang et al, 1999;2002a;Bast et al., 2001;Swerdlow et al.,<br />
2001b), the first objective of the present study was to assess whether 20 Hz stimulation of<br />
the vHip would induce a reversible disruption of PPI. In a complementary experiment,<br />
we examined the effect of 20 Hz electrical stimulation to the dHip on PPI, hypothesizing<br />
that if dHip NMDA infusions fail to disrupt PPI (Swerdlow et al., 2001b;Zhang et al,<br />
2002a), then electrical stimulation at this frequency should also fail to reduce PPI. The<br />
final behavioral experiment examined the effect of 2 Hz vHip stimulation on PPI.<br />
Additional microdialysis experiments addressed two issues related to the effects of 20 Hz<br />
stimulation of the hippocampus. The first assessed whether unilateral vHip stimulation<br />
40
would result in an increase in DA efflux bilaterally in the NAc. Secondly, as the effects<br />
of 20 Hz dHip stimulation on NAc DA are unknown, a similar microdialysis experiment<br />
assessed the effects of these stimulation parameters on NAc DA efflux. Male Long-<br />
Evans (LE) rats were used in all experiments in an effort to maintain consistency between<br />
the neurochemical data collected previously and the present behavioral experiments.<br />
Methods<br />
Subjects<br />
Male LE rats (Charles River Canada, St. Constant, Quebec, Canada) were pah-<br />
housed in Plexiglas cages and handled daily until surgery. The colony was kept on a<br />
12/12 hour light/dark cycle (lights on at 0700), at a temperature of 22±1°C. Rats were<br />
given food (Purina Rat Chow) and tap water ad libitum. Experiments were conducted in<br />
accordance with the standards of the Canadian Council on Animal Care and the<br />
Committee on Animal Care at the University of British Columbia approved all<br />
procedures.<br />
Surgery<br />
At the time of surgery, subjects weighed 330 to 370 g. Rats were anaesthetized<br />
with ketamine hydrochloride (100 mg/kg, i.p., MTC Pharmaceuticals) and xylazine (10<br />
mg/kg, i.p., Rompun), and placed in a stereotaxic frame. The dorsal surface of the skull<br />
was exposed, and holes were drilled. A bipolar stimulating electrode (Plastics One,<br />
Roanoke, VA) was implanted into either the vHip (AP -5.8 mm from bregma, ML ±5.5<br />
mm from midline, DV -6.0 mm from dura) or dHip (AP -3.0 mm, ML ±1.5 mm, DV -<br />
3.0 mm) of all rats. Guide cannulae were also implanted dorsal to the NAc in those rats<br />
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<br />
dental acrylic. Wire obdurators were inserted into the cannula to keep them patent.<br />
Animals were allowed at least 7 days to recover from surgery before testing began, and<br />
were regularly handled beginning three days following surgery.<br />
Prepulse Inhibition Testing<br />
Testing was conducted in a single sound-attenuating startle chamber (ambient<br />
noise level 62 dB), containing a transparent Plexiglas tube (8.2 cm in diameter, 20 cm in<br />
length), mounted on a Plexiglas frame (SR-LAB, San Diego Instruments, San Diego,<br />
CA). Noise bursts were presented through a speaker mounted 24 cm above the tube. An<br />
accelerometer below the frame of the apparatus measured whole body startle amplitude,<br />
defined as the average of 100 1 ms accelerometer readings collected beginning at<br />
stimulus onset. Each PPI test session began with a 5 min acclimatization period during<br />
which a 70 dB background noise level was presented, which remained constant for the<br />
entire test session. Following the acclimatization period, six pulse alone trials (120 dB,<br />
40 ms) were presented to achieve a relatively stable level of startle amplitude before<br />
presentation of the prepulse + pulse trials. The data from these pulse alone trials was not<br />
considered in the analysis of PPI. Immediately following the six initial pulse alone trials,<br />
presentation of the trials that were used in the calculation of PPI levels began. A total of<br />
84 trials of five different types were presented. Trials presented were of three types:<br />
pulse alone (12 trials; 120 dB, 40 ms), prepulse + pulse (10 trials X 3 prepulse intensities<br />
- discussed below), or no stimulus (42 trials). Prepulse + pulse trials consisted of the<br />
presentation of a 20 ms prepulse of 73, 76, or 82 dB 80 ms before the presentation of the<br />
pulse. The pulse and prepulse + pulse trials were presented in a pseudorandom order.<br />
42
One no stimulus trial was presented between each pair of pulse and prepulse + pulse<br />
trials. The inter-trial interval varied randomly from 3 to 12 s (average 7.5 s). The total<br />
length of each PPI session was approximately 18 min. Calibration of the apparatus was<br />
performed using a RadioShack Digital Sound Level Meter and adjustments were made as<br />
necessary.<br />
A within-subjects design was used in all PPI experiments. Experiments were<br />
conducted in two successive weeks; all animals were tested four times during these two<br />
weeks. The four PPI sessions were referred to as follows. The 'stim' PPI session<br />
occurred 2 min following electrical stimulation of either the vHip or dHip, 'stim+48' was<br />
the session 48 hr following stimulation, 'control' was the PPI session 2 min following<br />
exposure to the stimulation box without stimulation, and 'control+48' followed the<br />
'control' session <strong>by</strong> 48 hrs. Animals were randomly assigned to one of two sequences to<br />
control for potential order effects. Rats in sequence one received their PPI sessions in the<br />
following order: stim, stim+48, control, and control+48. Rats in sequence two received<br />
their PPI sessions in the control, control+48, stim, and stim+48 order.<br />
Following recovery from surgery, animals were habituated to the stimulation<br />
environment on the two days immediately preceding their first PPI test. Each rat was<br />
removed from the colony and taken to the stimulation room. This small room was<br />
immediately adjacent to the room used for PPI testing, and contained two transparent<br />
Plexiglas chambers (32 cm X 32 cm X 41 cm) used for stimulation. In the stimulation<br />
room, each rat was connected to a stimulation lead and placed in a Plexiglas chamber for<br />
10 min, following which they were returned to the colony. On the day of their first PPI<br />
test, the animals were treated exactly as on the two previous days except that after 10 min<br />
43
in the stimulation box, electrical stimulation (20 Hz: 200 cathodal, constant current<br />
pulses, 300 uA, 20 Hz, pulse width 0.5 ms; 2 Hz: identical except frequency of pulses<br />
was 2 Hz) was applied to half the rats, while the other half were given no current.<br />
Stimulation was delivered through an isolator (Iso-flex, A.M.P.I, Israel) via a Master-8<br />
stimulator (A.M.P.I.). The experimenter observed the animals for 90 sec following<br />
stimulation, disconnected them, and quickly placed them in the PPI chamber. This<br />
procedure took a total of 2 min. Immediately following the PPI session, the animals were<br />
returned to the colony. All rats were then tested for PPI 48 hours later. For this test, the<br />
rats were removed from the colony and immediately placed in the PPI apparatus.<br />
Following the second PPI session, the animals were handled daily for five days. On the<br />
sixth day following the first PPI test session, all rats were given one habituation session<br />
in the stimulation chamber for 10 min. The following day, the PPI testing procedure<br />
described above was repeated, with control animals receiving electrical stimulation and<br />
previously stimulated animals receiving no current. Forty-eight hours later, all rats<br />
received their last PPI session.<br />
Neurochemical Experiments<br />
The microdialysis procedure commonly employed in our laboratory has been<br />
described in detail elsewhere (Taepavarapruk et al, 2000;Howland et al., 2002). Briefly,<br />
concentric-style microdialysis probes (2 mm of exposed membrane) were constructed in<br />
our laboratory. Probes were attached to gas-tight syringes via a liquid swivel containing<br />
perfusion medium (147 mM NaCl, 3.0 mM KC1,1.3 mM CaCl 2.H 20, 1.0 mM<br />
MgCl 2.6H 20, 0.01 sodium phosphate buffer; pH 7.3-7.4) and flushed for 10 to 20 min<br />
using a syringe pump. Probes were then secured in a copper collar and inserted into the<br />
44
NAc (7.8 mm ventral to dura) and the rats were allowed to move freely in a Plexiglas box<br />
(32 cm x 32 cm x 41 cm high) with access to food and water for 12 to 18 hours before<br />
experimental testing began the following morning. The probes were continuously<br />
perfused at 1 pL/min overnight. Probes were inserted bilaterally for the vHip stimulation<br />
experiment and unilaterally ipsilateral to the electrode for the dHip stimulation<br />
experiments.<br />
Two high-performance liquid chromatography systems with electrochemical<br />
detection (HPLC-ED) consisting of an ESA 582 pump (ESA Inc., Bedford, MA),<br />
Rheodyne Inert manual injector (Rheodyne, Rohnert Park, CA), an Ultrasphere column<br />
(Beckmann, Fullerton, CA.; ODS 5 pm, 15 cm x 4.6 mm), an ESA 5011 analytical cell,<br />
and a Coulochem II EC detector (ESA Inc.) were used to quantify DA levels in all<br />
experiments. The working potentials were: +450 mV (electrode 1), -300 mV (electrode<br />
2), and +450 mV (guard cell). The mobile phase (pH 3.5) consisted of 6 g/L sodium<br />
acetate, 10 mg/L ethylenediaminetetra-acetic acid (EDTA), 150 mg/L octyl sulfate<br />
(adjustable), 35 ml/L glacial acetic acid and 865 mL Milli Q purified water.<br />
Chromatograms were registered on a dual-pen chart recorder (Kipp and Zonen, Bohemia,<br />
NY). All samples were injected immediately after collection and DA peak heights were<br />
measured manually.<br />
Dialysate samples were collected at 10 min intervals throughout the experiment.<br />
Once four baseline samples were collected that did not differ <strong>by</strong> more than ± 10%, the<br />
rats received 20 Hz electrical stimulation of either the vHip or dHip (same parameters<br />
described for the PPI experiments). Stimulation was timed to ensure that the next<br />
dialysis sample reflected only "stimulation-evoked" changes in DA efflux. The<br />
45
experimenter remained in the testing room following the stimulation to record any<br />
behavioral effects of the stimulation. Nine samples were collected following stimulation,<br />
after which the animals were disconnected and sacrificed.<br />
Histology<br />
Upon completion of each experiment, animals were sacrificed with an overdose of<br />
pentobarbital, and perfused transcardially with 0.9% saline followed <strong>by</strong> 10%<br />
formaldehyde. Brains were stored in 10% sucrose in 10% formaldehyde for at least 1<br />
week, after which they were sectioned (50 pm) using a cryostat and stained with cresyl<br />
violet. Placements of the electrodes and probes were confirmed under a light microscope<br />
with the assistance of a rat brain atlas (Paxinos and Watson, 1997).<br />
Data Analysis<br />
Data were analysed using repeated measures analyses of variance with the aid of<br />
SPSS (version 10.0). All post-hoc tests were performed separately. Variance is indicated<br />
on all graphs with the standard error of the mean (SEM). Significance levels for all<br />
statistical tests were set at 0.05.<br />
For the PPI experiments, two measures were calculated for each animal. The<br />
startle amplitude represented the mean startle amplitude of the 12 pulse alone trials<br />
presented after the six habituation trials. For calculation of PPI, startle amplitudes were<br />
averaged for each trial type. The percent PPI for each prepulse intensity was calculated<br />
using the formula: [100 - (100 X startle amplitude on prepulse + pulse trials) (startle<br />
amplitude on pulse alone trials)]. Two-way (prepulse intensity, test session as factors)<br />
repeated measures ANOVAs were performed on the data obtained from each stimulation<br />
site. As the prepulse intensity factor did not significantly interact with any of the other<br />
46
factors, percent PPI data was averaged across the three prepulse intensities for each rat,<br />
there<strong>by</strong> creating a global PPI score (Wan et al., 1995;Bakshi and Geyer, 1998;1999).<br />
Both startle amplitude and percent PPI were then analyzed using separate one-way<br />
ANOVAs with condition as a repeated measures factor. The repeated measures<br />
assumption of sphericity was met in all cases. Post-hoc analyses were performed using<br />
the Neuman-Keul's test where appropriate.<br />
Following the microdialysis experiments, baseline DA levels for each rat were<br />
calculated <strong>by</strong> averaging the four baseline samples. In all figures, DA levels are expressed<br />
as a percentage change from baseline (baseline is expressed as 0%). One-way repeated<br />
measures analyses of variance with time as a within-subjects factor were performed on all<br />
data and Dunnett's post-hoc tests were performed where appropriate. The baseline<br />
sample taken immediately before the first stimulation was used as the critical value<br />
during computation of the Dunnett's post-hoc tests.<br />
Results<br />
Immediate Behavioral Effects of Stimulation<br />
During the 2 min that the rats remained in the stimulation apparatus after<br />
stimulation was applied, a variety of behavioral effects were noted. The rats were<br />
generally awake and alert before administration of stimulation. At the onset of<br />
stimulation, behavioral arrest was observed in all animals. Higher frequency (20 Hz)<br />
stimulation of the vHip resulted in 5 to 20 wet dog shakes (WDS) that were restricted to<br />
45 s following initiation of the stimulation in 20 of the 21 animals (rats used in the<br />
dialysis studies are included in this description). Additionally, all rats engaged in<br />
vigorous forward locomotor behavior following cessation of the 20 Hz stimulation, as has<br />
47
een previously quantified (Taepavarapruk et al., 2000). Stimulation of the dHip resulted<br />
in an initial period of WDS activity in 4 of the 28 animals. All rats displayed a period of<br />
increased activity and rearing within 30 sec of the cessation of stimulation. Between 70<br />
and 100 s following the cessation of stimulation, 18 of the 28 rats had a second bout of<br />
WDS activity. One to 20 WDS were displayed during this period. Two Hz stimulation<br />
of the vHip caused an initial period of behavioral arrest followed <strong>by</strong> behavioral activation<br />
manifested <strong>by</strong> grooming and some rearing. Wet dog shakes were observed in 3 of 15<br />
animals during application of the 2 Hz stimulation.<br />
Differential Effects of 20 Hz Stimulation of the vHip or dHip on PPI and Startle<br />
Amplitude<br />
Twenty Hz stimulation of the vHip (n=14) resulted in a significant and reversible<br />
disruption of PPI (Fig. 2-1 A). A one-way repeated measures ANOVA performed on the<br />
PPI data for the vHip stimulation group revealed a significant main effect for the test<br />
condition factor (F(3, 39) = 9.32, p< 0.001). Post-hoc analyses revealed that percent PPI<br />
levels were significantly lower in the stim session (20.19 ± 6 %) than in the stim+48<br />
(41.73 ± 4%), control (42.39 ± 4 %), or control+48 (44.47 ± 4 %) sessions. This effect<br />
was replicated with rats tested previously with 2 Hz stimulation (data discussed in section<br />
3.3 below). In contrast, 20 Hz stimulation of the dHip (n=13) failed to induce any<br />
significant changes in percent PPI (Fig. 2-1C; F(3, 36) = 2.01, N.S.). Percent PPI levels<br />
were similar in the stim (39.40 ± 3%), control (46.94 ± 5 %), stim+48 (51.40 ± 4 %), and<br />
control+48 (48.67 ± 5 %) sessions.<br />
48
stim control stim control<br />
+48 +48<br />
Condition<br />
stim control stim: control<br />
+48 +48<br />
Condition<br />
startle<br />
stim control stim control<br />
+48 +48<br />
Condition<br />
dHip - startle<br />
stim control stim control<br />
+48 +48<br />
Condition<br />
Figure 2-1. Effects of 20 Hz stimulation of the hippocampus on PPI and acoustic startle<br />
amplitude. A, Percent PPI scores 2 min (stim) or 48 hr (stim+48) following 20 Hz<br />
stimulation (10 s) of the vHip (n=14), and respective control PPI scores (control and<br />
control+48). The asterisk denotes a significant reduction in PPI in the stim group when<br />
compared to all other groups (p
Presentation of the auditory pulse alone induced robust startle in all rats (Fig. 2-<br />
1B, D). A one-way repeated measure ANOVA revealed that average startle amplitudes<br />
for the 4 PPI test sessions of the vHip 20 Hz stimulation group did not differ significantly<br />
(F(3, 39) = 1.40, N.S.). Thus, stimulation of the vHip did not significantly affect startle<br />
amplitude (average startle amplitude 116.61 ± 20 arbitrary startle units). Although<br />
stimulation of the dHip did not alter percent PPI levels (Fig. 2-1C), a significant main<br />
effect of test session was observed when startle amplitude was examined (Fig. 2-1D, F(3,<br />
36) = 3.37, p < 0.03). Post-hoc analyses revealed that when tested in the stim+48<br />
condition, rats had significantly lower startle amplitudes (74.24 ± 10) than when tested in<br />
the control condition (101.30 ± 13). Although a similar trend was noted between the stim<br />
and stim+48 test sessions, it was not significant.<br />
Effects of 2 Hz Stimulation of the vHip on PPI and Startle Amplitude<br />
Two hertz stimulation of the vHip (n=15) did not significantly alter PPI levels<br />
(Fig. 2-2A, F(3, 42) = 0.34, N.S.). When tested in the stim (46.93 ± 4 %), control (42.35<br />
± 4 %), stim+48 (43.03 ± 4 %), and control+48 (43.52 ± 3 %) conditions, the 2 Hz group<br />
demonstrated similar levels of PPI. In order to verify that 20 Hz stimulation would<br />
disrupt PPI in this group of rats, one week following their last PPI session, nine of the<br />
rats received 20 Hz stimulation of the vHip, and were tested for PPI 2 min and 24 hr later.<br />
Results revealed that 2 min following stimulation, PPI scores were significantly lower<br />
(30.01 ± 6 %) than 24 hr following stimulation (46.96 ± 4 %, F(l, 8) = 5.88, p < 0.05,<br />
data not shown). These data confirmed the short-term disruptive effect of 20 Hz<br />
stimulation of the vHip on PPI previously described.<br />
Stimulation (2 Hz) of the vHip produced an unexpected significant increase in<br />
50
A<br />
vHip-PPI<br />
stim control stim control<br />
+48 +48<br />
Condition<br />
B<br />
300, vHip - startle<br />
stim control stim control:<br />
+48 +48<br />
Condition<br />
Figure 2-2. Effects of 2 Hz stimulation of the vHip on PPI and acoustic startle<br />
amplitude. A, Percent PPI scores resulting from 2 Hz stimulation (100 s) of the vHip<br />
(n=15) either 2 min (stim) or 48 hr (stim+48) prior to testing, and respective control PPI<br />
scores (control and control+48). Control testing sessions were run exactly the same as<br />
'stim' sessions; however, no stimulation was applied to the vHip. B, Amplitude of the<br />
startle response during the same test conditions depicted in panel A. The cross denotes a<br />
significant difference between the stim condition and the stim+48 and control+48 groups<br />
(p
startle amplitude (Fig. 2-2B, F(3, 42) = 9.04, p < 0.001). Post-hoc analyses revealed that<br />
following 2 Hz stimulation, startle amplitudes were significantly higher (213.68 ± 29)<br />
than during the stim+48 (137.04 ± 15) or control+48 (134.71 ± 19) test sessions.<br />
Effects of Stimulation of the vHip and dHip on NAc DA Efflux<br />
As depicted in Fig. 2-3, baseline levels of DA in the NAc were relatively stable in<br />
all groups prior to stimulation. Stimulation of the vHip (20 Hz, n=7) induced a robust,<br />
long-lasting 35.41 ±4% increase in DA efflux in the NAc that was restricted to the<br />
hemisphere ipsilateral to the stimulating electrode. Separate one-way repeated measures<br />
ANOVA's confirmed that the increase in DA efflux in the ipsilateral hemisphere was<br />
significantly greater than baseline for 20 min (F(12, 72) = 4.04, p < 0.001, Dunnett's p <<br />
0.05), whereas no significant changes in DA efflux were observed in the contralateral<br />
hemisphere following vHip stimulation (F(12, 72) = 1.23, N.S.). Stimulation of the dHip<br />
(n=7) failed to evoke an increase in DA efflux in the NAc (F(12, 72) = 1.52, N.S.).<br />
Histology<br />
The locations of representative stimulation electrodes and the active tips of<br />
microdialysis probes are depicted in Fig. 2-4A-C. Ventral hippocampal placements (Fig.<br />
4A) were similar to those previously reported in the CAl/ventral subiculum (Wan et al.,<br />
1996;Blaha et al., 1997;Klarner et al, 1998;Zhang et al., 1999;2002a;Taepavarapruk et<br />
al, 2000;Bast et al., 2001;Swerdlow et al., 2001b). Electrodes aimed at the dHip (Fig.<br />
4B) were similarly positioned to dHip cannulae reported <strong>by</strong> a number of different groups<br />
(Swerdlow et al., 2001b;Zhang et al., 2002a) in the CA3/dentate gyrus region of the<br />
dHip. The placements of the active tips of the microdialysis probes aimed at the NAc did<br />
52
^ -20 -I 1 1 1 1 1 1 1 1 1 1 1 1<br />
1 2 3 4 5 6 7 8 9 10 11 12 13<br />
Time (X 10 min)<br />
Figure 2-3. Unilateral stimulation (20 Hz, 10 s) of either the vHip (n=7, stim) or dHip<br />
(n=7), and its effect on NAc DA efflux in the ipsilateral (vHip - black diamonds; dHip -<br />
white triangles) or contralateral (vHip - white squares) hemisphere. Data points<br />
represent the mean level of DA obtained over a 10 min sampling period, and error bars<br />
represent SEM. Asterisks denote a significant difference from baseline (p
Figure 2-4. Schematic diagram of the placements of stimulating electrodes and<br />
microdialysis probes in all experiments. A, Representative placements of electrode tips<br />
(black circles) aimed at the vHip. B, Representative placements of electrode tips (black<br />
circles) aimed at the dHip. C, Representative placements of microdialysis probes aimed<br />
at the NAc. The black bars illustrate the position of the probes and are scaled to 2 mm in<br />
length to accurately reflect only the area of the brain from which each probe sampled.<br />
Numbers correspond to the anterior or posterior distance (in mm) of each plate from<br />
bregma. Plates adapted from Paxinos and Watson (Paxinos and Watson, 1997).<br />
54
not substantially differ between the vHip and dHip stimulation experiments and are<br />
depicted on the same panels (Fig. 2-4C).<br />
Discussion<br />
In the present study, the effects of electrical stimulation of the hippocampus on<br />
PPI were site and frequency specific. Unilateral 20 Hz stimulation of the vHip, but not<br />
the dHip, caused a significant reduction in PPI at all prepulse intensities when testing<br />
began 2 min, but not 24 or 48 hr, after stimulation. Two Hz stimulation of the vHip<br />
failed to produce any changes in PPI. Changes in startie amplitude were unpredictable<br />
and difficult to interpret. Higher frequency stimulation of the vHip did not change startle<br />
amplitude significantly, whereas 2 Hz stimulation significantly increased startle<br />
amplitude 2 min following stimulation. When tested in the control condition, rats in the<br />
dHip group showed significantly higher startle amplitudes than when tested in the<br />
stim+48 condition. Experiments performed with microdialysis in freely moving rats<br />
confirmed our previous report that 20 Hz stimulation of the vHip results in a significant<br />
increase in NAc DA efflux (Taepavarapruk et al., 2000;Bast et al., 2001), and showed<br />
further that this increase was restricted to the hemisphere ipsilateral to the stimulating<br />
electrode. Finally, 20 Hz stimulation of the dHip failed to significantly increase NAc DA<br />
efflux.<br />
Potent Disruption of Sensorimotor Gating Induced <strong>by</strong> 20 Hz Stimulation of the vHip, but<br />
not dHip<br />
Numerous studies have reported that prolonged activation of the vHip, but not<br />
dHip, induced <strong>by</strong> bilateral infusion of NMDA, disrupts PPI in both Sprauge-Dawley (SD)<br />
and Wistar (W) rat strains (Wan et al., 1996;Klarner et al., 1998;Zhang et al,<br />
55
1999;2002a;Bast et al., 2001;Swerdlow et al, 2001b). The present study demonstrated a<br />
similar disruption of PPI following 20 Hz electrical stimulation of the vHip in the LE<br />
strain. Although hooded rats, such as the LE strain, are less commonly used in PPI<br />
experiments than the SD and W strains, they have been employed in studies of the<br />
genetic determinants (Swerdlow et al., 2001c;2003) and other pharmacological aspects of<br />
PPI (Rasmussen et al., 1997;Faraday et al., 1999;Binder et al., 2001). Recent<br />
experiments suggest that the three strains are differentially sensitive to the disruptive<br />
effects of DA agonists (Swerdlow et al., 2001c;2003), which is interesting given the role<br />
of the vHip in modulating NAc DA efflux discussed below.<br />
The degree (~ 50 %) and duration (< 24 hr) of PPI disruption following the brief<br />
20 Hz electrical stimulation employed in these studies were similar to that observed<br />
following NMDA infusion (Wan et al, 1996;Zhang et al., 1999;2002a;Bast et al.,<br />
2001;Swerdlow et al., 2001b), although some inconsistencies have been reported<br />
(Klarner et al., 1998). By using electrical stimulation of the vHip, we have demonstrated<br />
the brevity (10 s) and specificity (20 Hz vs. 2 Hz) <strong>by</strong> which aberrant activity in the vHip<br />
results in disrupted PPI. Clearly, the mechanism <strong>by</strong> which vHip stimulation disrupts PPI<br />
must significantly outlast the duration of stimulation. Analysis of the startle amplitude<br />
data revealed that no significant changes in startle occurred following 20 Hz stimulation<br />
of the vHip. Inspection of the startle amplitude data following NMDA infusion into the<br />
vHip suggests that startle amplitude is typically either unchanged (Wan et al.,<br />
1996;Klarner et al., 1998;Bast et al., 2001) or reduced (Zhang et al.,<br />
1999;2002a;Swerdlow et al., 2001b) following chemical stimulation of the vHip. Given<br />
the inconsistent changes in startie amplitude relative to consistent disruption of PPI<br />
56
following stimulation of the vHip, it is unlikely that the reduction in PPI observed here is<br />
due to changes in startle amplitude.<br />
The role of the hippocampus in the modulation of PPI has also been assessed with<br />
a variety of other manipulations. Permanent lesions of either the vHip or dHip do not<br />
affect PPI (Swerdlow et al., 1995;2000b;Pouzet et al., 1999 but see also Caine et al.,<br />
2001), although lesions of the vHip increase the sensitivity of rats to apomorphine-<br />
induced disruption of PPI (Swerdlow et al., 1995;2000b). Interestingly, temporary<br />
inactivation of either the vHip or dHip reversibly disrupts PPI (Zhang et al., 2002b).<br />
Data regarding modulation of PPI <strong>by</strong> the dHip are inconsistent and difficult to interpret.<br />
Infusion of the noncompetitive NMDA antagonist MK-801 into the vHip does not affect<br />
PPI (Bakshi and Geyer, 1998), whereas MK-801 infusions into the dHip may (Bakshi and<br />
Geyer, 1998;1999) or may not (Bast et al., 2000;Zhang et al., 2000b) alter PPI<br />
responding. In summary, these previous data demonstrate a subtle role for the vHip in<br />
the modulation of PPI under normal conditions. However, the present 20 Hz stimulation<br />
data, and those of others groups using NMDA (Wan et al., 1996;Zhang et al.,<br />
1999;2002a;Bast et al., 2001;Swerdlow et al., 2001b), indicate that overactivity in the<br />
vHip can cause a potent reduction in PPI. Although the dHip may also modulate PPI<br />
under certain conditions, data from this study and others (Swerdlow et al., 2001b;Zhang<br />
et al., 2002a) clearly indicate that induced overactivity of this structure does not<br />
significantly disrupt PPI.<br />
Frequency Dependence of the vHip Stimulation-induced Disruption of PPI<br />
Two Hz stimulation of the vHip, which causes a significant reduction in NAc DA<br />
efflux that may be mediated <strong>by</strong> the activation of group 2/3 mGluR's (Taepavarapruk et<br />
57
al., 1998), failed to significantly affect PPI in this study (Fig. 2-2). Available<br />
pharmacological evidence is consistent with the observed lack of effect on PPI following<br />
2 Hz vHip stimulation. For instance, blockade of DA receptors with DA antagonists,<br />
such as haloperidol, does not alter levels of PPI in rats (Mansbach et al., 1988;Depoortere<br />
et al., 1997;Hart et al., 1998;Feifel and Priebe, 1999). Additionally, systemic<br />
administration of the group 2/3 mGluR agonist LY354740 has no effect on PPI levels<br />
(Schreiber et al., 2000). Thus, pharmacological treatments that mimic some of the effects<br />
of 2 Hz vHip stimulation are also without effect on PPI responding.<br />
Unexpectedly, 2 Hz stimulation of the vHip resulted in a significant increase in<br />
startle amplitude immediately following the stimulation when compared to the stim+48<br />
and control+48 conditions. Treatment with group 2/3 mGluR agonists or haloperidol<br />
decreased or had no effect on startle magnitude respectively (Mansbach et al.,<br />
1988;Depoortere et al., 1997;Hart et al., 1998;Feifel and Priebe, 1999;Schreiber et al.,<br />
2000). Given that these drugs were administered systemically, it is possible that their<br />
effects may result from actions in brain areas other than those affected <strong>by</strong> 2 Hz<br />
stimulation. However, it is interesting that stimulation of the vHip with two different<br />
frequencies had different effects on both startle and PPI. Given the inconsistencies in<br />
startle changes observed following NMDA stimulation (Wan et al., 1996;Zhang et al.,<br />
1999;2002a;Legault et al., 2000;Taepavarapruk et al., 2000;Bast et al., 2001;Swerdlow et<br />
al., 2001b), it will be important to replicate these findings in separate groups and/or<br />
strains of rats to ensure their reliability.<br />
58
Increases in NAc DA Efflux following Hip Stimulation are Site and Hemisphere Specific<br />
Stimulation of the vHip with either NMDA or brief periods of 20 Hz electrical<br />
stimulation produces a long-lasting increase in NAc DA efflux (Legault et al.,<br />
2000;Taepavarapruk et al., 2000). The effect of 20 Hz stimulation is mediated <strong>by</strong><br />
iGluR's in the NAc, there<strong>by</strong> implicating the direct glutamatergic projection from the<br />
vHip to NAc in this effect (Blaha et al, 1997;Taepavarapruk et al., 2000). The present<br />
neurochemical experiments (Fig. 3) extend our previous findings, as the increase in NAc<br />
DA efflux following 20 Hz electrical stimulation of the vHip was restricted to the<br />
hemisphere ipsilateral to the stimulating electrode. This finding is consistent with the<br />
fact that the vHip projections to forebrain areas such as the NAc are primarily unilateral<br />
(Kelley and Domesick, 1982;Groenewegen et al, 1987;Brog et al., 1993).<br />
In contrast to stimulation of the vHip, 20 Hz stimulation of the dHip failed to<br />
increase NAc DA efflux significantly. Although the dHip sends glutamatergic efferents<br />
to the NAc, it is important to note that the vHip projects primarily to the NAc shell<br />
whereas the dHip projects primarily to the NAc core (Swanson and Cowan, 1977;Kelley<br />
and Domesick, 1982;Whitter, 1986;Groenewegen et al., 1987;Brog et al., 1993). Probe<br />
placements adjacent to both the shell and core subregions of the NAc were employed in<br />
studies in which stimulation of the vHip increased NAc DA efflux (Fig. 2-4C); therefore,<br />
we monitored DA efflux in a similar region of the NAc following stimulation of the<br />
dHip. This raises the possibility that any effect of the dHip stimulation on NAc DA<br />
efflux was restricted to the NAc core. However, unpublished observations failed to<br />
confirm an increase in NAc DA efflux with dialysis probes located in the core of the NAc<br />
(Howland, Taepavararpuk, and Phillips).<br />
59
Microdialysis experiments have examined dynamic changes in NAc DA efflux<br />
during PPI test sessions. Zhang and colleagues (Zhang et al., 2000a) demonstrated that<br />
systemic administration of amphetamine disrupted PPI, whereas treatment with cocaine<br />
had no effect (but also see Martinez et al., 1999). Interestingly, both drugs increased<br />
NAc DA efflux <strong>by</strong> at least 100 % of baseline during the PPI test sessions. Consequently,<br />
these authors suggest that the behavioral effects of DA agonists on PPI are critically<br />
dependent on their mechanism of action. Other experiments have shown that<br />
presentation of startle pulses alone cause a significant decrease in NAc DA efflux that is<br />
not observed when prepulses of a moderate intensity are presented prior to the startle<br />
pulses (Hum<strong>by</strong> et al., 1996). These data suggest that during PPI, the presentation of the<br />
prepulses prevents perturbation of the mesoaccumbens DA system in response to the<br />
startling stimuli. Together, these data imply that certain drugs, such as amphetamine, can<br />
interfere with the effect of a prepulse on DA efflux in the NAc, there<strong>by</strong> disrupting PPI.<br />
Given these data, it is intriguing that two different methods of stimulating the<br />
vHip, both of which increase DA efflux in the NAc, can disrupt PPI. The fact that 20 Hz<br />
stimulation of the dHip or 2 Hz stimulation of the vHip failed to disrupt PPI and did not<br />
increase NAc DA efflux is consistent with the hypothesis that increased NAc DA efflux<br />
following 20 Hz vHip stimulation may be a factor in the disruption of PPI. However,<br />
systemic administration of the D 2 antagonist haloperidol (Wan et al., 1996;Zhang et al.,<br />
1999;Bast et al., 2001) or intra-NAc infusions of the non-NMDA receptor antagonist<br />
CNQX (Wan et al., 1996) fail to block the disruptive effects of intra-vHip NMDA<br />
infusion on PPI responding. Therefore, it is unlikely that the disruptive effects of<br />
60
stimulation of the vHip on PPI are mediated solely <strong>by</strong> activation of the mesoaccumbens<br />
DA system or non-NMDA receptors in the NAc (Wan et al., 1996;Bast et al., 2001).<br />
Potential Mechanisms Underlying vHip Stimulation-induced Disruption in PPI<br />
Normal DA and Glu transmission appears to be important for PPI (Zhang et al.,<br />
2000a;Geyer et al., 2001). For example, infusions of DA or DA agonists into the NAc<br />
disrupt PPI (Mansbach et al., 1988;Swerdlow et al., 1990;Wan et al., 1995), and infusions<br />
of Glu agonists and antagonists such as AMPA, APV, CNQX, and MK-801 into the NAc<br />
have complex and regionally specific effects (Reijmers et al., 1995;Wan et al., 1995;Wan<br />
and Swerdlow, 1996). Recent physiological experiments raise the possibility that the<br />
effects of vHip stimulation on PPI may involve interactions between multiple receptor<br />
subtypes in the NAc. Stimulation of the vHip shifts NAc neurons to an activated or 'up'<br />
state (O'Donnell and Grace, 1995) and recent experiments from our laboratory indicate<br />
that 20 Hz stimulation (10 s) of the vHip alters the responses of medium spiny neurons in<br />
the NAc shell to afferent input from the vHip and BLA (Floresco et al., 2001). These<br />
effects are dependent on the 20 Hz stimulation-induced increase in NAc DA and a<br />
complex pharmacology involving activation of both D t and NMDA receptors, but not D 2<br />
and non-NMDA receptors (Floresco et al., 2001). Importantly, the potentiating effect of<br />
20 Hz stimulation of the vHip on NAc neurons is maintained for at least 20 min, which<br />
corresponds to the duration of PPI trials in the present study.<br />
Stimulation of the vHip may also disrupt PPI via the widespread connections of<br />
the vHip to a number of areas in the CSPP circuitry critical for normal PPI (Klarner et al.,<br />
1998). Data obtained in the present study do not refute this hypothesis. The vHip is<br />
reciprocally connected to a number of areas other than the NAc, such as the mPFC,<br />
61
amygdala, thalamus, septum, and ventral pallidum (Groenewegen et al, 1987;Amaral and<br />
Whitter, 1995;Conde et al., 1995), all of which are important for the regulation of PPI<br />
(Swerdlow et al., 2001a). Stimulation of the vHip with doses of NMDA that disrupt PPI<br />
cause increased fos-protein expression in a number of limbic and cortical areas such as<br />
the NAc, lateral septum, mPFC, and dorsomedial striatum (Klarner et al., 1998).<br />
Stimulation of the vHip also induces various types of plasticity in the mPFC such as<br />
long-term potentiation and depression (Laroche et al., 2000). Therefore, subtle changes<br />
in multiple areas induced <strong>by</strong> vHip stimulation may disrupt PPI (Klarner et al., 1998).<br />
Although the dHip projects to the NAc core, it is not directly connected to other areas<br />
implicated in PPI (Swanson and Cowan, 1977;Kelley and Domesick, 1982;Whitter,<br />
1986;Amaral and Whitter, 1995), and therefore aberrant activity generated in the dHip<br />
may not have access to the neural substrates critical to disrupt PPI. This conjecture is<br />
consistent with the failure of 20 Hz stimulation of the dHip to increase NAc DA efflux in<br />
the NAc or to disrupt PPI in the present study.<br />
Conclusion<br />
Our data clearly demonstrate that brief, higher frequency stimulation of vHip, but<br />
not dHip, disrupts PPI and increases DA efflux in the NAc. Given the anatomical and<br />
functional abnormalities frequently observed in the hippocampal formation of patients<br />
with schizophrenia, these data support the assertion that abnormalities in limbic-cortico-<br />
striatal interactions may underlie deficits in sensorimotor gating in schizophrenia.<br />
Identification of the mechanism underlying the disruptive effects of vHip stimulation on<br />
PPI may provide insight into potential targets for pharmacological treatment strategies for<br />
schizophrenia.<br />
62
\<br />
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69
CHAPTER THREE: DELAYED ONSET OF PREPULSE INHIBITION DEFICITS<br />
FOLLOWING KAINIC ACID TREATMENT ON POSTNATAL DAY SEVEN IN<br />
<strong>RAT</strong>S. 2<br />
Introduction<br />
Given the heterogeneity and complexity of schizophrenia, its etiology has been<br />
difficult to define. The neurodevelopmental hypothesis of schizophrenia proposes that<br />
adverse events or genetic abnormalities may disrupt early brain development, and interact<br />
with environmental factors or influence subsequent brain development to cause the<br />
disorder (Weinberger, 1995;Benes et al., 2000;McClure and Weinberger, 2001). Once<br />
the illness is expressed, a diverse array of subde changes are found in temporal and<br />
frontal brain areas such as the hippocampus, striatum, thalamus, and frontal cortex<br />
(Pakkenberg, 1990;Suddath et al., 1990;Bogerts, 1997;Jones, 1997;Heckers etal.,<br />
1998;Harrison, 1999;Gothelf et al., 2000;McClure and Weinberger, 2001;Meyer-<br />
Lindenberg et al., 2002). Neurodegeneration in these areas is rarely observed in the<br />
brains of patients with schizophrenia (but see Stevens, 1994), therefore alterations in<br />
normal patterns of connectivity between affected brain areas may underlie the expression<br />
of some symptoms of schizophrenia (Harrison, 1999;Friston, 1999;Benes, 2000;Penn,<br />
2001).<br />
Accordingly, the consequences of disrupting the normal development of limbic<br />
and cortical areas on behaviors in rodents related to schizophrenia have been examined<br />
(for reviews see Lipska and Weinberger, 2000;Van den et al., 2003). Specific behavioral<br />
abnormalities have been observed in adult, but not prepubescent rats, that received<br />
2<br />
A version of this chapter has been published: Howland JG, Hannesson DK, Phillips AG (2004). Delayed<br />
onset of prepulse inhibition deficits following kainic acid treatment on postnatal day seven in rats.<br />
European Journal of Neuroscience 20:2639-2648.<br />
70
ventral hippocampal (vHip) lesions on postnatal day (PND) 7 (Lipska and Weinberger,<br />
2000). Behavioral changes in prepulse inhibition (PPI) and locomotor responses to<br />
novelty, which resemble certain positive symptoms in schizophrenia, are robust and can<br />
be exacerbated <strong>by</strong> acute administration of dopamine (DA) agonists immediately before<br />
testing in adulthood (Lipska et al., 1993; 1995;Le Pen and Moreau, 2002). Importantly,<br />
temporary reduction of neural activity within the vHip on PND7, with the sodium<br />
channel blocker tetrodotoxin (TTX) increased locomotor activity in response to novelty,<br />
injection stress, and amphetamine after puberty (Lipska et al., 2002). Similar results have<br />
been observed following neonatal lesions of the amygdala (Daenen et al., 2001; 2002;<br />
2003), but not the medial prefrontal cortex (Lipska et al., 1998;Brake et al., 2000;Van<br />
den et al., 2003 but see Flores et al., 1996). Additionally, systemic administration of the<br />
non-competitive NMDA receptor antagonist dizocilpine (MK-801) on PND7 can reduce<br />
PPI levels and increase locomotor activity in female rats (Harris et al., 2003; but see<br />
Beninger et al., 2002).<br />
The apparent sensitivity of limbic-cortical circuits on PND7 to functional<br />
disruption raises the possibility that increased neural activity induced <strong>by</strong> the ionotropic<br />
glutamate agonist kainic acid (KA) at this developmental age would result in behavioral<br />
changes in rats similar to those discussed above. Kainic acid has been used extensively<br />
to study limbic-motor seizures and excitotoxicity in adult rats (for reviews see Sperk,<br />
1994;Ben Ari and Cossart, 2000); however, its effects during early development are not<br />
as well characterized. During the first two postnatal weeks, systemic administration of<br />
KA results in tonic-clonic seizures and the selective activation of the hippocampus and<br />
lateral septum without the high levels of cell death normally observed in limbic and<br />
71
cortical areas following administration during or after the 3 r<br />
postnatal week (Nitecka et<br />
al., 1984;Tremblay et al., 1984;Stafstrom et al., 1992;Khalilov et al., 1999;Koh et al.,<br />
1999;Lynch et al., 2000;Silveira et al., 2002). Although few studies have analyzed<br />
behavioral changes in adulthood following a single administration of KA during the first<br />
week of life, there are reports of deficits in conditioned avoidance (de Feo et al., 1986)<br />
and spatial learning using the radial arm maze (Lynch et al., 2000), but not the water<br />
maze (Stafstrom et al., 1993;Koh et al., 1999).<br />
The present study sought to examine whether systemic KA administration on<br />
PND7 would alter the behavior of rats in a pattern similar to that observed in other animal<br />
models of schizophrenia (Lipska et al., 1993;Harris et al., 2003). Kainic acid or saline<br />
was administered to rat pups on PND7 and their PPI levels and locomotor responses to a<br />
novel environment were measured before and after puberty. To assess potential<br />
alterations of the DA system following KA administration, we tested the potential<br />
interactive effects of KA treatment and apomorphine, a direct DA agonist, on PPI and<br />
also with amphetamine-induced locomotion. Subgroups of KA- and saline-treated rats<br />
were also tested on a standard hippocampal-dependent Morris Water Maze task in<br />
adulthood to examine aspects of spatial learning and memory that are dependent on<br />
normal hippocampal function (Morris et al., 1990).<br />
Methods<br />
Subjects<br />
Two independent groups of animals (i.e. Group 1 and Group 2) were tested in this<br />
study. Testing protocols for both groups were similar with small procedural differences<br />
noted in the methods section where appropriate. Pregnant Long-Evans rats were obtained<br />
72
from Charles River (Quebec, Canada) at 13 to 15 days of gestation. They were singly<br />
housed and left undisturbed until giving birth. The colony was maintained on a 12/12<br />
hour light/dark cycle (lights on at 0700), at a temperature of 22+1 °C. All rats were given<br />
food (Purina Rat Chow) and tap water ad libitum. Experiments were conducted in<br />
accordance with the standards of the Canadian Council on Animal Care and were<br />
approved <strong>by</strong> the Committee on Animal Care at the University of British Columbia.<br />
Kainic Acid Administration<br />
The day of birth of the pups was designated post-natal day (PND) 0. On PND3,<br />
the litters were sexed and culled to include only males (6-9 rats per litter). On PND7, all<br />
but one of the pups were removed from the nest, weighed and individually placed in<br />
small compartments of a cardboard box for KA administration. They were then removed<br />
from the colony and taken to a small heated room. KA (1.5 mg/kg, Tocris) or saline was<br />
injected (i.p.) with a 30-gauge needle (10 ml/kg). Care was taken to ensure that both KA<br />
and saline was administered to members from each litter. Previous reports indicated that<br />
KA administration of 1-2 mg/kg on PND7 causes behavioral seizures in most pups<br />
without high mortality rates (Stafstrom et al., 1993;Lynch et al., 2000). The behavior of<br />
all rats was recorded with an overhead video camera for 180 minutes. Before being<br />
returned to their mothers, the pups were earmarked according to treatment condition.<br />
The litters were then left undisturbed (except for normal cage changing) until weaning on<br />
PND25. Weanling rats were housed in cages of 2 or 3 with members of their litter. All<br />
rats were handled for at least 5 days before behavioral testing.
Prepulse Inhibition<br />
On PND35 and 56, rats were removed individually from the colony and taken<br />
immediately to the PPI apparatus. Testing was conducted in a single sound-attenuating<br />
startle chamber (ambient noise level 64 dB), containing a transparent Plexiglas tube (8.2<br />
cm in diameter, 20 cm in length), mounted on a Plexiglas frame (SR-LAB, San Diego<br />
Instruments, San Diego). Noise bursts were presented through a speaker mounted 24-cm<br />
above the tube. An accelerometer below the frame of the apparatus measured whole<br />
body startle amplitude, defined as the average of 100 1-ms accelerometer readings<br />
collected from stimulus onset. Each PPI test session began with a 5-min acclimatization<br />
period during which a 70-dB background noise level was presented, which remained<br />
constant for the entire test session. Following the acclimatization period, six pulse alone<br />
trials (120 dB, 40 ms) were presented to achieve a relatively stable startle amplitude<br />
before PPI testing. Data from these pulse-alone trials was not considered in the analysis<br />
of PPI. Immediately following the six initial pulse-alone trials, presentation of the trials<br />
that were used in the calculation of PPI levels were initiated. Trials presented were of<br />
three types: pulse alone (12 trials, 120 dB, 40 ms), prepulse + pulse (10 trials X 3<br />
prepulse intensities - discussed below), or no stimulus (42 trials). Prepulse + pulse trials<br />
consisted of the presentation of a 20 ms prepulse of 73, 76, or 82 dB 80 ms before the<br />
presentation of the pulse. The pulse and prepulse + pulse trials were presented in a<br />
pseudorandom order. One no-stimulus trial was presented between each pair of pulse and<br />
prepulse + pulse trials. The inter-trial interval varied randomly from 3 to 12 s (average<br />
7.5 s). Calibration of the apparatus was performed using a RadioShack Digital Sound<br />
Level Meter and adjustments were made as necessary.<br />
74
Between PND98 and 105, a randomly selected subset of the rats from Group 2<br />
were re-tested on PPI following injection with vehicle (0.1% ascorbic acid) and<br />
apomorphine (Sigma; 0.2 mg/kg; s.e.) 72 hr later to assess: 1) the consistency of the PPI<br />
reduction in KA-treated animals and 2) the potential role of dopaminergic mechanisms in<br />
the observed PPI decreases in the KA-treated animals. The PPI sessions were conducted<br />
in a manner identical to that described above, except that immediately before the PPI<br />
session, all rats were weighed and injected with the appropriate volume of vehicle or<br />
drug.<br />
Locomotor Activity<br />
On PND36 and 57, rats were weighed, removed from the colony, and<br />
immediately placed in locomotor boxes. For Group 1, the locomotor boxes were in two<br />
similar, small adjacent rooms (4 boxes in one, 3 in the other) constructed from clear<br />
Plexiglas (32 cm X 32 cm X height 41 cm) and fitted with four pairs of infra-red beams<br />
positioned 10 cm apart and 2.5 cm above a metal grid floor. All sensors were interfaced<br />
to a computer-controlled system (MANX). Motor activity counts were collected in 10-<br />
min bins during testing. Care was taken to ensure that rats from each group were tested<br />
in both rooms and in all boxes. Spontaneous locomotor activity levels in a novel<br />
environment were measured for 60 min. Immediately following the spontaneous<br />
locomotor activity test, all rats were injected with D-amphetamine (1.5 mg/kg, i.p.) and<br />
returned to the locomotor boxes. Locomotor activity was then monitored for an<br />
additional 90 min before the rats were returned to the colony room. For Group 2, testing<br />
procedures were identical, except that a Med Associates System was used to collect the<br />
data. Eight Med Associates Test Chambers (ENV-008; 30.5 cm X 24.1cm X height 21.0<br />
75
cm) were fitted with 4 pairs of infrared photocells 3.5 cm from the floor evenly spaced on<br />
the long walls. The chambers had metal grid floors and two operant levers (which were<br />
retracted during locomotor activity testing). Each chamber was contained within a Med<br />
Associates sound attenuating cubicle with a house light that was illuminated during<br />
testing.<br />
Water Maze Testing<br />
A subgroup of the animals from Group 1 was tested between PND65 and 75 in a<br />
water maze. Testing was performed in a circular, white swimming pool 180 cm in<br />
diameter, 54 cm in height. The escape platform was 34 cm in height and 14 cm in<br />
diameter. The pool was filled with water such that the top of the platform was 3 cm<br />
below the surface of the water. For visible platform trials, an additional piece of wood<br />
and wire mesh was secured to the top of the invisible platform such that it extended 3 cm<br />
out of the water. The water was rendered opaque <strong>by</strong> the addition of white Tempura<br />
Powder paint. Data were recorded using a computerized tracking system (HVS Image,<br />
Hampton, England). Rats were transported to the pool room in groups and testing was<br />
conducted over 3 consecutive days. Rats were released facing the wall of the pool from<br />
one of the geographic poles in a pseudorandom order. Each rat was allowed 60 s to swim<br />
to the platform, after which they were left on the platform for 10 s. They were then<br />
removed from the water maze, and the next rat was tested. The intertrial interval for each<br />
rat was approximately 7 min. On the first day, rats were trained to swim to the visible<br />
platform over 5 trials to allow habituation to the testing environment and swimming. The<br />
location of the platform was varied for each trial, and was never in the location of the<br />
hidden platform used on day 2. Hidden platform training was performed on day 2. Rats<br />
76
were given 10 trials to learn to find the constant location of the hidden platform. Finally,<br />
on day 3, the rats were tested in a 30-s probe trial without the platform present. The<br />
percent time spent in the quadrant that contained the hidden platform on day 2 was taken<br />
as a measure of spatial memory.<br />
Histology<br />
Following behavioral testing, a subgroup of animals (n=5 from each group) was<br />
randomly selected, sacrificed with an overdose of pentobarbital, and perfused<br />
transcardially with 0.9% saline followed <strong>by</strong> 10% formalin. Brains were stored in 10%<br />
sucrose/10% formalin for at least 1 week, after which coronal sections (40 u.m)<br />
containing the hippocampus were taken using a vibratome. Every fourth section was<br />
saved and stained with cresyl violet. An experimenter blind to the treatment group<br />
examined each section using a light microscope for gross cell loss in the hippocampus<br />
with particular attention paid to the CA3 region due to its established sensitivity to the<br />
excitotoxic effects of KA in adult rats (Ben Ari and Cossart, 2000).<br />
Data Analysis<br />
For the PPI experiments, two measures were calculated for each animal. The<br />
startle amplitude represented the mean startle amplitude of the 12 pulse-alone trials<br />
presented after the 6 habituation trials. Startle amplitude data were compared with an<br />
independent samples t-test for each age. PPI was calculated <strong>by</strong> averaging startle<br />
amplitudes for each trial type. The percent PPI for each prepulse intensity was calculated<br />
using the formula: [100 - (100 X startle amplitude on prepulse + pulse trials) -s- (startle<br />
amplitude on pulse alone trials)]. A repeated measures ANOVA was performed on the<br />
data obtained from each age with prepulse intensity as a within subjects factor and<br />
77
treatment on PND7 as a between subjects factor. Results from the apomorphine<br />
experiment were also analyzed with a mixed ANOVA (prepulse intensity and test as<br />
within subjects factors, and treatment at PND7 as a between subjects factor). Locomotor<br />
activity data were compared using repeated measures ANOVA at each age with test type<br />
as a within subjects factor and treatment on PND7 as a between subjects factor. For the<br />
water maze task, trials were blocked into pairs to reduce variance; latencies and path<br />
lengths to find the hidden platform were analyzed using a repeated measures ANOVA<br />
(trial block as a within subjects factor, treatment on PND7 as a between subjects factor).<br />
Additionally, swim speeds were measured for all rats and compared using a repeated<br />
measures ANOVA. After the probe trials, the percent time spent in each quadrant was<br />
calculated <strong>by</strong> dividing the time spent in that quadrant <strong>by</strong> the total length of the probe trial<br />
(30 s). A repeated measures ANOVA was performed on the data with quadrant as a<br />
within subjects factor and treatment on PND7 as a between subjects factor. For all<br />
ANOVA's, post-hoc analyses were performed using the Neuman-Keuls test where<br />
appropriate. The significance level for all statistical tests was p < 0.05.<br />
Results<br />
Immediate Behavioral Effects ofPND7 KA Administration<br />
Ten to 20 min following KA administration, the majority of pups began to show<br />
behavioral signs of seizure activity as previously reported (Tremblay et al.,<br />
1984;Stafstrom et al, 1992;Lynch et al., 2000;Silveira et al, 2002). Initially, the<br />
majority of the pups showed evidence of a hunched posture and heavy breathing followed<br />
<strong>by</strong> the appearance of a continuous, clonic 'scratching' activity of the rear limbs. Many of<br />
the rats developed swimming seizures during which they rolled onto their sides during<br />
78
outs of forelimb and hindlimb clonus, which continued for 30 to 120 min. Some of the<br />
pups developed more severe tonic seizures in the second hour after KA administration,<br />
which lasted up to 60 min. Only data from pups that displayed behavioral signs of<br />
seizure activity for at least 30 minutes were analyzed in these experiments, and no<br />
differences in seizure severity between pups from Group 1 and Group 2 could be readily<br />
quantified. Six pups (11%) died as a result of the KA administration, while 10 (18%)<br />
were not used for the behavioral experiments due to mild, intermittent seizures. None of<br />
the saline-treated animals displayed seizure-related behaviors.<br />
Effects ofPND7 KA Administration on Body Weight<br />
On PND7, pups randomly selected for KA administration weighed 18.13 ± 0.37 g<br />
while those administered saline weighed 17.86 ± 0.46 g. On PND8, the pups were<br />
weighed again to ensure that suckling behavior was not altered between the groups after<br />
treatment on PND7. The KA- and saline-treated rats weighted 20.45 ± 0.45 g and 20.77<br />
± 0.55 g on PND8 respectively. An independent samples t-test revealed that both groups<br />
of animals gained a similar amount of weight during the 24 hr after treatment (t(50) = -<br />
1.28, N.S.). On PND36, the rats treated with KA on PND7 weighed slightly more than<br />
those treated with saline (KA: 181.90 ± 2 g; saline: 175.90 ± 2 g); however, when tested<br />
on PND57, the rats treated with KA weighed significantly more than those treated with<br />
saline (KA: 395.33 ± 5 g; saline: 379.66 ± 5 g) as revealed <strong>by</strong> a significant age <strong>by</strong> weight<br />
interaction (F(l, 68) = 3.98, p = 0.05).<br />
79
70-,<br />
A PND35<br />
PP3 PP6 PP12 average<br />
Prepulse Intensity<br />
PP3 PP6 PP12 average<br />
Prepulse Intensity<br />
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140 i<br />
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100<br />
80<br />
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1<br />
saline kainic<br />
Treatment<br />
Figure 3-1. Prepulse inhibition (PPI) scores from rats in Group 1 treated on postnatal day<br />
(PND) 7 with saline (white bars, n=17) or kainic acid (KA; black bars, n=20). A, Effects<br />
of.PND 7 KA treatment on PPI at PND35. B, Average starde amplitudes of the rats on<br />
PND35. C, Percent PPI scores for those rats tested at PND56. D, Average startle<br />
amplitudes of the rats on PND56. Asterisks indicate a significant difference between<br />
groups (p
Prepulse Inhibition is Reduced in Post-Pubescent, but not Pre-pubescent Rats, Following<br />
PND7 KA Administration<br />
Group 1: As shown in Fig. 3-1 A, rats administered either KA (n=20) or saline<br />
(n=17) on PND7 had similar levels of PPI on PND35. Percent PPI scores were similar in<br />
response to trials with 3 and 6 dB prepulses, whereas PPI was substantially increased for<br />
trials during which a 12 dB prepulse was presented.' A repeated measures ANOVA<br />
revealed a significant effect of prepulse (F(2, 70) = 78.92, p< 0.001), but no significant<br />
prepulse <strong>by</strong> treatment interaction (F(2, 70) = 0.26, N.S.) or main effect of treatment (F(l,<br />
35) = 2.04, N.S.). On PND35, rats in the KA-treated group demonstrated slightly lower<br />
starde amplitudes than those in the saline treated group to the pulse alone trials (55.27 ± 5<br />
vs. 67.51 ± 7 arbitrary startle units) groups (Fig. 3-1B). However, this difference was not<br />
significant (t(35) = -1.41, N.S.).<br />
In contrast to PND35, PPI levels obtained from KA- and saline-treated rats in<br />
young adulthood (PND56) were significantly different (Fig. 3-1C). A repeated measures<br />
ANOVA revealed a significant effect of prepulse intensity (F(2, 70) = 33.05, p < 0.001),<br />
as well as a significant prepulse <strong>by</strong> treatment interaction (F(2, 70) = 5.57, p < 0.01). Rats<br />
in the saline-treated condition showed a typical pattern of increasing percent PPI in<br />
response to prepulses of increasing intensity (24.80 ± 5% for PP3, 41.77 ± 4% for PP6,<br />
and 54.38 ± 4% for PP12 trials). When compared to the saline-treated animals, KA-<br />
treated rats responded with significantly lower percent PPI scores for the PP6 trials<br />
(19.17 ± 6% PPI, p < 0.05), and attenuated PPI in response to PP12 trials (47.15 ± 4%).<br />
As a result, animals in the KA-treated group demonstrated a 25% reduction in average<br />
percent PPI scores when compared with the saline-treated group (KA-treated = 30.18 ±<br />
81
3%; saline-treated = 40.32 ± 4%), which approached significance (F(l, 35) = 3.93, p =<br />
0.055). The average startle amplitude of the KA-treated rats (77.20 ± 12 arbitrary startle<br />
units) was lower than that of the saline-treated group (102.11 ± 18), although this<br />
difference was not significant (t(35) = -1.17, N.S.).<br />
Group 2: As in Group 1, rats in both the KA- (n=19) and saline- (n=14) treated<br />
groups tested on PND35 had similar levels of PPI (Fig. 3-2A). A repeated measures<br />
ANOVA revealed a significant main effect of prepulse intensity (F(2, 62) = 28.62, p<br />
£-•30<br />
CD<br />
OL 20 A<br />
A PND35<br />
PP3 PP6 PP12 average<br />
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Prepulse Intensity<br />
PP3 PP6 PP 12 average<br />
Prepulse Intensity<br />
140<br />
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•a-<br />
CO<br />
60<br />
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saline kainic<br />
Treatment<br />
Figure 3-2. Prepulse inhibition (PPI) scores from rats in Group 2 treated on postnatal day<br />
(PND) 7 with saline (white bars, n=14) or kainic acid (black bars, n=19). A, Effects of<br />
postnatal day (PND) 7 KA treatment on PPI at PND35. B, Average startle amplitudes of<br />
the rats on PND35. C, Percent PPI scores for those rats tested at PND56. D, Average<br />
startle amplitudes of the rats on PND56. The asterisk indicates a significant difference<br />
between groups (p
shown). Additionally, a significant main effect of test (F(l, 16) = 16.94, p < 0.005) and a<br />
test <strong>by</strong> prepulse interaction (F(2,32) = 4.82, p < 0.02) were observed. Inspection of the<br />
data confirmed that apomorphine treatment significantly disrupted PPI in both groups of<br />
rats (Fig. 3-3A) while post-hoc analyses revealed that apomorphine disrupted PPI<br />
significantly more during the 6 dB prepulse trials than during either the 3 or 12 dB trials<br />
(p < 0.05, data not shown).<br />
Importantly, the absence of a significant test <strong>by</strong> treatment interaction (F(l,16) =<br />
0.09, N.S.) does not support the hypothesis that animals treated with KA on PND7 are<br />
more sensitive to the disruptive effects of low doses of apomorphine on PPI. A separate<br />
analysis of the data obtained following the vehicle injection revealed that those animals<br />
treated with KA (n=10) on PND7 had lower PPI scores (38.36 ± 4%) than those treated<br />
with saline (n=8; 50.00 ± 6%, Fig. 3-3 A). Although this difference was of a similar<br />
magnitude to the differences in PPI reported at PND56 for groups 1 and 2 above, it failed<br />
to reach significance perhaps due to insufficient power (t(16) = -1.65, p = 0.12).<br />
Apomorphine treatment significantly decreased startle amplitude in both groups (Fig. 3-<br />
3B). This is reflected <strong>by</strong> a significant main effect of test (F(l,16) = 6.15, p < 0.05), but<br />
not group (F(l, 16) = 0.13, N.S.) or a group <strong>by</strong> test interaction (F(l, 16) = 0.07, N.S.).<br />
Locomotor Activity in Response to Novelty and Amphetamine in Rats Following PND7<br />
KA Administration<br />
Group 1: As depicted in Fig. 3-4A and B, KA- (n=20) and saline- (n=17) treated<br />
rats in this group showed similar levels of spontaneous locomotor activity in a novel<br />
environment (60 min) or following amphetamine administration (1.5 mg/kg, 90 min) on<br />
84
S-V K-V S-A K-A<br />
Treatment<br />
S-V K-V S-A K-A<br />
VTreatment<br />
Figure 3-3. A, Effects of pretreatment with vehicle (0.1% ascorbic acid, A) or<br />
apomorphine (0.2 mg/kg, B) on average percent PPI scores in adulthood. Animals were<br />
administered either saline (n=8, white bars) or kainic acid (n=10, black bars) on postnatal<br />
day 7. B, Average startle amplitudes of those rats depicted in panel A. In both panels,<br />
asterisks indicate a significant difference between groups. The cross indicates a between<br />
group difference that approached significance (p
1 4<br />
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C Nov PND57 D Amph PND57<br />
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: Treatment<br />
Figure 3-4. Locomotor activity levels in response to novelty (A, C) or amphetamine (1.5<br />
mg/kg; B, D) of all rats tested on either postnatal day (PND) 36 (A, B) or PND57 (C, D).<br />
Group labels on the x-axis of each panel reflect the treatment of the rats on PND7 (5 =<br />
saline, K - kainic acid) and their group (1 = group 1,2 = group 2). The asterisk indicates<br />
a significant difference between the denoted groups (p
PND36. A repeated measures ANOVA failed to confirm a significant treatment effect<br />
(F(l, 35) = 0.16, N.S.) or treatment <strong>by</strong> test interaction (F(l, 35) = 0.12, N.S.). At PND57,<br />
a repeated measures ANOVA did not reveal a main effect of treatment (F(l, 35) = 2.01,<br />
N.S.), but did reveal a significant test <strong>by</strong> treatment interaction (F(l, 35) = 4.10, p = 0.05).<br />
Post-hoc analyses revealed that although both groups showed similar locomotor<br />
responses in the novelty condition (Fig. 3-4C), rats treated with KA on PND7 had<br />
significantly higher locomotor responses (2640.25 ± 225 beam crosses) following<br />
injection with amphetamine than rats treated with saline (2145.06 ± 143; Fig. 3-4D).<br />
Group 2: Similar to the data presented for Group 1, the locomotor responses of<br />
rats treated with either KA (n=19) or saline (n=14) on PND7 did not differ in either the<br />
spontaneous activity or amphetamine conditions on PND36 (Fig. 3-4A and B). This is<br />
reflected in an insignificant main effect of treatment (F(l, 31) = 1.30, N.S.) and treatment<br />
<strong>by</strong> test interaction (F(l, 31) = 2.29, N.S.). In contrast to the data obtained from Group 1,<br />
when tested on PND57, rats in Group 2 failed to show significant differences in<br />
locomotor activity in either condition (main effect of treatment: F(l, 31) = 1.70, N.S.;<br />
treatment <strong>by</strong> test interaction: F(l, 31) = 1.66, N.S.).<br />
Spatial Learning and Memory in the Morris Water Maze is not Altered in Rats Following<br />
PND7 KA Administration<br />
On the first test day, all rats learned to escape <strong>by</strong> swimming to the visible<br />
platform (data not shown). On the second day of testing, rats in both groups learnt to<br />
swim to the hidden platform (Fig. 3-5A). A repeated measures ANOVA performed on<br />
the latency data revealed a significant main effect of trial block (F(4, 52) = 17.43, p <<br />
0.001), but not a significant effect of group (F(l, 13) = 0.279, N.S.) or a significant block<br />
87
E<br />
F<br />
50<br />
40<br />
30<br />
20<br />
10<br />
A<br />
0 -n———r———i •'•.- i-<br />
B<br />
Trial Block<br />
1 • ' / .2 . . 3 ;<br />
Quadrant<br />
Figure 3-5. A, Average latencies (s) to locate the hidden platform in the water maze of<br />
rats treated with saline (white diamonds, n=7) or kainic acid (black squares, n=8) on<br />
postnatal day 7. Trial blocks represent the average search time of 2 trials for each rat.<br />
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<br />
KA- (n=8) and saline-treated (n=7) rats were significantly slower at finding the platform<br />
during block 1 (i.e. trials 1 and 2) than the subsequent 4 blocks (average search time -<br />
block 1: 38.66 ± 6 s; blocks 2 to 5: 14.38 ± 3 s; p < 0.05). Path lengths were significantly<br />
reduced across trial blocks in both groups (F(4, 52) = 14.33, p < 0.01; data not shown);<br />
however, neither total path length (KA-treated = 4,029.10 ± 483 cm, saline-treated =<br />
4597.04 ± 609 cm; F(l, 13) = 1.56, N.S.) nor swim speed (KA-treated = 22.33 ± 1 cm/s,<br />
saline-treated = 25.14 ± 1 cm/s; F(l, 13) = 3.21, N.S.) differed significandy between the<br />
groups. When retention of the platform location was tested 24 hr later using a 30 s probe<br />
trial, both groups spent most of their time searching in the quadrant which had contained<br />
the platform the previous day (Fig. 3-5B). A repeated measures ANOVA revealed a<br />
significant main effect of quadrant (F(3, 39) = 20.88, p < 0.001), but no significant group<br />
<strong>by</strong> quadrant interaction (F(3, 39) = 0.581, N.S.). Post-hoc analyses revealed that both<br />
groups spent significandy more time in quadrant 2 (41.65 ± 5 %), which contained the<br />
platform, than in either quadrants 3 (19.22 ± 4 %) or 4 (8.24 ± 3 %). Inspection of the<br />
data presented in Fig. 3-5B reveals that the animals also spent more time in quadrant 2<br />
than quadrant 1 (30.89 ± 3 %), although this difference failed to reach significance.<br />
Histology<br />
In agreement with previous studies (Nitecka et al., 1984;Stafstrom et al.,<br />
1992;Koh et al., 1999;Lynch et al., 2000), histological examination of the hippocampi of<br />
rats which had received either KA or saline on PND7 revealed no signs of gross cell loss<br />
or hippocampal damage (Fig. 3-6).<br />
89
Figure 3-6. A representative cresyl violet stained section of the dorsal hippocampus of a<br />
KA-treated (A) and saline-treated rat (B).<br />
90
Discussion<br />
The present study demonstrates that treatment with KA on PND7 results in the<br />
delayed emergence of behavioral changes in adulthood not seen before puberty.<br />
Following treatment with KA on PND7, rats in two independent groups showed disrupted<br />
PPI on PND56, but not PND35. When re-tested 6 weeks later, a subgroup of the KA-<br />
treated animals continued to exhibit relatively lower PPI than saline-treated controls.<br />
Additionally, administration of a low dose of apomorphine (0.2 mg/kg) disrupted PPI in<br />
both KA- and saline-treated rats; however, KA-treated rats failed to show enhanced<br />
sensitivity to the disruptive effects of apomorphine. When the locomotor responses of<br />
these animals in response to a novel environment and amphetamine challenge were<br />
analyzed, an inconsistent pattern emerged. KA- and saline-treated rats had similar<br />
locomotor responses to both novelty and amphetamine on PND36, as well as novelty on<br />
PND57. Following amphetamine administration (1.5 mg/kg) on PND57, locomotor<br />
activity was significantly increased in the KA-treated rats from Group 1, but not from<br />
Group 2. Furthermore, neither spatial learning nor memory were affected <strong>by</strong> the<br />
administration of KA on PND7 as assessed in the Morris water-maze, confirming<br />
previous studies (Stafstrom et al., 1993;Koh et al., 1999).<br />
Effects of Neonatal KA Administration on PPI<br />
In two separate groups of rats, we observed that KA treatment on PND7<br />
significantly disrupted PPI in postpubescence, but not before puberty. It is important to<br />
note that PPI has been reliably measured in rat pups as young as 16 days of age (Martinez<br />
et al., 2000;Swerdlow et al., 2000), and that the rats tested in the present experiments on<br />
PND35 showed consistent levels of PPI with relatively low variance. Post-pubescent rats<br />
91
treated with KA on PND7 had mean PPI levels that were 75 to 80% of those shown <strong>by</strong><br />
saline-treated animals. This effect was observed consistently in two groups of animals<br />
tested independendy (Fig. 3-1C and 3-2C) and was evident without changes in startle<br />
amplitude (Fig. 3-1D and 3-2D). Previous studies have reported the effects of neonatal<br />
manipulations on PPI in adult rats. Data presented <strong>by</strong> Lipska and colleagues (Lipska et<br />
al, 1995; Fig. 4) from rats that had sustained permanent lesions of the vHip on PND7,<br />
indicate PPI levels that were approximately 70% of their control littermates, although a<br />
significant difference between groups was noted only at the lowest prepulse intensity (4<br />
dB above baseline). Other studies report slightiy larger effects over a wider range of<br />
prepulse intensities following neonatal lesions of the vHip (Le Pen and Moreau,<br />
2002;Daenen et al., 2003) and the amygdala (Daenen et al., 2003) or treatment with MK-<br />
801 on PND7 (Harris et al., 2003). Given that these treatments result in relatively subtle<br />
disruptions in PPI under baseline testing conditions, it is perhaps not surprising that KA<br />
treatment on PND7, which does not cause gross brain damage (Nitecka et al.,<br />
1984;Stafstrom et al., 1992;Koh et al., 1999;Lynch et al, 2000; present data), causes<br />
relatively small disruptions in PPI.<br />
Rats with neonatal vHip lesions also show heightened sensitivity to the disruptive<br />
effects of apomorphine (0.1 mg/kg), there<strong>by</strong> supporting the hypothesis that mesolimbic<br />
DA transmission is altered in the lesioned rats (Lipska et al., 1995). In the present study,<br />
KA treatment on PND7 failed to enhance the disruptive effects of apomorphine treatment<br />
on PPI. However, administration of 0.2 mg/kg of apomorphine immediately before<br />
testing disrupted PPI in both the KA- and saline-treated animals (Fig. 3-3A). This result<br />
is surprising because Long-Evans rats are reported to be less sensitive than Sprague<br />
92
Dawley rats (the strain used <strong>by</strong> Lipska et al., 1995) to the disruptive effects of<br />
apomorphine on PPI. Indeed, it has been demonstrated that Long-Evans fats fail to show<br />
disrupted PPI following administration of doses of apomorphine as high as 0.5 mg/kg<br />
(Swerdlow et al., 2001). Although no clear explanation exists for these discrepant data,<br />
complex interactions between rat strain and PPI have been studied extensively (Varty and<br />
Geyer, 1998;Ellenbroek and Cools, 2000;Swerdlow et al., 2000). In this context, further<br />
examination of the effect of early KA-treatment's potentially interactive effects with<br />
apomorphine may be profitable in other rat strains.<br />
Using C57BL6 mice, Yee and colleagues (Yee et al., 2004a; 2004b) elegandy<br />
demonstrated that pretreatment with dopamine agonists such as apomorphine or non<br />
competitive A/-methyl-D-aspartate antagonists, such as dizocilpine and phencyclidine,<br />
disrupts PPI, a finding that has previously been well documented (Geyer et al., 2001).<br />
However, when prepulse-elicited reactivity was examined, a dissociation between the two<br />
classes of drugs was observed where<strong>by</strong> apomorphine increases and dizocilpine and<br />
phencyclidine decrease prepulse-elicited reactivity (Yee et al., 2004a; 2004b).<br />
Incorporation of a prepulse alone condition in future experiments may allow for parallels<br />
to be drawn between these pharmacological studies and KA treatment. For example, if<br />
KA administration on PND7 decreased prepulse-elicited startle, it could be argued that<br />
KA administration has effects similar to dizocilpine or phencyclidine pretreatment, but<br />
not apomorphine pretreatment.<br />
Effects of Neonatal KA on Locomotor Activity<br />
Increased locomotor activity in response to treatments that activate the<br />
mesolimbic DA system is a cardinal feature of many animal models of schizophrenia<br />
93
(Lipska et al., 1993;Hall, 1998;Weiss et al., 2000;Lipska et al., 2002;Harris et al, 2003).<br />
Neonatal KA treatment in the present study failed to increase locomotor behavior<br />
consistently following exposure to a novel environment or an amphetamine injection,<br />
although Group 1 did show a significant increase in locomotor activity following<br />
treatment with amphetamine (Fig. 3-4D). It is unclear why KA-treated rats in Group 2<br />
failed to show elevated locomotor responses following amphetamine treatment on<br />
PND57 given that both groups displayed similar seizure-related behaviors following KA<br />
administration on PND7 and similar deficits in PPI when tested at PND56. These results,<br />
considered in conjunction with the failure of apomorphine to affect PPI, question the role<br />
of postsynaptic alterations in the mesolimbic DA system in the observed effects of KA<br />
treatment on PPI. Furthermore, the absence of consistent changes in locomotor behavior<br />
in early adulthood following KA-induced aberrant activity in developing cortico-limbic<br />
circuits on PND7, underscores important differences between these data and the<br />
decreased activity induced <strong>by</strong> the TTX administration into the vHip (Lipska et al., 2002)<br />
and systemic MK-801 administration (Harris et al., 2003).<br />
Potential Mechanisms Underlying the Observed PPI Changes<br />
TTX infusions into the vHip on PND7 potentiate locomotor activity in early<br />
adulthood (Lipska et al., 2002) and systemic administration of the NMDA antagonist<br />
MK-801 on PND7 may also increase locomotor activity and disrupt PPI (Harris et al.,<br />
2003, but see Beninger et al., 2002). Lipska and colleagues failed to detect any gross<br />
morphological changes in the adult brains of animals injected with TTX on PND7<br />
(Lipska et al., 2002). In contrast, administration of MK-801 on PND7 increases<br />
apoptosis in limbic and cortical brain areas such as the hippocampus and thalamus<br />
94
(Harris et al., 2003), and this effect may underlie the observed behavioral changes in<br />
adult animals. Taken together, these data suggest that neural circuits subserving<br />
locomotor behavior and PPI are particularly sensitive to alterations in neural activity<br />
during the first postnatal week.<br />
All rats included in this study displayed generalized tonic-clonic seizures for 1 to<br />
2 hr following KA treatment on PND7, therefore it is reasonable to assume that aberrant<br />
activity would have occurred in neural circuits that regulate PPI and locomotor activity in<br />
adulthood (i.e. the hippocampus and septum; (Tremblay et al., 1984;Khalilov et al.,<br />
1999;Silveira et al., 2002). This raises the question of whether the behavioral effects<br />
observed in this study are the result of the activation of glutamate receptors and/or the<br />
consequence of seizures early in postnatal development. Obviously, these possibilities<br />
cannot be resolved based on the present experiments. However, it is well known that<br />
ionotropic glutamate receptor activity is critical for the normal formation of cortico-<br />
limbic circuits (Ben Ari et al., 1997;Feldman and Knudsen, 1998;Luthi et al., 2001).<br />
Although most of this research has focused on NMDA and AMPA receptor mediated<br />
events, some recent studies suggest that kainate receptors are dynamically regulated<br />
during critical periods of experience-dependent plasticity in thalamocortical synapses<br />
(Kidd and Isaac, 1999;Kidd et al., 2002). As a result, it is conceivable that<br />
hyperstimulation of kainate receptors could result in developmental alterations of the<br />
neural circuitry known to regulate PPI. Given that KA was administered systemically in<br />
the present study and genes encoding kainate receptor subunits are widely expressed in<br />
the brain during early postnatal development (Bahn et al., 1994), KA administration<br />
95
could have subtly altered a number of brain regions, there<strong>by</strong> producing the observed<br />
behavioral effects.<br />
The dose of KA (1.5 mg/kg) used in the present study induced seizures in the<br />
neonatal rats, therefore the effects of seizures, and not ionotropic glutamate receptor<br />
hyperstimulation per se, may also underlie the behavioral effects observed. Seizures<br />
induced <strong>by</strong> systemic administration of KA on PND7 increase glucose metabolism<br />
primarily in the dorsal hippocampus and lateral septum (Tremblay et al., 1984) and<br />
transiendy increase c-fos expression in the hippocampus (Silveira et al., 2002).<br />
Significant changes in the morphology of the hippocampus observed routinely following<br />
KA administration post-weaning are not evident after acute systemic treatment with KA<br />
during postnatal weeks 1 and 2 (Nitecka et al., 1984;Stafstrom et al., 1992;Koh et al.,<br />
1999;Lynch et al., 2000).<br />
More subtle alterations in the excitability of limbic circuits have been noted<br />
following administration of KA on PND7 including a reduction in long-term potentiation<br />
in the dentate gyrus, slower kindling rates, and increased paired pulse inhibition in the<br />
perforant path in adult rats, accompanied <strong>by</strong> impairments of spatial learning as assessed<br />
on a radial arm maze (Lynch et al., 2000). Exposure to KA on PND15 may also<br />
exacerbate cognitive impairment and cell loss in the hippocampus induced <strong>by</strong> seizures in<br />
adulthood (Koh et al., 1999). Recently, i.c.v. administration of KA (10-50 nmol) on<br />
PND7 has been demonstrated to cause a relatively subtle loss of hippocampal neurons,<br />
particularly in the CA3 and CA1 subregions of the hippocampus (Montgomery et al.,<br />
1999;Dong et al., 2003a). Although some cell loss is observed immediately following<br />
KA administration on PND7, greater cell loss occurs in adulthood (Humphrey et al.,<br />
96
2002). Interestingly, dynamic alterations in neurogenesis are correlated with this cell<br />
loss; when more hippocampal cells are lost, the rate of neurogenesis in the dentate gyrus<br />
is increased (Dong et al., 2003b). These results obtained with i.e.v. administration<br />
suggest that hippocampal circuits may be particularly vulnerable to alterations caused <strong>by</strong><br />
KA during the end of the first postnatal week. It is important to note that the histological<br />
methods used in the present study preclude a detailed analysis of subtle changes in<br />
hippocampal circuits such as those discussed above following i.e.v. administration.<br />
However, given the findings of Lynch and colleagues (2000) and those using i.e.v.<br />
administration of KA, the changes in PPI observed in the present study may be attributed<br />
to alterations in the development of the hippocampus, particularly the dentate gyrus.<br />
This hypothesis receives further support from studies in adult rats that confirm a<br />
role for the hippocampus and dentate gyrus in the neural circuitry that regulates PPI. For<br />
example, stimulation or deactivation of the hippocampus disrupts PPI (for review see<br />
Bast and Feldon, 2003), and infusion of the cholinergic agonist carbachol into the dentate<br />
gyrus disrupts PPI (Caine et al., 1991; 1992), an effect that is not reversed <strong>by</strong><br />
pretreatment with the D2 antagonist spiperone (Caine et al., 1991). Additionally, reduced<br />
synaptophysin immunoreactivity (Varty et al., 1999) and alterations in specific<br />
interneurons (Greene et al., 2001) are observed in the dentate gyri of socially-isolated rats<br />
that exhibit disrupted PPI.<br />
Conclusion<br />
Attempts to model the etiologies of psychiatric disorders such as schizophrenia in<br />
animals are challenging. All current models have drawbacks, mainly attributable to the<br />
specific brain manipulations performed (Lipska and Weinberger, 2000;Penn, 2001 ;Van<br />
97
den Buuse et al., 2003). The data reported here demonstrate that a single period of<br />
intense cortico-limbic activity induced <strong>by</strong> the systemic administration of KA on PND7<br />
can cause a selective disruption of PPI that is only observed in adult rats. This provides<br />
further support for the hypothesis that in rats, the end of the first postnatal week is a<br />
critical period for the development of cortico-limbic circuits that mediate behaviors such<br />
as PPI and locomotor activity (Lipska et al., 1993; 1995; Wood et al., 1997;Daenen et al.,<br />
2002; 2003;Harris et al., 2003). However, the present apomorphine-PPI experiment and<br />
locomotor activity experiments do not support the notion that acute administration of KA<br />
on PND7 results in the expression of an array of behavioral changes consistent with<br />
symptoms of schizophrenia.<br />
98
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104
CHAPTER FOUR: BEHAVIORAL CONVULSIONS INDUCED BY EARLY<br />
POSTNATAL ADMINIST<strong>RAT</strong>ION OF <strong>THE</strong> NR2B ANTAGONIST R025-6981<br />
FAIL <strong>TO</strong> AFFECT SENSORIMO<strong>TO</strong>R GATING OR LOCOMO<strong>TO</strong>R BEHAVIOR<br />
IN PRE-AND POST-PUBESCENT <strong>RAT</strong>S. 3<br />
Introduction<br />
Schizophrenia is a complex disorder resulting from multiple etiological risk<br />
factors. Adverse events during early development have been proposed to contribute to<br />
the etiology of schizophrenia (Weinberger, 1987). Supporting evidence includes<br />
increased risk of developing schizophrenia following perinatal events such as maternal<br />
infection and obstetrical complications (for review see Lewis and Levitt, 2002).<br />
Additionally, subtle signs of cognitive and behavioral impairment are present during<br />
childhood and early adolescence in those who go on to develop schizophrenia in<br />
adulthood (Lewis and Levitt, 2002). These observations, among others, led to the<br />
proposal that the primary pathology of schizophrenia occurs during early development<br />
and its effects somehow remain largely silent until adulthood (Weinberger, 1987;Lewis<br />
and Levitt, 2002). The effects of disturbing the early neural development of rats on<br />
behavioral measures relevant to schizophrenia are being examined to test this hypothesis.<br />
During early postnatal life, glutamate neurotransmission, particularly via NMDA<br />
receptors, is critical for the formation of neural circuits due to its established role in<br />
processes such as synaptogenesis (Rabacchi et al., 1992;Fox et al., 1996;Luthi et al.,<br />
2001), cell birth (Gould et al., 1994), and cell survival (Vallano, 1998;Ikonomidou et al,<br />
1999). Abnormalities of the glutamate system, with an emphasis On NMDA<br />
hypofunction, have been linked to patients with schizophrenia (Jentsch and Roth,<br />
3<br />
A version of this Chapter will be submitted for publication: Howland JG, Choi FY, Phillips AG (2005)<br />
Behavioral convulsions induced <strong>by</strong> early postnatal administration of the NR2B antagonist Ro25-<br />
6981 fail to affect sensorimotor gating or locomotor behavior in pre- and post-pubescent rats.<br />
105
1999;01ney et al., 1999;Goff and Coyle, 2001;Coyle and Tsai, 2004). Accordingly,<br />
previous studies have administered non-competitive NMDA antagonists such as<br />
phencyclidine, ketamine, and MK-801 to neonatal rats and demonstrated alterations in<br />
several behavioral paradigms including prepulse inhibition (PPI; (Wang et al.,<br />
2001;Harris et al., 2003), locomotor activity (Facchinetti et al, 1993;Wang et al.,<br />
2001;Harris et al., 2003;Fredriksson et al., 2004), stereotypy (Semba et al., 2001), set-<br />
shifting (Stefani and Moghaddam, 2005), and spatial memory (Gorter and de Bruin,<br />
1992;Wang et al., 2001;Fredriksson et al., 2004;Stefani and Moghaddam, 2005).<br />
Importandy, blocking NMDA receptor function at the end of the first postnatal week<br />
causes extensive apoptosis throughout forebrain areas implicated in schizophrenia such as<br />
the hippocampus, prefrontal cortex, striatum, and thalamus (Ikonomidou et al.,<br />
1999;Wang et al., 2001;Harris et al., 2003;Fredriksson et al, 2004) there<strong>by</strong> providing a<br />
potential mechanism that may underlie the behavioral changes observed in adulthood.<br />
Recently, an explosion in knowledge surrounding the composition of NMDA<br />
receptors and their subunit composition has emerged (Cull-Candy et al, 2001). This<br />
provides a unique opportunity to examine the potential role of various subclasses of the<br />
receptors in disorders such as schizophrenia. NMDA receptors are heterodimers<br />
composed of a variety of subunits (Fig. 4-1) and over the course of development, the<br />
expression levels of some subunits undergo dynamic change. At birth, NR2B subunits<br />
are expressed at high levels, whereas expression of NR2A subunit levels is low. Over the<br />
first two to three weeks of development, this pattern reverses, and during late adolescence<br />
and into adulthood, NMDA receptors containing NR2A subunits predominate (Monyer et<br />
al, 1994;Guilarte and McGlothan, 1998;Ritter et al., 2002;Zhang et al., 2002). As the<br />
106
Figure 4-1. Cartoon illustrating an NMDA receptor within the cell membrane. NMDA<br />
receptors are assemblies of various subunits including NR1, NR2A to NR2D, and NR3A<br />
and NR3B. Functional receptors contain two NR1 subunits and some combination of two<br />
additional subunits. As is depicted, the binding site for non-competitive NMDA<br />
antagonists such as MK-801 and phencyclidine is located within the pore of the receptor.<br />
Thus, the receptor must be open in order for these antagonists to bind. In the present<br />
experiments, the NR2B-specific antagonists Ro25-6981 and ifenprodil were used, which<br />
have been reported to bind in the region of the polyamine site. Interestingly, these drugs<br />
only bind to receptors termed diheteromers that contain two NR1 subunits and two NR2B<br />
subunits. In this manner, their high degree of specificity is achieved.<br />
107
subunit composition of NMDA receptors determine their precise kinetics, the unique<br />
subunit composition of NMDA receptors during the neonatal period is likely to be an<br />
important factor in normal development (Cull-Candy et al., 2001).<br />
Interestingly, numerous lines of evidence suggest that subunit-specific changes of<br />
the NMDA receptor may exist in schizophrenia: (1) significant increases in the<br />
expression of NR2B subunits have been noted in the hippocampus (Gao et al., 2000),<br />
thalamus (Clinton and Meador-Woodruff, 2004), and temporal cortex (Grimwood et al.,<br />
1999) of schizophrenic patients, (2) increased expression of NR2D in the prefrontal<br />
cortex (Akbarian et al., 1996) and decreases in NR1 expression in the hippocampus (Gao<br />
et al., 2000) have been demonstrated, (3) novel polymorphisms and variants in the<br />
promoter regions of the NR2A (Itokawa et al, 2003) and NR2B genes (Miyatake et al.,<br />
2002; Qin et al., 2005) may also exist in schizophrenia, (4) behavioral and neurochemical<br />
abnormalities consistent with the disorder are observed in mice with genetically altered<br />
NR1 (Mohn et al., 1999;Duncan et al., 2004) and NR2A (Miyamoto et al., 2001)<br />
subunits. Therefore, in an effort to further understand the effects of transiently disrupting<br />
glutamate receptors containing specifically NR2B subunits, we administered the subunit-<br />
selective NMDA antagonist [(+/-)-(R*,S*)-alpha-(4-hydroxyphenyl)-beta-methyl-4-<br />
(phenylmethyl)-l-piperidine propanol] (Ro25-6981) to neonatal rats on postnatal days<br />
(PND) 6 and 7.<br />
Ro25-6981 is a high affinity non-competitive antagonist of receptors containing<br />
the NR2B subunit (Fischer et al., 1997). As NR2B-containing NMDA receptors are<br />
overexpressed in the neonatal period, administration of Ro25-6981 provides a unique<br />
opportunity to assess the importance of these specific receptors in the maturation of the<br />
108
neural circuits that mediate behaviors with potential relevance to schizophrenia.<br />
Importantly, Ro25-6981 has low affinities for a-adrenergic and serotoninergic receptors,<br />
a problem recognized for other NR2B antagonists such as ifenprodil (Mutel et al., 1998).<br />
Following neonatal treatment, rats were tested for PPI and locomotor responses to a novel<br />
environment and amphetamine challenge both before and after puberty. These behavioral<br />
tests are extensively used to model the information processing deficits and striatal<br />
dopamine elevations observed in patients with schizophrenia (Lipska and Weinberger,<br />
2000;Braff et al., 2001 ;van den et al., 2003;Bast and Feldon, 2003). Finally, the<br />
disruptive effect of the dopamine agonist apomorphine on PPI was also assessed in a sub<br />
group of adult rats.<br />
Unexpectedly, the dose of the Ro25-6981 used (15 mg/kg) caused behavioral<br />
convulsions when administered on PND 6. As is detailed in the methods section, this<br />
dose has been used extensively in adult rats without reports of convulsive activity, and<br />
was chosen based on "affinity estimates for NR2B-containing receptors. Due to this<br />
observation, we also performed a series of parametric studies to further examine the<br />
convulsive effects of a number of NMDA antagonists during early development.<br />
Additionally, previous work performed in our laboratory demonstrated that seizures<br />
induced <strong>by</strong> kainic acid on PND 7 resulted in the delayed expression of PPI deficits in rats<br />
(Howland et al., 2004). From this standpoint, the long-term behavioral effects of<br />
convulsions following Ro25-6981 were of considerable interest.<br />
109
Methods<br />
Experiment 1 - Incidence and Characteristics of Behavioral Convulsions in Postnatal<br />
Rats Following Antagonism of NMDA Receptors Containing the NR2B Subunit<br />
Subjects<br />
Long-Evans or Sprague-Dawley rat pups were used in all experiments. Pregnant<br />
Long-Evans rats were obtained from Charles River (Quebec, Canada) at 13 to 15 days of<br />
gestation. They were singly housed and left undisturbed until giving birth. The colony<br />
was maintained on a 12/12 hour light/dark cycle (lights on at 0700), at a temperature of<br />
22±1°C. All rats were given food (Purina Rat Chow) and tap water ad libitum. Sprague-<br />
Dawley rat pups were obtained from the University of British Columbia Animal Care<br />
Center. The pups were delivered to our animal colony on the desired postnatal day<br />
without their mother. All experiments were initiated within 60 min of the arrival of the<br />
rat pups in the laboratory. Experiments were conducted in accordance with the standards<br />
of the Canadian Council on Animal Care and were approved <strong>by</strong> the Committee on<br />
Animal Care at the University of British Columbia.<br />
Experimental Procedures<br />
The day of birth of the pups was designated PND 0. Rat pups of the appropriate<br />
age (PND 6, 9, 12,15) were weighed and separated into individual compartments of a<br />
cardboard box for drug administration. The pups were removed from the colony and<br />
taken to a room where the behavioral effects of the administered drugs were videotaped<br />
and scored <strong>by</strong> an observer. The subunit-selective NMDA-NR2B antagonists Ro25-6981<br />
(7.5, 15, and 30 mg/kg) and ifenprodil (15 and 30 mg/kg) were injected (10 ml/kg; i.p.)<br />
and the behavior of the animals was videotaped for 60 minutes. Reports indicate that 15<br />
110
mg/kg of Ro25-6981 is in the dose range necessary to block most NR2B-containing<br />
receptors in the adult rodent brain (Murray et al., 2000;Lee and Rajakumar, 2003), and<br />
doses of 20 to 30 mg/kg have been used in vivo (Chaperon et al., 2003;Higgins et al.,<br />
2005). Similar data exists for ifenprodil (Murray et al., 2000). Following the<br />
experiment, the pups were sacrificed with carbon dioxide. All drugs were obtained from<br />
Sigma.<br />
Experiment 2 - Long-term Behavioral Effects of Convulsions Resulting from Antagonism<br />
of NMDA Receptors Containing the NR2B Subunit<br />
Subjects<br />
Two independent groups of Long-Evans rats (i.e. Group 1 and Group 2) were<br />
tested in this study. The animals were obtained, housed and cared for in a manner<br />
identical to that described in Experiment 1. Testing protocols for both groups were<br />
similar with small procedural differences noted in the methods section where appropriate.<br />
Ro25-6981 Administration<br />
On PND3, the litters were sexed and culled to include only males (4-9 rats per<br />
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<br />
pups were removed from the nest, weighed and placed individually in small<br />
compartments of a cardboard box. They were then removed from the colony and taken to<br />
a small heated room. Ro25-6981 (15 mg/kg) or saline was injected (i.p.) with a 30-gauge<br />
needle (10 ml/kg). Care was taken to ensure that both treatments were administered to<br />
members of each litter. The behavior of all rats was recorded with an overhead video<br />
camera for 60 min. Before being returned to their mothers, the pups were earmarked<br />
according to treatment condition. The litters were then left undisturbed (except for<br />
111
normal cage changing) until weaning on PND 25. Weanling rats were housed in cages of<br />
2 or 3 with members of their litter and these groups remained together for the duration of<br />
the experiment. All rats were handled before behavioral testing.<br />
Prepulse Inhibition<br />
On PND 35 and 56, rats were removed individually from the colony and taken<br />
immediately to the PPI apparatus. Testing was conducted in a single sound-attenuating<br />
startle chamber (ambient noise level 64 dB), containing a transparent Plexiglas tube (8.2<br />
cm in diameter, 20 cm in length), mounted on a Plexiglas frame (SR-LAB, San Diego<br />
Instruments, San Diego). Noise bursts were presented through a speaker mounted 24-cm<br />
above the tube. An accelerometer below the frame of the apparatus measured whole<br />
body startle amplitude, defined as the average of 100 1-ms accelerometer readings<br />
collected from stimulus onset. Each PPI test session began with a 5-min acclimatization<br />
period during which a 70-dB background noise level was presented, which remained<br />
constant for the entire test session. Following the acclimatization period, six pulse alone<br />
trials (120 dB, 40 ms) were presented to achieve a relatively stable startle amplitude<br />
before PPI testing. Data from these pulse-alone trials was not considered in the analysis<br />
of PPI. Immediately following the six initial pulse-alone trials, presentation of the trials<br />
that were used in the calculation of PPI levels were initiated. Trials presented were of<br />
four types: pulse alone (12 trials, 120 dB, 40 ms), prepulse + pulse (12 trials X 3 prepulse<br />
intensities - discussed below), prepulse alone (12 trials X 3 prepulse intensities) or no<br />
stimulus (12 trials). Prepulse + pulse trials consisted of the presentation of a 20 ms<br />
prepulse of 73, 76, or 82 dB 80 ms before the presentation of the pulse. Prepulse alone<br />
trials consisted only of a 20 ms prepulse (73, 76, 82 dB). All trials were presented in a<br />
112
pseudorandom order. After the trials used for calculation of PPI were presented, 6<br />
additional pulse-alone trials were presented. These, along with the first 6 pulse-alone<br />
trials, were used to calculate the level of habituation over the testing period. As no<br />
significant differences were observed between the groups in habituation and or for the<br />
prepulse-alone trials, these data are not presented. The inter-trial interval varied<br />
randomly from 3 to 12 s (average 7.5 s). Calibration of the apparatus was performed<br />
using a RadioShack Digital Sound Level Meter and adjustments were made as necessary.<br />
Spontaneous Locomotor Activity<br />
On PND 36, rats were weighed, removed from the colony, and immediately<br />
placed in 1 of 8 Med Associates Test Chambers (ENV-008; 30.5 cm X 24.1cm X height<br />
21.0 cm) to measure spontaneous locomotor activity for 60 min. The chambers were<br />
fitted with 4 pairs of infrared photocells 3.5 cm from the floor evenly spaced on the long<br />
walls and had metal grid floors and two operant levers (which were retracted during<br />
locomotor activity testing). Each chamber was contained within a Med Associates sound<br />
attenuating cubicle with a house light illuminated during testing.<br />
Dopaminergic Challenges of PPI and Locomotor Activity<br />
For those rats in Group 1, 7 to 14 days after PPI testing in early adulthood, the<br />
responses of the animals to administration of dopamine agonists were measured in both<br />
the PPI and locomotor activity tests. Some rats were tested for PPI first followed <strong>by</strong><br />
locomotor activity while others were tested in the reverse order. A minimum of 7 days<br />
separated locomotor activity and PPI testing. The PPI sessions were conducted in a<br />
manner identical to that described above, except that immediately before the PPI session,<br />
all rats were weighed and injected with the appropriate volume of vehicle (ascorbic acid,<br />
113
0.1%) or drug (apomorphine, Sigma, 0.2 mg/kg; s.e.). Each rat was tested twice (i.e.<br />
following either vehicle or apomorphine injection), and the PPI tests were at least 5 days<br />
apart. For locomotor activity testing, the rats were weighed, removed from the colony,<br />
and placed in the locomotor activity boxes for 60 min (as described above for PND 36).<br />
All rats were then immediately injected with D-amphetamine (1.5 mg/kg, i.p.) and<br />
returned to the locomotor boxes. Locomotor activity was monitored for an additional 90<br />
min before the rats were returned to the colony room.<br />
For rats in Group 2, PPI responses following apomorphine challenge were not<br />
measured. However, locomotor responses to D-amphetamine were examined at both<br />
PND 36 and PND 57. Rats were weighed and placed in the locomotor boxes as<br />
previously described, however, at both ages, they were injected with D-amphetamine (1.5<br />
mg/kg) immediately following the spontaneous locomotor activity test. Locomotor<br />
activity following D-amphetamine was measured for 90 min in all cases.<br />
Data Analysis<br />
For the PPI experiments, two measures were calculated for each animal. The<br />
startle amplitude represented the mean startle amplitude of the 12 pulse-alone trials<br />
presented after the 6 habituation trials. Startle amplitude data were compared using an<br />
independent samples t-test for each age. PPI was calculated <strong>by</strong> averaging startle<br />
amplitudes for each trial type. The percent PPI for each prepulse intensity was calculated<br />
using the formula: [100 - (100 X startle amplitude on prepulse + pulse trials) (startle<br />
amplitude on pulse alone trials)]. A repeated measures ANOVA was performed on the<br />
data obtained from each age with prepulse intensity as a within-subjects factor and<br />
treatment on PND 6 and 7 as a between-subjects factor. Results from the apomorphine<br />
114
experiment were also analyzed using a repeated measures ANOVA (prepulse intensity<br />
and test as within-subjects factors, and treatment at PND 7 as a between-subjects factor).<br />
Locomotor activity data were compared using repeated measures ANOVA at each age<br />
with test type as a within-subjects factor and treatment on PND 7 as a between-subjects<br />
factor. For all ANOVA's, post-hoc analyses were performed using the Neuman-Keuls<br />
test where appropriate. The significance level for all statistical tests was p < 0.05.<br />
Results<br />
Experiment 1 - Behavioral Effects of Ro25-6981 Administration in the Early Postnatal<br />
Period<br />
Approximately 5 min following injection with Ro25-6981, the majority of PND 6<br />
pups displayed behavioral convulsions. The convulsions were similar those typically<br />
described following administration of kainic acid (Howland et al., 2004) or bicuculline<br />
(Lai et al., 2002) in neonatal rats. Generally, they consisted of loss of posture,<br />
vocalizations, and rapid bursts of motor activity (especially with the hind legs). The<br />
majority of the animals showed bouts of bilateral forelimb and hindlimb clonus and/or<br />
tonus on their backs, in what has been called a 'swimming' posture <strong>by</strong> other authors<br />
(Stafstrom et al., 1993). In most cases, the behavioral convulsions subsided within 30 to<br />
60 min following injection. Interestingly, following their initial bout of convulsions,<br />
some animals would have another period of convulsive activity if they were handled <strong>by</strong><br />
the experimenter. Those pups injected repeatedly with Ro25-6981 (see Experiment 2)<br />
showed no evidence of tolerance to the convulsive effects of the drug.<br />
As depicted in Table 4-1, the convulsive effects of Ro25-6981 were age and dose<br />
dependent. Whereas all pups treated with 15 mg/kg of the drug on PND 6 had<br />
115
convulsions, only 25% of PND 9 pups, and 0% of PND 12 or 15 (using either 15 or 30<br />
mg/kg) pups displayed evidence of convulsions. However, pups injected with Ro25-<br />
6981 were more active than saline-injected controls. Additionally, none of the rats<br />
treated with 7.5 mg/kg of Ro25-6981 displayed behavioral convulsions. In an effort to<br />
rule out the possibility that the convulsions observed in the PND 6 pups were caused <strong>by</strong><br />
an unknown property of Ro25-6981 unrelated to NR2B antagonism, ifenprodil was also<br />
administered to some animals on PND 6. Similarly to the results obtained with Ro25-<br />
6981, almost all pups (89%) treated with ifenprodil had behavioral convulsions.<br />
Experiment 2 - Effects of Neonatal Ro25-6981 Administration on Body Weight<br />
As described in detail for experiment 1, administration of Ro25-6981 (15 mg/kg;<br />
Group 1: n=25; Group 2: n=16) resulted in behavioral convulsions for 30 to 60 minutes.<br />
Some individual differences in the severity of the convulsions were noted; although these<br />
did not correlate with performance on the behavioral tests performed. As a result, data<br />
from all rats is included.<br />
All rats were weighed on PND 6, twice on PND 7, and once on PND 9, 36 and 57.<br />
Repeated measures ANOVA's performed on these data indicate that administration of<br />
Ro25-6981 to the pups had no significant effect on body weight at any age (Group 1: F(l,<br />
41)=0.90, N.S.-; Group 2: F(l, 30)=0.49, N.S.). However, comparing the average weights<br />
of rats in Group 1 and Group 2 revealed that the animals in Group 1 were significandy<br />
lighter than those in Group 2 (F(l, 73)=48.97, p < 0.001). On PND 6 and 7, rats in Group<br />
1 weighed approximately 70% of those in Group 2 (PND 6: Group 1 mean = 12.16 g;<br />
Group 2 = 17.00 g). During puberty and early adulthood, the difference in weights<br />
116
Drug Postnatal Dose (i.p., Total Number Total Number<br />
Day mg/kg) Injected Exhibiting<br />
Convulsions<br />
Ro25-6981 6 7.5 8 0<br />
Ro25-6981 6 15 8 8<br />
Ro25-6981 9 15 4 1<br />
Ro25-6981 12 15 5 0(A)<br />
Ro25-6981 15 15 4 0(A)<br />
Ro25-6981 15 30 3 0(A)<br />
Ifenprodil 6 15 4 4<br />
Ifenprodil 6 30 5 4<br />
Table 4-1. The incidence of behavioral convulsions in rats administered NR2B-selective<br />
NMDA antagonists at various postnatal ages. i.p. = intraperitoneal.<br />
117
narrowed to a 10 to 15% difference (PND 36: Group 1 = 159.77 g; Group 2 = 173.58 g;<br />
PND 57: Group 1 = 339.26 g; Group 2 = 398.96 g).<br />
Neonatal Ro25-6981 Administration Does not Alter PPI Responding Either before<br />
Puberty or in Early Adulthood<br />
Before Puberty (PND 35): As is shown in Figure 4-2A and 4-3A, comparable<br />
levels of PPI was elicited in both saline- and Ro25-6981-treated rats for Groups 1 (saline:<br />
n=19, average PPI: 27.86±2%; Ro25-6981: n=25, average PPI: 26.53±3%) and 2 (saline<br />
n=16, average PPI: 32.24±3%; Ro25-6981: n=16, average PPI: 26.38±4%). Repeated<br />
measures ANOVA's performed separately for each Group revealed a significant effect of<br />
prepulse (Group 1: F(2, 84)=96.62, p < 0.001; Group 2: F(2, 60)=49.92, p < 0.001), but<br />
no significant treatment effects (Group 1: F(l, 43)=0.13, N.S.; Group 2: F(l, 30)=1.45,<br />
N.S.) or treatment <strong>by</strong> prepulse interactions (Group 1: F(2, 84)=0.08, N.S.; Group 2: F(2,<br />
60)=0.14, N.S.). Figures 4-2B and 4-3B depict the average startie amplitudes for all<br />
animals tested in Groups 1 and 2. Independent samples t-tests revealed no significant<br />
differences between saline- and Ro25-6981-treated rats in either Group (Group 1:<br />
t(42)=1.35, N.S.; Group 2: t(30)=0.03, N.S.).<br />
Early Adulthood (PND 56): Similar to before puberty, all groups tested<br />
demonstrated robust PPI in early adulthood (Fig. 4-2C and 4-3C), and no significant<br />
differences were observed between animals treated with either saline (average PPI, Group<br />
1: 30.60±3%; Group 2: 31.74±3%) or Ro25-6981 (average PPI, Group 1: 28.33±3%;<br />
Group 2: 37.00±5%). Repeated measures ANOVA's confirmed these observations with<br />
no significant treatment (Group 1: F(l, 42)=0.29, N.S.; Group 2: F(l, 30)=0.68, N.S.) or<br />
treatment <strong>by</strong> prepulse interactions (Group 1: F(2, 84)=0.63, N.S.; Group 2: F(2, 60)=0.69,<br />
118
Q_<br />
Q.<br />
:
pp3 pp6 pp12 average<br />
Prepulse Intensity :<br />
pp3 pp6 pp12<br />
Prepulse Intensity average<br />
B<br />
160i<br />
D<br />
160!<br />
1120<br />
I 80]<br />
I 40]<br />
saline Ro<br />
Group<br />
saline Ro<br />
Group<br />
Figure 4-3. Prepulse inhibition (PPI) scores from rats in Group 2 treated on postnatal day<br />
(PND) 6 and 7 with saline (white bars, n=16) or Ro25-6981 (Ro, 15mg/kg; black bars,<br />
n=16). A, Effects of postnatal day (PND) 7 KA treatment on PPI at PND 35. B, Average<br />
starde amplitudes of the rats on PND 35. C, Percent PPI scores for those rats tested at<br />
PND 56. D, Average startle amplitudes of the rats on PND 56.<br />
120
N.S.)- As expected, significant main effects for prepulse intensity were found for both<br />
groups (Group 1: F(2, 84)=70.90, p < 0.001; Group 2: F(2, 60)=32.71, p < 0.001).<br />
Additionally, startle amplitudes did not differ as a result of treatment in either Group 1<br />
(Fig. 4-2D; t(42)=l .05, N.S.) or Group 2 (Fig. 4-3D; t(30)=0.73, N.S.)<br />
Apomorphine Challenge in Adulthood has Similar Effects on PPI in Neonatally Saline- or<br />
Ro25-6981 -treated Rats<br />
To examine the potential effects of neonatal Ro25-6981 treatment on the<br />
dopaminergic regulation of PPI, 16 saline-treated and 18 Ro25-6981 -treated rats from<br />
Group 1 were retested for PPI following injection with either vehicle (0.1% ascorbic acid<br />
in saline) or apomorphine (0.2 mg/kg). Figure 4-4A summarizes the results of these tests.<br />
Following vehicle injection, PPI levels in both saline- and Ro25-6981-treated rats were<br />
similar to those observed when PPI was tested on PND 56. Apomorphine (0.2 mg/kg)<br />
significantly disrupted PPI in both the saline- and Ro25-6981-treated rats, as reflected <strong>by</strong><br />
a significant main effect of test (vehicle versus apomorphine; F(l, 32)=31.18, p < 0.01)<br />
and a significant test <strong>by</strong> prepulse interaction (F(2, 64)=10.75, p < 0.001, post-hoc, p <<br />
0.05). Additional analyses revealed that significant effects only existed for trials with 6<br />
and 12 dB prepulses in both groups (p < 0.05). As shown in Fig. 4-4B, startle amplitude<br />
was also significantly reduced <strong>by</strong> apomorphine administration in both groups (F(l,<br />
32)=8.59, p < 0.01).<br />
Neonatal Treatment with Ro25-6981 and Amphetamine-Induced Locomotor Activity in<br />
Adult Rats<br />
Group 1: Spontaneous locomotor activity in response to a novel environment was<br />
assessed for 60 min in the saline- and Ro25-6981-treated rats. Both before puberty (Fig.<br />
121
GL<br />
D-:<br />
a><br />
70-<br />
60-<br />
50-<br />
40-<br />
30-<br />
20 \<br />
lO-<br />
CI<br />
A<br />
asal+veh<br />
•Ro+veh<br />
•sal+apol<br />
oRo+apo<br />
Condition<br />
average<br />
sal+ Ro+ sal+ Ro+<br />
veh veh apo apo<br />
Group<br />
Figure 4-4. A, Effects of pretreatment with vehicle (0.1% ascorbic acid) or apomorphine<br />
(0.2 mg/kg) on percent PPI scores in adulthood. Animals were administered either saline<br />
(n=16) or Ro25-6981 (15 mg/kg; n=18) on postnatal day 6 and 7. B, Average starde<br />
amplitudes of those rats depicted in panel A. In both panels, asterisks indicate a<br />
significant difference between groups.<br />
122
Figure 4-5. Locomotor activity levels (photobeam breaks) in response to novelty (A, B<br />
left side) or amphetamine (1.5 mg/kg; B right side) of rats tested on either postnatal day<br />
(PND) 36 (A) or PND 57 (B) in Group 1. Rats were treated with either saline (white<br />
bars) or Ro25-6981 (black bars, 15 mg/kg) on PND 6 and 7. The asterisk denotes a<br />
significant difference between amphetamine-treated groups (p
Figure 4-6. Locomotor activity levels (photobeam breaks) in response to novelty or<br />
amphetamine (1.5 mg/kg) of rats tested on either postnatal day (PND) 36 (A) or PND 57<br />
(B) in Group 2. Rats were treated with either saline (white bars, n = 12) or Ro25-6981<br />
(black bars, 15 mg/kg, n = 12) on PND 6 and 7. Note the difference in scale for the units<br />
of activity levels (y-axis) for each panel.<br />
124
4-5A) and in early adulthood (Fig. 4-5B), exploration levels were similar in both groups<br />
(before puberty: t(42)=0.45, N.S.; early adulthood: see ANOVA below). Interestingly,<br />
administration of the dopamine agonist D-amphetamine (1.5 mg/kg) further increased<br />
locomotor activity <strong>by</strong> approximately 28% in rats neonatally treated with Ro25-6981<br />
(2193.75 ± 143 beam breaks) than saline (1712.67 ± 133 beam breaks) during the 90 min<br />
test (Fig. 4-5B). This effect was confirmed <strong>by</strong> a significant treatment <strong>by</strong> test interaction<br />
(F(l, 22)=7.83, p < 0.05), and post-hoc analyses (p < 0.05).<br />
Group 2: Spontaneous and amphetamine-induced locomotor activity was compared<br />
between treatment groups both before puberty (Fig. 4-6A) and in early adulthood (Fig. 4-<br />
6B) for those rats in Group 2. Unexpectedly, the groups did not significandy differ on<br />
either test at either age. Statistical analyses revealed insignificant main effects of<br />
treatment (PND 36: F(l, 22)= 0.27, N.S.; PND 57: 0.18, N.S.) and treatment <strong>by</strong> test<br />
interactions (PND 36: F(l, 22)=0.12, N.S.; PND 57: F(l, 22)=0.18, N.S.).<br />
Discussion<br />
The present experiments assessed the acute and delayed behavioral effects of the<br />
administration of Ro25-6981, an antagonist of NR2B-containing NMDA receptors, in<br />
rats. During PND 6 or 7, administration of Ro25-6981 resulted in behavioral convulsions<br />
that typically lasted 30 to 60 minutes. The convulsions were dose-dependent and could<br />
be elicited with a second NR2B antagonist, ifenprodil. Parametric experiments<br />
demonstrated that convulsions could not be reliably elicited in rats older than PND 7.<br />
The delayed behavioral effects of administering Ro25-6981 on PND 6 and 7 were<br />
inconsistent. Ro25-6981-treated rats in Group 1 performed similarly to saline-treated<br />
animals on PPI and spontaneous locomotion tests both before and after puberty.<br />
125
However, the Ro25-6981 -treated animals were significantly more active when treated<br />
with amphetamine in early adulthood, but failed to show increased sensitivity to the<br />
disruptive effects of apomorphine on PPI. In contrast, Ro25-6981-treated rats tested in<br />
Group 2 failed to show significant differences from saline-treated animals in any of the<br />
behavioral tests conducted.<br />
Antagonism of NR2B-containing NMDA Receptors Induces Behavioral Convulsions<br />
The convulsive effects of Ro25-6981 were unexpected in the present experiments;<br />
however, they appear to be reliable as they were dose-dependently elicited in two strains<br />
of rats <strong>by</strong> both Ro25-6981 and ifenprodil. Interestingly, some groups (Fischer et al.,<br />
1997;Mutel et al., 1998;Mallon et al., 2005) have described agonist-like properties of<br />
Ro25-6981 when the drug is applied in conjunction with low concentrations of NMDA.<br />
In Xenopus oocytes, Ro25-6981 (1 uM) profoundly inhibits the current elicited <strong>by</strong> 100<br />
u.M of NMDA, but surprisingly potentiates the current elicited <strong>by</strong> 1 uM of NMDA<br />
(Fischer et al., 1997). In another study using hippocampal slices from pubescent rats,<br />
Ro25-6981 (3 uM) potentiated the effects of NMDA (4 to 10 uM) on CA1 field<br />
potentials and paired-pulse interactions (Mallon et al., 2005). As also reported <strong>by</strong> Fisher<br />
et al. (1997), these effects were more significant with lower concentrations of NMDA<br />
(Mallon et al., 2005). Interestingly, the NR2A specific antagonist NVP-AAM077<br />
(Auberson et al., 2002) effectively blocked the effects of Ro25-6981 in the hippocampal<br />
slice suggesting that the NR2B subunit may reduce NMDA receptor activity <strong>by</strong> exerting<br />
tonic inhibitory effects on receptors containing the NR2A subunit (Mallon et al., 2005).<br />
A number of issues make integrating the present data with other reports in the<br />
literature challenging. Most importantly, the subunit composition of NMDA receptors<br />
126
dynamically changes over development; therefore, care must be taken when comparing<br />
data sets gathered from animals of different ages (for example, comparing the present<br />
data to those of Mallon et al., 2005). During the first postnatal week, NR2A subunits are<br />
expressed at very low levels in the rodent brain, whereas in adulthood NR2A subunits<br />
predominate (Monyer et al., 1994;Guilarte and McGlothan, 1998;Cull-Candy et al.,<br />
2001;Ritter et al., 2002;Zhang et al., 2002). Given these expression differences, it is<br />
unlikely that the observations made <strong>by</strong> Mallon and colleagues (2005) apply during the<br />
early neonatal period since very few NR2A subunits are expressed at that age.<br />
Additionally, numerous reports indicate that in vivo Ro25-6981 antagonizes NR2B<br />
containing NMDA receptors at doses within the range used in the present study (Lee and<br />
Rajakumar, 2003;Chaperon et al., 2003;De Vry and Jentzsch, 2003;Boyce-Rustay and<br />
Holmes, 2005). Therefore, the most likely explanation of the present data is that the<br />
behavioral convulsions observed resulted from the antagonist action of Ro25-6981 on<br />
NR2B-containing NMDA receptors.<br />
Although convulsions resulting from NMDA receptor antagonism are<br />
counterintuitive, they are not without precedence in the literature. For example, systemic<br />
administration of the non-competitive NMDA antagonist MK801 (0.1, 0.5, 1.0 mg/kg) to<br />
neonatal rat pups induces behavioral and electrographic seizures and exacerbates seizures<br />
caused <strong>by</strong> kainic acid (Stafstrom et al., 1997). Given that MK-801 would primarily act<br />
on NMDA receptors containing NR2B subunits at this developmental age, these results<br />
support our assertion that the neonatal brain may be prone to convulsions following the<br />
blockade of receptors containing NR2B subunits.<br />
127
Further experiments are necessary to understand the mechanism underlying the<br />
convulsive effects of neonatal NMDA receptor antagonism. Seizures are more common<br />
neonatally than later in development, an effect that may be related to differences in the<br />
net ratio of excitation and inhibition early in development (Ben Ari et al., 1997;Holmes et<br />
al, 2002). As previously described, intricate developmental regulation of NMDA<br />
receptor expression exists during the first few weeks of life. The expression patterns and<br />
function of other receptors, including GABA A and AMPA receptors, are also<br />
developmentally regulated. For instance, GABA A receptors are expressed early in the<br />
embryonic period and provide much of the excitatory drive in the developing brain until<br />
the end of the first postnatal week when NMDA and AMPA currents begin to<br />
predominate (Ben Ari et al., 1997;Holmes et al., 2002;Khazipov et al., 2004). The<br />
excitatory action of GABA is primarily due to delayed expression of the K +<br />
/Cf co-<br />
transporter KCC2, resulting in increased intracellular Cf concentrations. Thus, when<br />
GABA A channels open under these conditions, Cl" ions exit the neuron resulting in<br />
depolarization (Holmes et al., 2002). However, under some conditions, GABA A<br />
activation may also increase the resting membrane conductance sufficiently that the net<br />
effect of opening these channels is inhibitory (Leinekugel et al., 1999;Khalilov et al.,<br />
1999a;Holmes et al., 2002). As resting membrane conductance at this age is also<br />
critically dependent on glutamate receptor conductances (Khalilov et al., 1999a), one<br />
possible explanation of our observations is that antagonism of NMDA receptors with<br />
Ro25-6981 may 'tip the balance' in favor of excitation, there<strong>by</strong> triggering a convulsion.<br />
128
The Long-Term Behavioral Effects of Ro25-6981 on PPI and Locomotor Activity<br />
The levels of PPI and locomotor activity observed before and after puberty in the<br />
present study are similar to previous experiments from our group (Howland et al., 2004).<br />
Accordingly, percent PPI increased with higher prepulse intensities (Fig. 4-2 and 4-3).<br />
Average PPI levels were also lower in prepubescence than in early adulthood.<br />
Interestingly, a relatively low dose of apomorphine (0.2 mg/kg) disrupted PPI in the<br />
present study (Fig. 4-4A). This effect is consistent with previous results using the Long-<br />
Evans strain in our laboratory (Howland et al., 2004), but not others (Swerdlow et al.,<br />
2001).<br />
Although the significantly potentiated locomotor activity following amphetamine<br />
administration in the Ro25-6981-treated rats of Group 1 (Fig. 4-5B) is intriguing, the<br />
failure to replicate this effect in Group 2 (Fig. 4-6B) questions the reliability of these<br />
data. However, an interaction between Ro25-6981 administration and the factors that led<br />
to significantly reduced body weight in Group 1 may explain these behavioral changes.<br />
When the present body weights are compared to previous studies from our laboratory<br />
(Howland et al., 2004) in which Long-Evans pups were reared under identical conditions<br />
to those described herein, it is clear that the pups tested in Group 1 had abnormally low<br />
body weights. The cause of these differences cannot be confirmed, although some<br />
renovations to our facility occurred during the period which the pregnant females arrived<br />
and gave birth to the pups in Group 1. Thus, the mothers of the rats tested in Group 1<br />
may have been abnormally stressed during the pre- and postnatal period. Indeed, stress<br />
during these developmental periods has been shown to regulate dopamine-dependent<br />
behaviors such as locomotor activity in adult rats (Kofman, 2002;Brake et al., 2004).<br />
129
The present experiments fail to provide convincing evidence that neonatal<br />
treatment with Ro25-6981 results in specific behavioral changes related to schizophrenia.<br />
However, these data stand in contrast to reports of significant behavioral effects of<br />
neonatal NMDA antagonism in adulthood using non-competitive antagonists. For<br />
example, neonatal treatment with phencyclidine (PND 7, 9, and 11) reduces PPI and<br />
significantly increases locomotor activity in response to a phencyclidine challenge, both<br />
of which were reversed following antipsychotic treatment (Wang et al., 2001). Increased<br />
spontaneous locomotor activity is also observed following ketamine administration to<br />
mice on PND 10 (Fredriksson et al., 2004). Finally, neonatal administration of MK-801<br />
alters PPI (Harris et al., 2003), locomotor activity (Harris et al., 2003), and set-shifting<br />
(Stefani and Moghaddam, 2005), although the PPI and locomotor effects were not<br />
independently replicated (Beninger et al., 2002) and were only found in female animals<br />
(Harris et al, 2003).<br />
A number of differences between non-competitive NMDA antagonists and Ro25-<br />
6981 may underlie the different behavioral effects observed following their<br />
administration. For example, non-competitive NMDA antagonists have actions at<br />
dopamine D 2 and serotonin 5-HT 2 receptors, in addition to their effects of NMDA<br />
receptors (Kapur and Seeman, 2002). Importantly, the neonatal administration of non<br />
competitive NMDA antagonists also causes significant apoptosis in areas such as the<br />
hippocampus, thalamus, cortex, and striatum 24 to 48 hours after treatment (Wang et al.,<br />
2001 ;Lai et al., 2002;Beninger et al., 2002;Harris et al., 2003-/Fredriksson et al., 2004).<br />
Given that cell loss or disarray in these areas is reported in schizophrenia (Pakkenberg,<br />
1990;Jones, 1997;Harrison, 1999;Gothelf et al., 2000), these findings strengthen the<br />
130
validity of neonatal non-competitive NMDA antagonist administration as a model of<br />
schizophrenia, and may explain their behavioral effects. It is not known whether Ro25-<br />
6981 administration causes similar neuropathological effects, although the potential<br />
exists given that various competitive and non-competitive NMDA antagonists cause<br />
apoptosis in the developing brain (Dconomidou et al., 1999).<br />
Conclusion<br />
The convulsions caused <strong>by</strong> Ro25-6981 on PND 6 and 7 failed to affect PPI or<br />
locomotor responding in the present experiments. In contrast, febrile seizures early in<br />
life are associated with increased risk for schizophrenia (Vestergaard et al., 2005) and<br />
psychosis (Kanemoto et al., 2001). Additionally, kainic acid-induced seizures on PND 7<br />
in rats result in the delayed emergence of subtle PPI disruptions in early adulthood<br />
(Howland et al., 2004) and have short and long-term effects on temporal lobe<br />
electrophysiology, especially within the hippocampus (Tremblay et al., 1984;Stafstrom et<br />
al., 1992;Khalilov et al., 1999b;Lynch et al., 2000;Silveira et al., 2002). As the<br />
electrographic characteristics of the convulsions induced <strong>by</strong> Ro25-6981 are presently<br />
unknown, experiments designed to address this issue may enable more specific<br />
hypotheses to be generated regarding the brain areas important for long-term behavioral<br />
changes following neonatal seizures and their relationship to schizophrenia.<br />
131
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137
CHAPTER FIVE: GENERAL DISCUSSION<br />
The experiments contained within the present dissertation were designed to<br />
address two main objectives. First, Chapter Two detailed a number of experiments<br />
conducted to test the effects of electrical stimulation of discrete sub-regions of the<br />
hippocampus on PPI. Results of these experiments suggest brief periods of higher<br />
frequency activity that result in increased locomotor activity and ventral striatal<br />
dopamine efflux reversibly disrupt PPI when applied to the ventral, but not dorsal,<br />
hippocampus. The experiments described in Chapters Three and Four were designed to<br />
further the hypothesis that a period at the end of the first postnatal week is particularly<br />
sensitive for the development of the circuits mediating PPI and locomotor activity<br />
(Lipska and Weinberger, 2000). The experiments summarized in Chapter Three support<br />
this hypothesis <strong>by</strong> demonstrating that a single exposure to the kainate receptor agonist<br />
kainic acid on postnatal day seven disrupted PPI in early adulthood, but not before<br />
puberty. In Chapter Four, the effects of administration of the NR2B-subunit selective<br />
NMDA receptor antagonist Ro25-6981 on postnatal day six and seven were also<br />
assessed. Serendipitously, Ro25-6981 was observed to induce convulsions at this age;<br />
however, no consistent effects of this treatment on PPI or locomotor activity were noted<br />
either before puberty or in early adolescence. Although the implications of these results<br />
have been discussed at some length in the proceeding chapters, the present chapter will<br />
serve to further integrate these data with contemporary strategies of modeling<br />
schizophrenia in rodents.<br />
138
The Role of the Hippocampus in the Regulation of Prepulse Inhibition and Locomotor<br />
Activity<br />
As detailed in the discussion section of Chapter Two, hippocampal regulation of<br />
PPI is complex and incompletely understood. However, a great deal of evidence using<br />
both NMDA (Wan et al., 1996;Klarner et al., 1998;Zhang et al, 1999;Swerdlow et al.,<br />
2001b;Zhang et al., 2002) and higher frequency electrical (Howland et al., 2004b)<br />
stimulation protocols, indicates that over-activity of the ventral, but not dorsal,<br />
hippocampus disrupts PPI. Numerous studies have also assessed the role of hippocampal<br />
activity in locomotor behavior patterns of adult rats. Similar to the effects described for<br />
PPI, stimulation of the ventral hippocampus with either NMDA (Mogenson and Nielsen,<br />
1984;Bardgett and Henry, 1999;Bast et al., 2001;Zhang et al., 2002) or higher frequency<br />
electrical current (Taepavarapruk et al., 2000) increases locomotion, whereas comparable<br />
stimulation of the dorsal hippocampus has no effect (Zhang et al., 2002). Here it is<br />
important to note that the dorsal hippocampus can influence locomotor activity under<br />
some conditions as dorsal hippocampal infusions of carbachol (Flicker and Geyer,<br />
1982a;Mogenson and Nielsen, 1984), picrotoxin (Flicker and Geyer, 1982b) or other<br />
agents (Bast and Feldon, 2003), can alter the behavior. Thus, although the regulation of<br />
PPI and locomotor activity <strong>by</strong> the dorsal and ventral hippocampus is complex, both<br />
behaviors are clearly more sensitive to stimulation of the ventral than the dorsal region<br />
(Zhang et al., 2002;Bast and Feldon, 2003;Howland et al., 2004b). Previous research<br />
supports functional differentiation within the hippocampus, especially in regards to<br />
spatial learning and memory (supported <strong>by</strong> the dorsal hippocampus) and fear-related<br />
behavior (ventral hippocampus) (Moser and Moser, 1998; Kjelstrup et al., 2002).<br />
139
When the roles of dopamine and glutamate in mediating the effects of either<br />
NMDA or electrical stimulation of the ventral hippocampus on PPI or locomotor activity<br />
are considered, an interesting dissociation has emerged (Bast and Feldon, 2003). As was<br />
previously described for PPI, neither dopamine antagonists (systemically administered)<br />
nor glutamate antagonists (infused into the nucleus accumbens) effectively block the<br />
disruption of PPI induced <strong>by</strong> infusion of NMDA into the ventral hippocampus (Wan et<br />
al., 1996;Bast et al., 2001). In contrast, both dopamine and glutamate antagonists block<br />
the increases in locomotion observed following stimulation of the ventral hippocampus<br />
(Bardgett and Henry, 1999;Taepavarapruk et al., 2000;Bast et al., 2001). Additionally,<br />
locomotor activity in a novel environment increases dopamine efflux in the nucleus<br />
accumbens, an effect that can be reversed <strong>by</strong> inactivation of the ventral hippocampus or<br />
administration of a glutamate receptor antagonist into the ventral tegmental area (Legault<br />
and Wise, 2001). Thus, these findings indicate that dopamine and glutamate transmission<br />
is critical for the ventral hippocampal modulation of locomotor activity, but not for PPI.<br />
Although the circuitry underlying the role of dopamine and glutamate in the<br />
ventral hippocampal stimulation-elicited increases in locomotor behavior is still being<br />
investigated, evidence suggests that dopamine and glutamate receptors in the nucleus<br />
accumbens (Taepavarapruk et al., 2000) and ventral tegmental area (Legault and Wise,<br />
2001) play an important role, although other brain regions may also be involved.<br />
Stimulation of the ventral hippocampus with NMDA increases dopamine efflux in the<br />
medial prefrontal cortex (Peleg-Raibstein et al., 2005), and co-administration of<br />
dopamine antagonists with NMDA into the ventral hippocampus blocks the expected<br />
increase in locomotor behavior observed when NMDA is administered alone (Gimenez-<br />
140
Llort et al., 2002). Thus, strong parallels exist between the neural circuitry and<br />
pharmacology underlying the regulation of locomotor activity <strong>by</strong> the ventral<br />
hippocampus and that commonly described for schizophrenia.<br />
On the other hand, the mechanisms underlying the disruption of PPI following<br />
stimulation of the ventral hippocampus are less clear. As neither dopamine nor glutamate<br />
antagonists reverse the PPI disruptions following ventral hippocampal stimulation (Wan<br />
et al., 1996;Bast et al., 2001), direct activation of glutamatergic afferents from the ventral<br />
hippocampus to the nucleus accumbens is unlikely to underlie these effects. A recent<br />
study provides further support for this notion <strong>by</strong> demonstrating that lesions of the fornix,<br />
a tract containing the ventral hippocampal efferents to the nucleus accumbens, fail to<br />
block the disruption of PPI following stimulation of the ventral hippocampus with<br />
NMDA (Swerdlow et al., 2004). Therefore, the modulation of PPI <strong>by</strong> the ventral<br />
hippocampus likely involves either (1) interactions with brain areas other than those<br />
directly implicated in locomotor control (i.e., areas other than the nucleus accumbens) or<br />
(2) poly-synaptic pathways to the nucleus accumbens that do not include projections via<br />
the fornix.<br />
One potential circuit that may underlie the effects of stimulation of the ventral<br />
hippocampus includes the ventral hippocampal projections to the perirhinal and<br />
entorhinal cortex (Swerdlow et al., 2004). These areas have been implicated in the<br />
modulation of PPI (Swerdlow et al., 2001b;Goto et al, 2002) and are connected, via non-<br />
fornical routes, to structures such as the nucleus accumbens (Totterdell and Meredith,<br />
1997), basolateral amygdala (Pitkanen et al., 2000), and medial prefrontal cortex (Insausti<br />
et al., 1997) that are also involved in the modulation of PPI (Swerdlow et al., 2001a).<br />
141
Importantly, given that the mechanisms through which stimulation of the ventral<br />
hippocampus disrupts PPI are currendy unknown, it has been suggested that further<br />
understanding of this phenomenon may aid in the development of novel therapeutic<br />
strategies for schizophrenia (Bast and Feldon, 2003).<br />
The Utility of Adult Animal Models of Schizophrenia<br />
As was discussed in Chapter One, animal models of schizophrenia created <strong>by</strong><br />
manipulations performed during adulthood generally have poor construct validity given<br />
numerous lines of evidence that support a developmental component to the etiology of<br />
the disorder. However, adult models of schizophrenia can still contribute to a better<br />
understanding of the disorder in a number of different ways. For example, novel insights<br />
into the neural substrates underlying the behavioral symptoms of schizophrenia can be<br />
gained <strong>by</strong> performing experiments using adult animals. Support for this assertion is<br />
provided <strong>by</strong> data presented in Chapter Two that suggest increased ventral hippocampal<br />
activity in adulthood may underlie some symptoms of schizophrenia. Interestingly,<br />
results from brain imaging studies confirm the importance of these preclinical findings <strong>by</strong><br />
demonstrating that basal levels of hippocampal activity are increased in schizophrenia<br />
(Heckers et al., 1998;Benes, 2000;Heckers, 2001).<br />
Additionally, the present results (Howland et al., 2004b), and those of other<br />
researchers (Zhang et al., 2002), suggest that the ventral hippocampus may be more<br />
involved in the regulation of sensorimotor processes such as PPI and locomotor activity<br />
than the dorsal hippocampus. As a result, examining alterations in specific sub-regions<br />
of the hippocampus in patients with schizophrenia may reveal previously unappreciated<br />
characteristics of the neurobiology of the disorder (Goldman and Mitchell, 2004). In<br />
142
humans, the anterior portion of the hippocampus corresponds to the rodent ventral<br />
hippocampus, and some studies indicate that this region may be preferentially altered in<br />
schizophrenia (Csernansky et al., 1998;Szeszko et al., 2003;Narr et al., 2004; but see also<br />
Narr et al., 2001;Velakoulis et al., 2001).<br />
Furthermore, experiments with adult rats enable basic knowledge of the neural<br />
mechanisms underlying performance of behavioral tasks to be generated quickly. This<br />
knowledge can then be used to assess the potential mechanisms underlying behavioral<br />
deficits following developmental manipulations. This type of strategy was used to<br />
demonstrate that the PPI disruptions observed in socially isolated rats are dependent on<br />
dopamine levels in the nucleus accumbens (Powell et al., 2003) and extensively <strong>by</strong><br />
Lipska and colleagues regarding various aspects of the altered neural circuitry and<br />
pharmacology underlying changes in the neonatal ventral hippocampal lesion model<br />
(Lipska et al., 1998;Lipska and Weinberger, 2000). Thus, combining information gained<br />
from both adult and developmental approaches will likely enable a more rapid and<br />
thorough understanding of the disorder (Floresco et al., 2005).<br />
Developmental Models of Schizophrenia - Effects of Early Postnatal Glutamate<br />
Manipulations<br />
Clearly, the results gained from both the neonatal kainic acid and Ro25-6981<br />
manipulations are not as strong as was desired at the outset of the experiments. However,<br />
a number of interesting observations have been gained from these data, many of which<br />
should be verified in future experiments. The most important result was the subtle and<br />
reliable decrease in PPI observed following kainic acid administration in Chapter Three.<br />
Consistent with the ventral hippocampal lesion model, the PPI deficits were observed<br />
143
only after the animals had reached early adulthood. However, these deficits were not<br />
augmented <strong>by</strong> apomorphine administration or accompanied consistently <strong>by</strong> changes in<br />
locomotor activity, as is the case with the PPI deficits following neonatal ventral<br />
hippocampal lesions (Lipska et al., 1993;Lipska et al., 1995).<br />
As was discussed in the introduction, some first degree relatives of patients with<br />
schizophrenia exhibit lower PPI responses than matched controls (Cadenhead et al.,<br />
2000). Thus, disrupted PPI may be considered a candidate marker for a 'trait' (or<br />
underlying predisposition) to develop schizophrenia. Accordingly, the kainic acid model,<br />
while not reproducing the full-blown expression of schizophrenia, may be useful as a<br />
method of producing an underlying predisposition for the disorder in rodents. Further<br />
support for this assertion is gained from studies suggesting that the hippocampus is also<br />
altered in first degree relatives of schizophrenic patients (Seidman et al., 2002) and in<br />
adult rats <strong>by</strong> neonatal kainic acid administration (Lynch et al., 2000).<br />
Assessing the interaction of neonatal kainic acid administration with additional<br />
manipulations may support the notion that schizophrenia results from multiple 'hits' over<br />
the course of development (McCarley et al., 1999;Bayer et al., 1999;Lewis and Levitt,<br />
2002;Wong and Van Tol, 2003;Ellenbroek, 2003). To date, such strategies have been<br />
rarely used in an attempt to model the disorder although in one report, rats with neonatal<br />
ventral hippocampal lesions treated repeatedly with PCP in early adulthood displayed<br />
enhanced locomotor responses when compared to lesioned rats that had not been treated<br />
with PCP (Hori et al., 2000). Support for this experimental design is provided <strong>by</strong><br />
observations indicating that neonatal kainic acid administration renders rats more<br />
144
sensitive to morphological damage and behavioral impairments following kainic acid<br />
administration in adulthood (Koh et al., 1999).<br />
It is disappointing that the long-term behavioral experiments performed with<br />
Ro25-6981 failed to yield positive results. However, investigating novel mechanisms of<br />
brain development in the context of schizophrenia will improve understanding of the<br />
disorder. The rational for the use of Ro25-6981 was conceived from detailed knowledge<br />
surrounding the dynamic changes of specific NMDA subunits during early development,<br />
and similar experiments developed to test mechanistically-driven hypotheses will likely<br />
be an important component of preclinical schizophrenia research in the future.<br />
Further consideration of the null results of the experiments assessing the<br />
behavioral effects of neonatal administration of the NR2B antagonist Ro25-6981 can be<br />
focused in two main directions: (1) why the experiments performed fail to yield<br />
significant results and (2) what conclusions may be drawn from these experiments with<br />
respect to the existing literature regarding early developmental animal models of<br />
schizophrenia.<br />
Although the experiments using Ro25-6981 were carefully designed, the effects<br />
of this drug have not been extensively studied, especially in regard to the developing<br />
mammalian brain. In contrast, the acute effects of kainic acid administration are well<br />
known during development, and fit logically with some conceptions of the etiology of<br />
schizophrenia (Howland et al., 2004a). For example, kainic acid administration early in<br />
the neonatal period alters the activity of the hippocampus (Stafstrom et al., 1992;Khalilov<br />
et al., 1999;Lynch et al., 2000;Silveira et al., 2002) and additional limbic and cortical<br />
areas including the entorhinal cortex (Khalilov et al., 1999) implicated in schizophrenia.<br />
145
Additionally, rats treated neonatally with kainic acid exhibit subtle physiological<br />
alterations in the hippocampus during adulthood, which may underlie some of the<br />
behavioral alterations observed in these animals (Lynch et al., 2000). To my knowledge,<br />
the present experiments are the first to assess the effects of Ro25-6981 administration in<br />
neonatal rats. In retrospect, it may have been profitable to more carefully assess the acute<br />
effects of Ro25-6981 administration on the neonatal brain before the long-term<br />
behavioral experiments described herein were performed.<br />
Two main avenues will likely be especially profitable for understanding both the<br />
acute and long-term effects of neonatal Ro25-6981 administration on brain and behavior.<br />
Initially, determining which brain areas are activated with either convulsive or sub-<br />
convulsive doses of the drug will enable the development of a more detailed hypothesis-<br />
driven approach regarding the potential long-term behavioral effects of the convulsions<br />
induced <strong>by</strong> Ro25-6981. I expect that the activity of structures such as the hippocampus,<br />
cortex, and striatum will be altered <strong>by</strong> administration of Ro25-6981 due to high levels of<br />
NR2B expression in those areas neonatally (Monyer et al., 1994;Loftis and Janowsky,<br />
2003), and the well documented role of these areas in seizures (Ben Ari and Cossart,<br />
2000). However, high levels of NR2B-containing NMDA receptors are also expressed in<br />
other areas including the thalamus and cerebellum at this developmental age (Loftis and<br />
Janowsky, 2003). Interestingly, connections between these areas and the frontal cortex<br />
have been implicated in schizophrenia (Andreasen et al., 1999;Konarski et al., 2005).<br />
Thus, altered activity patterns in distributed neural circuits may underlie the convulsions<br />
produced <strong>by</strong> Ro25-6981.<br />
146
Secondly, an assessment of the effects of Ro25-6981 administration on cell<br />
survival in the developing brain is essential. As detailed in Chapter Four, a number of<br />
competitive and non-competitive NMDA receptor antagonists induce massive cell death<br />
in the developing brain, a factor which may be important in the long-term behavioral<br />
effects of their administration (Ikonomidou et al., 1999;Wang et al, 2001;Harris et al.,<br />
2003;Fredriksson et al., 2004). The influence of Ro25-6981 on cell survival in the<br />
developing brain is currently unknown, however the high proportion of NR2J3-containing<br />
NMDA receptors early in development supports the hypothesis that treatment with Ro25-<br />
6981 may cause apoptosis during this developmental age.<br />
Although a detailed understanding of the effects of Ro25-6981 administration is<br />
lacking, the results of the behavioral experiments conducted prior to and after puberty are<br />
still surprising as the induction of three strong convulsions during the end of the postnatal<br />
week failed to alter PPI or locomotor responding. One factor that could be especially<br />
important in the different behavioral effects observed between the convulsions elicited <strong>by</strong><br />
kainic acid and Ro25-6981 is that convulsions elicited <strong>by</strong> kainic acid are significantly<br />
longer in duration than those elicited with Ro25-6981. Longer convulsions may disturb<br />
developing neural networks to a significantly greater extent than shorter ones (Jensen and<br />
Baram, 2000;Holmes, 2004). Previous studies using other convulsive agents such as<br />
flourothyl, which have a short duration of action (10 to 15 minutes), support this<br />
assertion. These experiments indicate that a series of 25 to 50 convulsions during the<br />
first week of life must be elicited for long-term alterations in behavior to be observed<br />
(Holmes et al., 1998;Huang et al., 1999). Further support for this hypothesis is gained<br />
from data suggesting that a history of prolonged febrile seizures is a significant risk factor<br />
147
for psychosis in adulthood epileptics (Kanemoto et al., 2001) and that prolonged febrile<br />
seizures result in long-term changes in hippocampal excitability in rodents (Chen et al.,<br />
1999;Jensen and Baram, 2000). Therefore, it is tempting to speculate that repeated<br />
convulsions induced <strong>by</strong> Ro25-6981 during the first postnatal week would be related to<br />
alterations in PPI, locomotor activity, and cognitive deficits in adult rats.<br />
Developmental Models of Schizophrenia - The Time Course of Symptom Emergence<br />
Although developmental animal models of schizophrenia are likely more<br />
ecologically valid than adult models, they are much more difficult to establish. One of<br />
the cardinal features of the majority of patients with schizophrenia is the delayed onset of<br />
many symptoms of the disorder (Marcotte et al., 2001;Wong and Van Tol, 2003),<br />
however, this feature of the disorder remains elusive in animal models (Marcotte et al.,<br />
2001). Research examining recovery of function after brain injury generally supports the<br />
assertion that behavioral impairment is less severe if the injury is suffered early in life - a<br />
phenomenon commonly termed the Kennard principle (Kolb et al., 2000;Marcotte et al.,<br />
2001). However, it is important to note that the pattern of recovery from injuries<br />
sustained during early brain development show some exceptions to the Kennard<br />
principle. For example, lesions of the frontal cortex induced at specific developmental<br />
stages (i.e. late during the embryologic period or after the first postnatal week) result in<br />
significant recovery over time, while other periods exist (i.e. the first postnatal week)<br />
where recovery from similar injuries is significantly poorer than expected (Kolb and<br />
Whishaw, 1989;Kolb et al., 2000).<br />
In the case of schizophrenia, the relation between adverse events early in<br />
development and subsequent manifestation of symptoms suggest a delayed response in<br />
148
which behavioral deficits emerge after a period of relative normality. The neonatal<br />
ventral hippocampal lesion model provides important proof that alterations in the neural<br />
substrates implicated in schizophrenia during certain developmental stages may result in<br />
the delayed onset of various behavioral and electrophysiological alterations related to the<br />
disorder (Lipska et al., 1993;Lipska et al., 1995;Wood et al., 1997;Lipska and<br />
Weinberger, 2000;Lipska et al, 2002;Goto and O'Donnell, 2002). Additional research<br />
suggests that manipulations other than ventral hippocampal lesions such as immune<br />
activation (Zuckerman et al., 2003), administration of the anti-mitotic agent arabinoside<br />
(Elmer et al., 2004), or kainic acid (Howland et al., 2004a) result in the delayed onset of<br />
behavioral abnormalities associated with schizophrenia (unfortunately, unlike the design<br />
employed in this thesis, the behavioral effects of many early neonatal treatments<br />
developed have not been tested before adulthood). Taken together, these data suggest<br />
that certain critical periods exist in the neonatal brain for the normal development of the<br />
neural circuits mediating behaviors commonly ascribed to schizophrenia in animals<br />
(Lipska and Weinberger, 2000;Marcotte et al., 2001;Howland et al., 2004a). Future work<br />
related to the mechanisms underlying these changes will likely provide novel insights<br />
into the etiology of the disorder.<br />
Criteria for Establishing Validity in Behavioral Models of Schizophrenia<br />
The present experiments relied heavily on PPI and locomotor behavior in<br />
assessing the experimental manipulations performed in modeling schizophrenia.<br />
Although these behaviors have been used extensively <strong>by</strong> researchers in the field, they<br />
have a number of shortcomings. Importantly, PPI is disrupted in a number of<br />
neurological and psychiatric disorders in addition to schizophrenia including<br />
149
Huntington's disease, Tourette's syndrome, obsessive-compulsive disorder, and<br />
pathological gambling (Braff et al., 2001). Given the complexity of the neural circuitry<br />
mediating PPI this is not particularly surprising; however, it is problematic in that<br />
disrupted PPI may not be characteristic of schizophrenia per se, but rather of alterations<br />
in the distributed circuitry mediating it (Swerdlow et al., 2001a).<br />
As previously discussed, increased locomotor activity is often used as an indirect<br />
measure of ventral striatal dopamine activity, especially when combined with the<br />
administration of dopamine agonists (Kelly et al., 1975;Castall et al., 1977;Porrino et al.,<br />
1984;Lipska and Weinberger, 2000;Marcotte et al., 2001). Unfortunately, altered<br />
locomotor behavior is a very general phenomenon and can be influenced <strong>by</strong> a number of<br />
factors including anxiety levels, alterations in sensory processing, and memory<br />
disruptions (Bast and Feldon, 2003). Although some parallels between increased<br />
locomotor activity and certain symptoms of schizophrenia have been suggested (Bast and<br />
Feldon, 2003), these assertions are clearly speculative (Marcotte et al., 2001).<br />
As a result of these shortcomings, testing PPI and locomotor activity levels should<br />
be viewed as an important 'first-pass' in the assessment of potential animal models of<br />
schizophrenia. Future experiments with an array of behaviors relevant to schizophrenia<br />
will serve to strengthen the validity of putative models of schizophrenia (Lipska and<br />
Weinberger, 2000). Recent interest has been generated around preclinical correlates of<br />
the negative and cognitive symptoms of schizophrenia as they have traditionally been<br />
ignored in much of the literature (Ellenbroek and Cools, 2000;Floresco et al., 2005).<br />
Tests assessing social interaction, reward sensitivity, and executive functions such as<br />
cognitive set shifting and working memory are likely to be especially useful in further<br />
150
understanding the more complicated and treatment-resistant symptoms of the disorder<br />
(Ellenbroek and Cools, 2000;Robbins, 2004;Floresco et al., 2005). It is worth pointing<br />
out, however, that many individuals with schizophrenia do not exhibit all symptoms of<br />
the disorder (Wong and Van Tol, 2003). Therefore, reliance on strategies that<br />
successfully model many aspects of the disorder in animals may be counterproductive in<br />
some instances.<br />
Interestingly, some manipulations, such as neonatal ventral hippocampal lesions,<br />
produce changes in many behaviors resembling schizophrenia (Lipska and Weinberger,<br />
2000), while others, such as certain neonatal NMDA antagonist treatment regimes,<br />
produce selective deficits on certain behavioral tasks relevant to one class of symptoms<br />
(e.g. cognitive symptoms) (Stefani and Moghaddam, 2005). The present data do not<br />
indicate how animals treated with either kainic acid or Ro25-6981 may respond if further<br />
testing was conducted. However, in some studies, rats treated neonatally with kainic acid<br />
display deficits in learning and memory (Lynch et al., 2000), although null findings have<br />
also been reported (Stafstrom et al., 1993). Therefore, in the future, it may be profitable<br />
to greatly extend the test battery beyond the tests presently employed.<br />
Finally, testing the efficacy of antipsychotic drugs at reversing the behavioral<br />
disturbances observed following neonatal kainic acid administration could greatly aid in<br />
assessing its validity as a model of schizophrenia. As has been noted throughout the<br />
present dissertation, both typical and atypical antipsychotics are effective at ameliorating<br />
the behavioral disturbances, including PPI deficits, in numerous animal models of<br />
schizophrenia (Lipska and Weinberger, 2000;Marcotte et al., 2001;Bast et al., 2001 ;Le<br />
Pen and Moreau, 2002;Bast and Feldon, 2003;Van den et al., 2003;Le Pen et al., 2003).<br />
151
Conclusion<br />
In the introduction, three main goals for developing animal models of<br />
schizophrenia were introduced: (1) advancing the understanding of the symptoms of the<br />
disorder, (2) furthering the understanding of schizophrenia's etiology, and (3) aiding in<br />
the development of pharmaceutical therapies (Lipska and Weinberger, 2000). The<br />
present dissertation explored two general strategies for developing behaviorally-oriented<br />
animal models of schizophrenia. Although the results were somewhat mixed, the<br />
experiments were successful at providing novel insights into the symptoms and etiology<br />
of the disorder. In general, they support the assertion that short periods of altered activity<br />
in the limbic system, and hippocampus in particular, at different points during<br />
development may underlie the expression of some of the most basic symptoms of<br />
schizophrenia. These data also suggest that the nature and anatomical location of these<br />
alterations critically determines their long-term functional effects.<br />
152
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