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Approaches to modeling schizophrenia in the rat Howland, John George 2005

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APPROACHES TO M O D E L I N G SCHIZOPHRENIA FN THE R A T by JOHN GEORGE H O W L A N D B A . (Hons.), University of Saskatchewan, 1999 M . A . , The University of British Columbia, 2001 A THESIS SUBMITTED IN P A R T I A L FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Psychology) THE UNIVERSITY OF BRITISH C O L U M B I A October 2005 © John George Howland, 2005 A B S T R A C T Schizophrenia is a complicated and variable disorder that is notoriously difficult to study. Converging lines of evidence support the hypothesis that schizophrenia is characterized by a diverse array of distributed changes in limbic and cortical areas of the brain involving abnormalities in dopamine and glutamate transmission. Furthermore, genetic and behavioral studies indicate that abnormalities in normal development contribute to the etiology of the disorder. The present dissertation used two general strategies in an attempt to model some of the basic characteristics of the disorder. In Chapter Two, experiments were conducted that demonstrate short periods of higher frequency stimulation applied to the ventral, but not dorsal, hippocampus in adult rats reversibly reduce prepulse inhibition, a pre-attentive processing mechanism that is disrupted in schizophrenic patients. In Chapters Three and Four, the behavioral effects of reversible pharmacological manipulation of glutamate receptors early in development on prepulse inhibition and locomotor activity were assessed both before and after puberty. Additional experiments tested the putative role of dopaminergic abnormalities following these manipulations. Results from Chapter Three demonstrate that administration of a convulsive dose of the glutamate receptor agonist kainic acid to neonatal rats on postnatal day seven results in the delayed emergence of PPI deficits in early adulthood. Levels of locomotor activity were not reliably altered in a novel environment or following amphetamine administration. The experiments conducted in Chapter Four were designed to assess alterations in prepulse inhibition and locomotor activity following administration of the NR2B-subunit selective N M D A antagonist Ro25-6981. Unexpectedly, Ro25-6981 administration resulted in behavioral convulsions when administered during the first postnatal week; however, no consistent behavioral abnormalities were revealed in rats treated with the drug. Although the present results are somewhat mixed, the experiments were successful at providing novel insights into the symptoms and etiology of schizophrenia. In general, they support the assertion that short periods of altered activity in the limbic system, and hippocampus in particular, at different points during development may underlie the expression of some of the most basic symptoms of schizophrenia. These data also suggest that the nature and anatomical location of these alterations critically determines their long-term functional effects. i i i TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS iv LIST OF TABLES viii LIST OF FIGURES ix ACKNOWLEDGMENTS xi CO-AUTHORSHD? STATEMENT xii CHAPTER ONE: GENERAL INTRODUCTION 1 Schizophrenia 2 Brief Overview of the Disorder 2 Etiological Theories 7 Animal Models 8 Genetic Models 11 Adult Pharmacological Models 12 Adult Lesion Models 14 Developmental Animal Models 16 Maternal Separation and Social Isolation Models 16 Neonatal Lesion Models 18 Neonatal Pharmacological Models 22 References 24 CHAPTER TWO: ELECTRICAL STIMULATION OF THE HIPPOCAMPUS DISRUPTS PREPULSE INHIBITION IN RATS: FREQUENCY AND SITE DEPENDENT EFFECTS 37 Introduction 37 Methods 41 Subjects 41 Surgery 41 Prepulse Inhibition Testing 42 Neurochemical Experiments 44 Histology 46 Data Analysis 46 Results 47 Immediate Behavioral Effects of Stimulation 47 Differential Effects of 20 Hz Stimulation of the vHip or dHip on PPI and Startle Amplitude : 48 iv Effects of 2 Hz Stimulation of the vHip on PPI and Startle Amplitude 50 Effects of Stimulation of the vHip and dHip on NAc DA Efflux 52 Histology 52 Discussion 55 Potent Disruption of Sensorimotor Gating Induced by 20 Hz Stimulation of the vHip, but not dHip 55 Frequency Dependence of the vHip Stimulation-induced Disruption of PPI 57 Increases in NAc DA Efflux following Hip Stimulation are Site and Hemisphere Specific 59 Potential Mechanisms Underlying vHip Stimulation-induced Disruption in PPI 61 Conclusion 62 References 63 CHAPTER THREE: DELAYED ONSET OF PREPULSE INHIBITION DEFICITS FOLLOWING KAINIC ACID TREATMENT ON POSTNATAL DAY SEVEN IN RATS 70 Introduction 70 Methods 72 Subjects 72 Kainic Acid Administration 73 Prepulse Inhibition 74 Locomotor Activity 75 Water Maze Testing 76 Histology ". 77 Data Analysis 77 Results ., 78 Immediate Behavioral Effects ofPND7 KA Administration 78 Effects of PND7 KA Administration on Body Weight 79 Prepulse Inhibition is Reduced in Post-Pubescent, but not Pre-pubescent Rats, Following PND7 KA Administration 81 Locomotor Activity in Response to Novelty and Amphetamine in Rats Following PND7 KA Administration 84 Spatial Learning and Memory in the Morris Water Maze is not Altered in Rats Following PND7 KA Administration 87 Histology 89 Discussion 91 Effects of Neonatal KA Administration on PPI 91 Effects of Neonatal KA on Locomotor Activity 93 Potential Mechanisms Underlying the Observed PPI Changes 94 Conclusion 97 References 99 CHAPTER FOUR: BEHAVIORAL CONVULSIONS INDUCED BY E A R L Y POSTNATAL ADMINISTRATION OF THE NR2B ANTAGONIST R025-6981 FAIL TO AFFECT SENSORIMOTOR GATING OR LOCOMOTOR BEHAVIOR IN PRE- AND POST-PUBESCENT RATS..... : 105 Introduction 105 Methods 110 Experiment 1 - Incidence and Characteristics of Behavioral Convulsions in Postnatal Rats Following Antagonism of NMDA Receptors Containing the NR2B Subunit.... 110 Subjects 110 Experimental Procedures 110 Experiment 2 - Long-term Behavioral Effects of Convulsions Resulting from Antagonism of NMDA Receptors Containing the NR2B Subunit 1111 Subjects I l l Ro25-6981 Administration I l l Prepulse Inhibition 112 Spontaneous Locomotor Activity 113 Dopaminergic Challenges of PPI and Locomotor Activity 113 Data Analysis 114 Results 115 Experiment 1 - Behavioral Effects ofRo25-6981 Administration in the Early Postnatal Period 115 Experiment 2 - Effects of Neonatal Ro25-6981 Administration on Body Weight 116 Neonatal Ro25-6981 Administration Does not Alter PPI Responding Either before Puberty or in Early Adulthood 118 Apomorphine Challenge in Adulthood has Similar Effects on PPI in Neonatally Saline- or Ro25-6981 -treated Rats 121 Neonatal Treatment with Ro25-6981 and Amphetamine-Induced Locomotor Activity in Adult Rats 121 Discussion 125 Antagonism of NR2B-containing NMDA Receptors Induces Behavioral Convulsions 126 The Long-Term Behavioral Effects of Ro25-6981 on PPI and Locomotor Activity.. 129 Conclusion 131 References 132 CHAPTER FIVE: GENERAL DISCUSSION 138 The Role of the Hippocampus in the Regulation of Prepulse Inhibition and Locomotor Activity 139 The Utility of Adult Animal Models of Schizophrenia 142 Developmental Models of Schizophrenia - Effects of Early Postnatal Glutamate Manipulations 143 Developmental Models of Schizophrenia - The Time Course of Symptom Emergence 148 Criteria for Establishing Validity in Behavioral Models of Schizophrenia 149 Conclusion 152 References 153 vi LIST OF TABLES Table 4-1. The incidence of behavioral convulsions in rats administered NR2B-selective N M D A antagonists at various postnatal ages 117 vii L I S T O F F I G U R E S Figure 2-1. Effects of 20 Hz stimulation of the hippocampus on PPI and acoustic startle amplitude 49 Figure 2-2. Effects of 2 Hz stimulation of the vHip on PPI and acoustic startle amplitude. 51 Figure 2-3. Unilateral stimulation (20 Hz, 10 s) of either the vHip or dHip, and its effect on NAc D A efflux in the ipsilateral or contralateral hemisphere 53 Figure 2-4. Schematic diagram of the placements of stimulating electrodes and microdialysis probes in all experiments 54 Figure 3-1. Prepulse inhibition scores from rats in Group 1 treated on postnatal day 7 with saline or kainic acid 80 Figure 3-2. Prepulse inhibition scores from rats in Group 2 treated on postnatal day 7 with saline or kainic acid 83 Figure 3-3. Effects of pretreatment with vehicle or apomorphine on average percent PPI scores in adulthood 85 Figure 3-4. Locomotor activity levels in response to novelty or amphetamine of all rats tested on either postnatal day 36 or 57 86 Figure 3-5. Average latencies to locate the hidden platform in the water maze of rats treated with saline or kainic acid on postnatal day 7 88 Figure 3-6. A representative cresyl violet stained section of the dorsal hippocampus of a KA-treated and saline-treated rat 90 Figure 4-1. Cartoon illustrating an N M D A receptor within the cell membrane 107 Figure 4-2. Prepulse inhibition scores of rats in Group 1 treated on postnatal day 6 and 7 with saline or Ro25-6981 119 Figure 4-3. Prepulse inhibition scores from rats in Group 2 treated on postnatal day 6 and 7 with saline or Ro25-6981 120 Figure 4-4. Effects of pretreatment with vehicle or apomorphine on percent PPI scores in adulthood 122 Figure 4-5. Locomotor activity levels in response to novelty or amphetamine of rats tested on either postnatal day 36 or 57 in Group 1 123 viii Figure 4-6. Locomotor activity levels in response to novelty or amphetamine of rats tested on either postnatal day 36 or 57 in Group 2. 124 ACKNOWLEDGMENTS The work contained within this dissertation would not have been completed without the help of many people. Those who directly assisted me with these projects are co-authors on my publications. Many of those who indirectiy assisted me in the lab are listed alphabetically below. I would like to especially acknowledge the wisdom and expert advise of Drs. Tony Phillips, Stanley B. Floresco, and Darren 'Twinkle-Toes' Hannesson throughout my graduate career and Dr. Karen Brebner for providing many helpful comments on the General Introduction and Discussion Sections of my dissertation. Finally, I would like to acknowledge NSERC, CIHR, and the Michael Smith Foundation for Health Research for funding. Thank you: Soyon Ahn, Steven Barnes, Alasdair Barr, Karen Brebner, Deanna Chavez, Christina Cheng, Michael Corcoran, Carine Dias, Brennan Eadie, Rachel Genn, Rosie Gilham, Natalia Gorelova, Lucy Greggorios-Pippas, Rebecca Harrison, Fred Lepiane, Sarah C. Lidstone, Brandi Ormerod, Julie Pongrac, Kitty So, Aline Stephan, Christina Thorpe, Giada Vacca, and liana Winrob. To my friends and family who may not have helped directly with my academic life, but in many other ways, don't worry, your thanks is coming (and you're likely not reading this anyway). USB U B C . CO-AUTHORSHIP STATEMENT I am the first author on all manuscripts presented in this thesis. I was directly involved in all stages of the research including, but not limited to, conception of the ideas, designing the experiments, conducting the studies, analyzing the data, and writing the manuscripts. Research assistants and other students helped in gathering some of the data presented in this dissertation and they are included as authors on the manuscripts with which they were involved. Dr. Phillips was also involved in all aspects of the research included in this dissertation. He also acted as my PhD supervisor and is included as an author on all manuscripts. C H A P T E R O N E : G E N E R A L INTRODUCTION The present dissertation summarizes several novel approaches to the development of rodent models of schizophrenia that are reliable and valid. As will become clear in the introduction, schizophrenia is an incredibly complicated and variable disorder (or group of disorders). Therefore, it is difficult to put forth a unitary set of criteria by which to verify the success of putative models of schizophrenia. The following pages detail a number of strategies aimed at reproducing some characteristics of the disorder. As the experiments of Chapter Two demonstrate, acute changes in hippocampal activity in adulthood reversibly alter behavior in a manner consistent with schizophrenia. Additionally, Chapters Three and Four show that under some circumstances, transient disruption of normal activity patterns in the developing brain is sufficient to subtiy change adult behavior in a manner consistent with schizophrenia. Finally, the implications of these studies and a number of directions for future research are discussed in Chapter Five. This dissertation is presented in a manuscript-based format, rather than the traditional thesis format. Chapters Two through Four are stand-alone manuscripts, containing their own Introduction and Reference sections, while Chapter One and Five are the General Introduction and Discussion sections of the entire dissertation. While this format has numerous practical advantages, it has two clear limitations. First, there is inevitably a certain amount of repetition between chapters, and second, individual chapters do not necessarily flow into the next. In an effort to address the second problem, I will include a brief statement of the objectives of each chapter in the General Introduction, and summarize the findings from all chapters in the General Discussion. 1 Schizophrenia Brief Overview of the Disorder Schizophrenia is a debilitating mental illness with a lifetime prevalence of 1.4-4.6 per 1000 people worldwide (Jablensky, 2000). Symptoms usually appear in late adolescence or early adulthood, although cases with earlier and later onset certainly exist (Wong and Van Tol, 2003). For many patients, schizophrenia has a chronic course with unpredictable relapses that require repeated hospitalizations. Although antipsychotic drugs effectively reduce symptom severity in many patients, their side effects are severe, and the drugs often fail to improve many of the negative and cognitive symptoms of the disorder (see below). It is certainly worth noting that the complexities and inconsistencies of the disorder are far from understood. As a result, the behavioral symptoms and neuropathological characteristics of the disorder described below have been simplified. Within the following chapters, pertinent details related to relevant aspects of schizophrenia are described where necessary. As the pharmacology underlying antipsychotics drug action has been used to support hypotheses related to the neural bases of the symptoms of schizophrenia, a brief review of their main classes is required. Typical antipsychotic drugs, which antagonize dopamine receptors, were discovered in the 1950's and revolutionized treatment of schizophrenia (Wong and Van Tol, 2003). During the 1980's and 1990's, psychiatrists began prescribing numerous atypical antipsychotic drugs to patients who did not respond to typical antipsychotics (either due to symptom profiles, side effects, or unknown reasons). Atypical antipsychotics have unique pharmacological effects, including 2 affinities for serotonin, adrenergic, dopamine, histamine, and muscarinic receptors, which may underlie their effectiveness in some patients (Wong and Van Tol, 2003). The behavioral symptoms of schizophrenia are varied and involve numerous advanced functions of the human brain. Generally, symptoms can be divided into three main categories: the classic psychotic or 'positive' symptoms, deficit or 'negative' symptoms, and cognitive impairment (Wong and Van Tol, 2003). Positive symptoms are those typically associated with the illness - disturbances such as hallucinations, delusions, and thought disorder. Importantly, the appearance of positive symptoms in late adolescence or early adulthood generally leads to the diagnosis of schizophrenia. A number of researchers have argued that altered activity of the dopamine system is the likely cause of positive symptoms, a hypothesis largely based on the effectiveness of antipsychotic drugs, which are dopamine D2 receptor antagonists, in reducing positive symptoms and dopamine agonists at inducing or aggravating positive symptoms (Snyder, 1973;Kapur and Mamo, 2003). Whereas positive symptoms may be thought of as the presence of abnormal behaviors, negative symptoms may be regarded as a reduction or loss in a number of normally occurring behaviors. Symptoms such as social withdrawal, apathy, reduced expression of affect, and the ability to experience pleasure are common examples of negative symptoms. The causes and treatment of negative symptoms are more poorly understood than the positive symptoms, although some atypical antipsychotic drugs, with actions at receptor subtypes other than the D2 receptor, may be more effective than typical antipsychotics at improving these symptoms (Meltzer et al., 1994;Wong and Van Tol, 2003). 3 Finally, impairments in cognitive domains such as pre-attentive processing, executive function, attention, learning, memory, and general intellectual function are an important component of schizophrenia (Elvevag and Goldberg, 2000;Sharma and Antonova, 2003;Lewis, 2004). Intense interest has focused recently on these symptoms because of studies showing that the cognitive symptoms are the most enduring and consistently debilitating of the disorder (Elvevag and Goldberg, 2000). Additionally, impairments in cognition may precede the development of the positive symptoms of the disorder, thereby providing a potential tool with which to identify those individuals at risk for expressing full-blown psychosis in early adulthood (Walker, 1994;Walker et al., 1994;Jones et al., 1994;Lewis, 2004). In addition to the behavioral changes observed in those suffering from the disorder, many alterations in the normal structure and function of the brain have been reported. For example, an overwhelming array of neuroanatomical changes is reported to exist in schizophrenia, many of which occur in the frontal and temporal lobes (McCarley et a l , 1999;Wright et al., 2000). Ventricular enlargement (Stevens, 1997;Harrison, 1999) and reductions in temporal (Lawrie and Abukmeil, 1998;Nelson et al., 1998) or frontal lobe volumes (Wright et al., 1999) are observed in the brains of schizophrenic patients. Reduced volume of the hippocampus (Suddath et al., 1990;Noga et al., 1996;Bogerts, 1997;Heckers, 2001), reduced cell numbers in the hippocampus (Gothelf et al., 2000;Heckers, 2001), nucleus accumbens (NAc; Pakkenberg, 1990), and thalamus (Jones, 1997;Clinton and Meador-Woodruff, 2004), and altered neuronal size and density in certain frontal cortical areas (Harrison, 1999) have been observed in patients with schizophrenia. Abnormalities in patterns of hippocampal (Heckers et al., 1998;Benes, 2000;Heckers, 2001;Holt et al., 2005), striatal (Meyer-Lindenberg et al., 2002) and prefrontal activation (McClure and Weinberger, 2001;Meyer-Lindenberg et al., 2002) during the performance of a variety of cognitive tasks are also characteristic of schizophrenic patients (Wong and Van Tol, 2003). Taken together, these neuroanatomical changes may be considered an enduring trait for schizophrenia in adulthood. Interestingly, relatives of patients with schizophrenia may also have reduced cortical volumes (Cannon et al., 1993), altered hippocampi (Seidman et al., 2002) and enlarged ventricles (Honer et al., 1994;Lawrie et al., 1999), thereby supporting the assertion that an underlying genetic abnormality may predispose individuals to develop the disorder, although this underlying 'trait' for schizophrenia may have to interact with other environmental factors to result in the expression of a schizophrenic 'state' in early adulthood. Such conceptions of the etiology of schizophrenia are termed 'two-hit' hypotheses by some authors (McCarley et al., 1999;Bayer et al., 1999;Wong and Van Tol, 2003;Ellenbroek, 2003). The neurochemical changes present in schizophrenia are also complex and incompletely understood. The affinity of typical antipsychotics for the D2 receptor and psychotomimetic agents such as amphetamine and phencyclidine for the dopamine transporter and the N-methyl-D-aspartate (NMDA) receptor, respectively, are suggestive of the importance of dopamine and glutamate neurotransmission in schizophrenia (Angrist et al., 1974;Jentsch and Roth, 1999). More direct evidence of abnormal dopamine-glutamate interactions is gained from studies suggesting that alterations in glutamate and glutamate receptor levels may be present in schizophrenia (for review, see Goff and Coyle, 2001) and that schizophrenic patients show elevated synaptic levels of 5 dopamine (Abi-Dargham et al., 2000) and potentiated increases in striatal dopamine release in response to amphetamine administration during episodes of acute psychosis (Breier et a l , 1997;Abi-Dargham et al., 1998;Laruelle et al., 1999;Laruelle et al., 2003) and periods of relative remission (McGowan et al., 2004). Current theories of cortico-striatal-limbic function and schizophrenia have begun to delineate mechanisms by which these neurochemical changes and developmental abnormalities may manifest themselves as the symptoms of schizophrenia. One such example is the tonic-phasic model of ventral striatal dopamine release proposed by Grace (Grace, 1991). The tonic-phasic model posits that in schizophrenia, reduced activity from cortical glutamatergic afferents to the ventral striatum reduces basal (or tonic) levels of dopamine in the ventral striatum. Tonic levels of dopamine are thought to regulate the responsivity of the ventral striatum to short-lasting (or phasic) changes in dopamine efflux in response to environmental stimuli. As a result, patients with schizophrenia are predicted to exhibit increased dopaminergic responsivity to salient environmental stimuli, which may be a partial explanation of the positive symptoms (Grace, 1991). This model, along with others like it (Carlsson et al., 1999), illustrates the importance of dopamine-glutamate interactions in the neurobiology of schizophrenia. Additionally, the therapeutic efficacy of atypical antipsychotic drugs (Emsley and Oosthuizen, 2003; A wad and Voruganti, 2004) and a variety of additional neuropathological findings (Costa et al., 2004;Lewis et al., 2005) underlie the importance of other neurotransmitter systems in schizophrenia. In particular, alterations in the G A B A and serotonin systems may exist in schizophrenia (Costa et al., 2004;Roth et al., 2004), although they will not be the focus of the present review. 6 Etiological Theories Currently, the cause(s) of schizophrenia remain poorly defined. Twin and gene linkage studies provide convincing evidence that a genetic predisposition to develop schizophrenia exists, although environmental factors must also contribute as concordance rates between monozygotic twins are approximately 50 percent, whereas for dizygotic twins and siblings, the risk is approximately 10 percent (Kennedy et al., 2003;Owen et al., 2004). One particularly prominent etiological theory of the last 15 to 20 years is the neurodevelopmental theory (Weinberger, 1987;Lewis and Levitt, 2002;Church et al., 2002;Eastwood, 2004;Rapoport et al., 2005). In its original form, the theory suggests that genetic abnormalities may alter early brain development, or make the brain more susceptible to disruption. This 'compromised brain' then interacts with environmental factors or later occurring events of brain development and ultimately results in the expression of the symptoms typically observed in schizophrenic patients (Benes, 2000;Benes et al., 2000;McClure and Weinberger, 2001;Lewis and Levitt, 2002). The neurodevelopmental theory is supported by evidence suggesting that people who develop schizophrenia have an increased incidence of adverse gestational and perinatal events, cognitive and behavioral abnormalities before diagnosis, congenital physical abnormalities, and alterations in brain morphology (for reviews see McClure and Weinberger, 2001;Lewis and Levitt, 2002). Further support is gained from evidence suggesting that the neuroanatomical changes observed in schizophrenia are present from the first episode diagnosis and their severity usually does not increase over time (Weinberger, 1987), although recent data challenges this notion (Weinberger, 1996;Rapoport et al., 1999;Church et al., 2002;Rapoport et al., 2005). Additionally, signs 7 of active neurodegeneration, such as gliosis, are generally absent from the brains of schizophrenic patients (Roberts et al., 1986;McClure and Weinberger, 2001), although such observations do not exclude the possibility that active neurodegenerative processes such as increased rates of apoptosis occur in patients with schizophrenia (Church et al., 2002). Animal Models Animal models have been used extensively in medicine to simplify complex and poorly understood pathologies. In psychiatry, and specifically in regards to schizophrenia, the development of reliable and valid animal models has proven difficult. Obviously, many symptoms of psychiatric disorders are inherently human, and thus can never be fully modeled in animals. As a result, attempts at modeling disorders such as schizophrenia in animals have been met with skepticism. Recently, a number of authors (Geyer and Markou, 1995;Lipska and Weinberger, 2000;Robbins, 2004) have advanced the notion that valuable and unique insights can be gained from modeling psychiatric disorders in animals, including rodents. Paramount to this undertaking is a realistic notion of the goal of the animal model, and the criteria which will be used to verify whether or not this goal has been achieved. With respect to schizophrenia, animal models have been developed for at least three main goals: furthering our understanding of its etiology, advancing our understanding of the symptoms (both behavioral and biological), and finally, aiding in the development and understanding of pharmaceutical therapies (Lipska and Weinberger, 2000;Robbins, 2004). The evaluation criteria (or dependent measures) used to validate models of schizophrenia are quite varied, mostly due to the inherent difficulties of 8 measuring the disorder in animals. Generally, models are designed to replicate the abnormal neurochemistry/neuropathology or behavioral changes described for schizophrenia. Within the present thesis, two behavioral tests - prepulse inhibition and locomotor behavior - are used extensively to validate the experimental manipulations used to model schizophrenia. Prepulse inhibition (PPI) of the acoustic startle response is an operational measure of sensorimotor gating, and is a normal pre-attentive behavioral response in which a weak sensory event (or prepulse) inhibits, or gates, the motor response to a starding stimulus (or pulse) (Fig. 1-1; Swerdlow et al., 2000a). PPI is disrupted in patients with schizophrenia or schizotypal personality disorder (Braff et al., 2001;Hamm et al., 2001 ;Ludewig et al., 2003) and in the first degree relatives of patients with schizophrenia (Cadenhead et al., 2000). These data suggest that PPI deficits are not a result of acute psychosis (Braff et al., 2001; but see also Meincke et al., 2004). Typical antipsychotic drugs do not ameliorate PPI deficits in schizophrenic patients (Mackeprang et al., 2002;Duncan et al., 2003a;Duncan et al., 2003b), although some beneficial effects of atypical antipsychotic drugs have been reported (Braff et al., 2001;Leumann et al., 2002;Kumari et al., 2002). More sophisticated longitudinal designs are necessary to further address the efficacy of antipsychotic drugs in reversing the PPI deficits observed in patients with schizophrenia (Kumari and Sharma, 2002). Importantly, PPI deficits are correlated with cognitive and behavioral symptoms such as distractibility, social perception, and thought disorder (Perry and Braff, 1994;Karper et al., 1996;Braff et al., 1999;Perry et al., 1999;Swerdlow et al., 2000a;Meincke et al., 2004;Wynn et al., 2005). Deficits in information processing capabilities, such as those measured by PPI, are core 9 cognitive symptoms of schizophrenia and may relate to deficits in stimulus filtering commonly proposed to exist in the disorder (Swerdlow et al., 2000a). As PPI is easily and reliably measured in animals, it has been extensively used in cross-species work as valid behavioral test with relevance to schizophrenia (Swerdlow et al., 2000a). The use of PPI as an "index" of schizophrenia in animals is validated by pharmacological experiments demonstrating that D A agonists such as apomorphine and N M D A antagonists such as phencyclidine alter PPI responding in animals and healthy humans, and antipsychotic drugs ameliorate these deficits in animals (Braff et al., 2001;Geyer et al., 2001). Extensive experimentation has been conducted to understand the neural substrates underlying the dopaminergic and glutamatergic regulation of PPI (Swerdlow et al., 2001). These experiments reveal a distributed circuitry in cortical, limbic, striatal, and pallidal areas important in the regulation of PPI. The pertinent details of this literature are reviewed in Chapter Two, and therefore will not be elaborated upon here. Locomotor responses to a variety of arousing stimuli such as novel environments or stimulant drugs are commonly measured in rodents as a measure of mesolimbic dopamine activity (Kelly et al., 1975;Castall et al., 1977;Porrino et al., 1984). More vigorous locomotor behavior in response to these treatments correlates positively with elevated ventral striatal dopamine levels (Kelly et al., 1975;Castall et al., 1977;Hooks et al., 1992). As previously reviewed, ventral striatal dopamine levels are higher in patients with schizophrenia than controls (Laruelle et al., 2003). As a result, locomotor activity levels are often used as an indirect measure of ventral striatal dopamine levels in animal models of schizophrenia (Lipska and Weinberger, 2000;Bast and Feldon, 2003;Boksa, 10 2004). Additionally, some authors have argued that increased locomotor activity may be homologous to some cognitive abnormalities in schizophrenia patients during acute episodes (Bast and Feldon, 2003), although these accounts are debatable (Marcotte et al., 2001). In the present thesis, PPI and locomotor activity levels were used as the main endpoints to assess whether the novel experimental manipulations employed would produce effects resembling aspects of schizophrenia. These tests were selected for the following reasons: (1) Both tests have been used frequendy to assess previous preclinical models of schizophrenia. Thus, comparisons could easily be rnade between existing models and those developed within the present work. (2) These tests can be reliably conducted before and after puberty, enabling the developmental course of the behavioral effects of the neonatal manipulations performed in Chapters Three and Four to be assessed. (3) Pharmacological challenges are easily conducted in conjunction with these tests thereby allowing the neurochemical mechanisms of observed behavioral changes to be evaluated. Given the multi-dimensional etiology of schizophrenia, researchers have advanced numerous strategies to model the disorder in animals, each with its own strengths and limitations. A brief review of the models pertinent to the present discussion of PPI and locomotor activity will now be presented. Genetic Models As previously discussed, the etiology of schizophrenia is at least partly dependent on complex genetic risk factors, likely involving a number of different genes. Therefore, there have been numerous attempts to model the disorder with genetic strategies. 11 Knockout and knockdown strategies typically target genes for neurotransmitter systems implicated in schizophrenia. One well-known example includes dopamine transporter knockout mice, which show hyperlocomotion and deficits in PPI that are reversed with antipsychotics (Gainetdinov et al., 2001). Similar results have been gained with the NR-1 knockdown mouse, which has reduced N M D A receptor function (Mohn et al., 1999;Duncan et a l , 2004). Genetic models targeting genes involved in neurodevelopment and implicated in schizophrenia have also been developed. Reduced expression of reelin, a protein important for normal cell positioning in the brain, has been described in schizophrenia (Impagnatiello et al., 1998). Heterozygous reeler mice, which exhibit a down regulation of reelin similar to levels observed in schizophrenic patients, also have a variety of behavioral abnormalities similar to those in schizophrenia (Costa et al., 2002). These genetic strategies are useful in understanding the impact of genetic alterations on behavior, neurochemistry, and histology at a systems level, although these single gene approaches only model part of the complex etiology of schizophrenia. Clearly, the future challenge for genetic strategies lies in modeling the complex multi-gene abnormalities that will likely be implicated in schizophrenia (Wong and Van Tol, 2003). Additionally, combining genetic approaches with environmental factors in a 'two-hit' approach may be especially valuable in the future (Ellenbroek, 2003). Adult Pharmacological Models These models generally rely on producing acute or long-lasting behavioral changes relevant to schizophrenia by administering psychoactive drugs to animals. Dopamine agonists such as amphetamine and N M D A antagonists such as MK-801, 12 phencyclidine or ketamine are most commonly used. Acute administration of these drugs induces deficits in PPI and increased locomotor activity (for review see Marcotte et al., 2001). Not surprisingly, the changes induced by dopamine agonists are reversed by typical antipsychotics (Marcotte et al., 2001). Additionally, sensitization with amphetamine, but not phencyclidine, in rats induces long-lasting deficits in PPI, which may more faithfully model the stable PPI deficits in schizophrenia (Tenn et al., 2005). Interestingly, administration of N M D A antagonists may more accurately model schizophrenia by inducing a broader range of symptoms, including cognitive deficits and negative symptoms (Jentsch and Roth, 1999). The effects of N M D A antagonism are also reversed by atypical antipsychotics, thereby improving the potential of the model to identify novel antipsychotic compounds. The data gained from adult pharmacological models is limited for a number of reasons. First, the effects are typically induced in normally developed adult animals. A wide range of evidence indicates that people who develop schizophrenia have abnormalities before adulthood. Importantly, combining neonatal perturbations of brain development with pharmacological challenges has provided valuable data, and will be discussed in the next section. Secondly, the changes in the activity of neurotransmitters such as dopamine are also more regionally specific than can be modeled with systemic administration of drugs (Kilts, 2001). Nevertheless, some treatment paradigms have proven to model more faithfully the neurochemical and behavioral changes in schizophrenia. For example, chronic exposure to phencyclidine in non-human primates results in cognitive deficits and reduced dopamine turn-over in the prefrontal cortex, both of which persist after drug treatment in discontinued (Jentsch et al., 1997). These effects 13 can also be reversed following treatment with the atypical antipsychotic clozapine (Jentsch et a l , 1997). Finally, some researchers argue that the development of novel antipsychotic drugs is precluded by dopamine agonist models because the therapeutic drugs that have emerged from these models block dopamine receptors, thereby leading to a circular pattern of discovery (Lipska and Weinberger, 2000). Adult Lesion Models Adult lesion models focus on the effects of lesioning limbic and cortical areas commonly implicated in both subcortical dopamine regulation and schizophrenia, such as the prefrontal cortex, hippocampus, and thalamus (Lipska and Weinberger, 2000;Marcotte et al., 2001). Obviously, the construct validity of adult lesion models is compromised by their adult initiation and large degree of damage that occurs (at least compared to that observed in schizophrenic patients); however, such models do provide unique insights into the neural substrates that may underlie many of the behavioral and neurochemical abnormalities in schizophrenia. Adult lesions of the medial prefrontal cortex in rodents result in hyper-responsiveness to stress (Jaskiw et al., 1990b), increases in spontaneous locomotor activity (Braun et al., 1993), increased activity in response to amphetamine (Jaskiw et al., 1990a), and deficits in PPI after challenge with the dopamine agonist apomorphine (Swerdlow et a l , 1995). Similarly, lesions of the ventral, but not dorsal, hippocampus result in increased spontaneous activity and hyperactivity and PPI deficits following administration of dopamine agonists (Lipska et al., 1991 ;Lipska et al., 1992;Swerdlow et al., 1995;Mittleman et al., 1998). These data enable a detailed understanding of the neural substrates mediating PPI and locomotor activity to be developed, and will be 14 returned to in greater detail in Chapter Two. Importantly, they can be effectively integrated with neuroanatomical and neurochemical data, leading to new hypotheses regarding the mechanisms underlying the behavioral effects of the lesions. For example, the connectivity of the ventral, but not dorsal, hippocampus with areas such as the prefrontal cortex, amygdala, nucleus accumbens, and ventral tegmental area of the midbrain may be linked causally to the behavioral effects observed following its removal (Bast and Feldon, 2003). OBJECTIVE ONE: Numerous well-documented shortcomings of permanent lesions exist, such as compensatory alterations in brain areas other than the lesioned area (Schoenfeld and Hamilton, 1977;Stein, 1979;Bast and Feldon, 2003). Additionally, animals may develop strategies that aid in coping with the permanent loss of function normally carried out by the lesioned area (Lomber, 1999;Bast and Feldon, 2003). Specifically in the context of PPI and locomotor behavior, differences between permanent and temporary manipulations of the hippocampus have been described (Swerdlow et al., 1995;Pouzet et al., 1999;Swerdlow et al., 2000b;Zhang et al., 2002). Therefore, in Chapter Two, I examine the effect of brief stimulation of discrete sub-regions of the hippocampus on PPI. Given the important role of the hippocampus in schizophrenia, these experiments may provide further insights into the pathophysiology of the disorder. 15 Developmental Animal Models As previously discussed, the neurodevelopmental hypothesis of schizophrenia has received considerable attention. Therefore, it is not surprising that numerous strategies have been developed to model schizophrenia using manipulations at distinct periods from embryological development until the time of weaning. As the present experiments focus on manipulations in the early neonatal period, most of the studies detailed below will focus on this time point. Numerous excellent reviews of experiments examining the effects of prenatal stress, obstetric complications, prenatal infection, and other developmental manipulations are available (Van den Buuse et al., 2003;Boksa, 2004). Before discussing developmental models, it is important to note that rodents are born at later stage of development than humans. Thus, the first week of postnatal life in rodents is typically described as corresponding to the third trimester of human fetal brain development (Lipska and Weinberger, 2000). Additionally, the surge of gonadal hormones that coincide with the onset of puberty occurs during the 5 t h and 6 t h postnatal weeks of the rodent life span. Thus, assessments of prepubertal function are generally conducted at approximately postnatal day 35 and assessments of adult function are performed after postnatal day 56. Maternal Separation and Social Isolation Models These two types of models involve removal of developing rats from their normal sources of social interaction, whether it be from their mothers early in development (maternal separation) or from their peers immediately following weaning (social isolation). Stress likely plays a major role in producing the behavioral changes observed in both models (Hall, 1998;Lipska and Weinberger, 2000;Weiss and Feldon, 2001 ;Van 16 den et al., 2003). In one example of a maternal separation paradigm, a 24 hour separation period on postnatal day three, six, or nine is sufficient to produce deficits in PPI and increased sensitivity to dopamine agonists that occur only after puberty (Ellenbroek et al., 1998;Husum et al., 2002). Interestingly, these deficits can be reversed with typical or atypical antipsychotics (Ellenbroek et al., 1998). Strain differences are also observed, with effects present in Wistar rats, but not the Lewis or Fisher 344 strains (Ellenbroek and Cools, 2000). These data provide important support for the assertion that early life experiences interact with genetic background to alter adult patterns of behavior with relevance to schizophrenia (Lipska and Weinberger, 2000;Ellenbroek and Cools, 2000). The social isolation model involves rearing weanling rat pups individually without access to their peers, thereby depriving them of social contact and considerable sensory stimulation (Hall, 1998;Weiss and Feldon, 2001 ;Van den et al., 2003). This model is distinct from maternal separation discussed above in that early development of the animals is not disturbed. Considerable research has been conducted using the social isolation model showing fairly consistent reductions in PPI, alterations in spontaneous locomotor activity, and locomotor responses to dopamine agonists and N M D A antagonists (Hall, 1998;Lipska and Weinberger, 2000;Weiss and Feldon, 2001). Parametric studies of the duration of rearing reveal that the pups must be in isolation for greater than three weeks starting at weaning (postnatal day 21 to 28) for behavioral alterations to be observed (Bakshi and Geyer, 1999;Varty et al., 1999). The behavioral effects are also sensitive to the strain of rats used (Weiss and Feldon, 2001), and are usually reversed with either typical or atypical antipsychotic drug treatment (Varty and Higgins, 1995;Bakshi et al., 1998). Thus, the social isolation model is an important 17 nonpharmacological tool that can be used to identify novel antipsychotic compounds (Weiss and Feldon, 2001). With respect to both the maternal separation and isolation rearing paradigms, reliability is a concern. For example, some groups have failed to observe PPI deficits in maternally separated rats using similar, but not identical, methods (Lehmann et al., 2000;Weiss and Feldon, 2001). Factors such as the temperature at which the pups are maintained and the exact conditions under which they are separated (i.e. separated and kept in a group versus kept individually) may help explain the conflicting results (Weiss and Feldon, 2001). With regard to isolation rearing, the robustness of the behavioral effects has been questioned, as some reports indicate that the model is quite sensitive to handling or other testing procedures (Weiss and Feldon, 2001). However, a long-term evaluation of the reliability of PPI deficits in the social isolation model indicate that PPI deficits occur in approximately 85 percent of groups tested against matched controls (Cilia et al., 2005). Neonatal Lesion Models A great deal of work in the last ten to fifteen years has examined the utility of performing neonatal lesions of cortical and limbic areas in modeling schizophrenia (Lipska and Weinberger, 2000). This procedure has a number of distinct advantages over those discussed above: (1) The normal maturation of specific brain areas is altered and this simplifies the search for mechanisms underlying observed changes in the model. (2) The consequences of the lesion can be examined over the course of development thereby allowing the timing of any changes to be compared to the normal course of the disease. (3) Manipulating the brain at an early age is consistent with the neurodevelopmental 18 theory of schizophrenia. (4) Further manipulations can be performed later in development to test the 'two-hit' hypothesis of schizophrenia. Most prominent among these models is the ventral hippocampal lesion model initially described by Lipska and colleagues (Lipska et al., 1993). In this model, rats are subjected to bilateral lesions of the ventral hippocampus with the excitotoxin ibotenic acid on postnatal day seven. The animals are then allowed to develop normally and can be tested before or after puberty on an array of behavioral tests relevant to schizophrenia. Numerous reports indicate that rats with neonatal ventral hippocampal lesions display PPI deficits and increased spontaneous locomotor activity (Lipska et al., 1993;Lipska et al., 1995). The PPI deficits are potentiated by a low dose of apomorphine (Lipska et al., 1995) and reversed by atypical antipsychotic drugs, but not by haloperidol (Le Pen and Moreau, 2002). Locomotor activity is also increased following stress (Lipska et al., 1993), dopamine agonist administration (Lipska et al., 1993), or treatment with N M D A antagonists (Al Amin et al., 2000;A1 Amin et al., 2001). The alterations in locomotor behavior can be normalized with haloperidol, but not atypical antipsychotic drugs (Al Amin et al., 2000). Treatment with ionotropic glutamate receptor antagonists (Al Amin et al., 2000) and increasing glycine availability (Le Pen et al., 2003) also reverses some of the behavioral impairments, thereby indicating a role of glutamate transmission in the underlying effects of neonatal ventral hippocampal lesions. Consistent with the typical course of schizophrenia, the PPI deficits and locomotor activity changes are not expressed until after the rats reach puberty and persist at least until mid-adulthood (Lipska and Weinberger, 1993;Lipska et al., 1995;Lipska and Weinberger, 2000;A1 Amin et al., 2001). Interestingly, lesioned rats show reduced social 19 interaction before and after puberty, a test which may have some relevance to the abnormalities observed in schizophrenic patients before their first episode (Sams-Dodd et al., 1997). Parametric experiments also suggest that the delayed onset of behavioral changes in the animals only occurs if the lesions are created during the first postnatal week of life (Wood et al., 1997), which corresponds to the third trimester in human development (Lipska and Weinberger, 2000). Finally, in an important paper, Lipska and Weinberger (Lipska and Weinberger, 1995) demonstrated that the effects of the neonatal lesion depend on the genetic background of the animals. Rats highly responsive to stress (the Fischer 344 strain) showed exaggerated effects of the lesion, whereas rats hypo-responsive to stress (the Lewis strain) showed reduced lesion effects. Thus, this model supports the theory that schizophrenia may develop from an interaction between genetic predisposition to the disorder and adverse environmental events (Lipska and Weinberger, 1995;Benes, 2000;Benes et al., 2000;McClure and Weinberger, 2001;Ellenbroek, 2003). The results gained from the ventral hippocampal model have led others to examine the effects of neonatally lesioning other areas such as the medial prefrontal cortex (Flores et al., 1996;Schneider and Koch, 2005), amygdala (Hanlon and Sutherland, 2000;Daenen et al., 2001;Daenen et al., 2002a;Daenen et a l , 2002b;Daenen et al., 2003a;Daenen et al., 2003b) and thalamus (Lipska et al., 2003) on a similar range of behaviors. Many of these experiments have yielded results similar to, or less consistent than, those already discussed. As a result, they will not be elaborated on further. While it is clear that many characteristics of schizophrenia have been modeled with great success using neonatal ventral hippocampal lesions, the severity and permanent nature of the hippocampal lesion limits the construct validity of this model for 20 schizophrenia (Lipska and Weinberger, 2000). As a result, Lipska and colleagues (2002) demonstrated that reversible temporary inactivation of the ventral hippocampus with tetrodotoxin on postnatal day seven is sufficient to induce significant changes in locomotor behavior in adulthood, but not before puberty. This manipulation produces limited gross morphological damage to the ventral hippocampus, and is short-lasting (24-48 hr) (Lipska et al., 2002). These data clearly highlight the fragility of the hippocampal-cortical circuit during this developmental period. Additional data demonstrate that a single infusion of the N M D A antagonist (±)-2-amino-5-phosphonopentanoic acid (APV) into the rat medial prefrontal cortex on postnatal day seven results in exaggerated amphetamine-induced locomotor activity in adulthood (Lafleur et al., 2001). These data support the hypothesis that an abnormality in activity-dependent plasticity involving glutamatergic hippocampal-prefrontal interactions may underlie some of the symptoms observed following neonatal ventral hippocampal lesions (Lipska et al., 2002). Additionally, these data support the hypothesis that the first postnatal week is a critical period for the development of cortico-limbic circuits mediating behaviors such as locomotor activity and PPI. Although the neonatal ventral hippocampal lesion model provides unique insight into the consequences of damaging a particular area early in development, it is unlikely that schizophrenia results from damage to a single brain area. Thus, the efficacy of altering the activity of the brain pharmacologically during the first postnatal week has also been tested in an effort to model schizophrenia. These experiments are discussed in the following section. 21 Neonatal Pharmacological Models As previously discussed, alterations of the glutamatergic system are likely to be involved in the expression of the symptoms of schizophrenia. A number of lines of evidence support the hypothesis that the glutamatergic system may also be involved in the development of the disorder. For example, adverse events during early development such as febrile seizures, fetal alcohol exposure, and hypoxia/ischemia involve the glutamatergic system (Morimoto et al., 1995;01ney et al., 1999;Zornberg et al., . 2000;Olney, 2004) and are risk factors for psychosis and/or schizophrenia (Cannon, 1997;Famy et al., 1998;01ney et al., 1999;Kanemoto et al., 2001;Vestergaard et al., 2005). The normal maturation and development of limbic and cortical areas relevant to schizophrenia is also dependent on normal glutamatergic function. For example, processes such as cell birth, survival and synaptogenesis are critically dependent on glutamate transmission, particularly at N M D A receptors (Rabacchi et al., 1992;Gould et al., 1994;Fox et al., 1996;Vallano, 1998;Ikonomidou et al., 1999;Luthi et al., 2001). Given the established role of glutamate neurotransmission in neonatal development during periods implicated in schizophrenia, a number of studies have examined the behavioral effects of manipulating glutamate transmission pharmacologically early in life. Most of these experiments have tested the effects of systemically administered N M D A receptor antagonists such as MK-801, ketamine or phencyclidine during the neonatal period. A number of groups have demonstrated deficits in PPI and alterations in locomotor behavior following treatment with these drugs (Facchinetti et al., 1993;Wang et al., 2001;Ffarris et al., 2003;Fredriksson et al., 2004). As will be elaborated further in Chapter Four of the present thesis, the long-lasting 22 morphological effects of administering non-competitive N M D A antagonists early in the neonatal period are also relevant to schizophrenia (Wang et al., 2001 ;Harris et al., 2003). OBJECTIVE TWO: Given that the end of the first postnatal week is a sensitive period for the development of neural circuits mediating PPI and locomotor activity (Wood et al., 1997;Wang et al., 2001;Lipska et al., 2002;Harris et a l , 2003), the second objective of this thesis was to extend these findings in two novel directions. In Chapter Three, a series of experiments examined the long-term behavioral effects of administering the glutamate receptor agonist kainic acid to postnatal day seven rats. Chapter Four summarizes an independent set of experiments designed to test the effects of administration of the highly specific N M D A receptor antagonist [(+/-)-(R*,S*)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-l-piperidine propanol] (Ro25-6981) during the same time period. These pharmacological manipulations were designed to subtly alter the normal development of the cortico-limbic circuitry involved in schizophrenia - kainic acid by selectively activating the hippocampus and Ro25-6981 by blocking NR2B subunit-containing N M D A receptors, which are highly expressed during the first postnatal week in rodents. 23 References Abi-Dargham A , Gi l R, Krystal J, Baldwin R M , Seibyl JP, Bowers M , van Dyck C H , Charney DS, Innis RB, Laruelle M (1998) Increased striatal dopamine transmission in schizophrenia: confirmation in a second cohort. A m J Psychiatry 155: 761-767. Abi-Dargham A , Rodenhiser J, Printz D, Zea-Ponce Y , Gi l R, Kegeles LS, Weiss R, Cooper TB, Mann JJ, Van Heertum R L , Gorman J M , Laruelle M (2000) Increased baseline occupancy of D2 receptors by dopamine in schizophrenia. Proc Natl Acad Sci U S A 97: 8104-8109. A l Amin HA, Shannon WC, Weinberger DR, Lipska B K (2001) Delayed onset of enhanced MK-801-induced motor hyperactivity after neonatal lesions of the rat ventral hippocampus. Biol Psychiatry 49: 528-539. A l Amin HA, Weinberger DR, Lipska B K (2000) Exaggerated MK-801-induced motor hyperactivity in rats with the neonatal lesion of the ventral hippocampus. Behav Pharmacol 11: 269-278. Angrist B , Sathananthan G, Wilk S, Gershon S (1974) Amphetamine psychosis: behavioral and biochemical aspects. J Psychiatr Res 11: 13-23. Awad A G , Voruganti L N (2004) Impact of atypical antipsychotics on quality of life in patients with schizophrenia. CNS Drugs 18: 877-893. Bakshi VP, Geyer M A (1999) Ontogeny of isolation rearing-induced deficits in sensorimotor gating in rats. Physiol Behav 67: 385-392. Bakshi VP, Swerdlow NR, Braff DL , Geyer M A (1998) Reversal of isolation rearing-induced deficits in prepulse inhibition by Seroquel and olanzapine. Biol Psychiatry 43: 436-445. Bast T, Feldon J (2003) Hippocampal modulation of sensorimotor processes. Prog Neurobiol 70: 319-345. Bayer TA, Falkai P, Maier W (1999) Genetic and non-genetic vulnerability factors in schizophrenia: the basis of the "Two hit hypothesis". Journal of Psychiatric Research 33: 543-548. Benes F M (2000) Emerging principles of altered neural circuitry in schizophrenia. Brain Res Brain Res Rev 31: 251-269. Benes F M , Taylor JB, Cunningham M C (2000) Convergence and plasticity of monoaminergic systems in the medial prefrontal cortex during the postnatal period: implications for the development of psychopathology. Cereb Cortex 10: 1014-1027. Bogerts B (1997) The temporolimbic system theory of positive schizophrenic symptoms. Schizophr Bull 23: 423-435. 24 Boksa P (2004) Animal models of obstetric complications in relation to schizophrenia. Brain Res Brain Res Rev 45: 1-17. Braff DL, Geyer M A , Swerdlow NR (2001) Human studies of prepulse inhibition of startle: normal subjects, patient groups, and pharmacological studies. Psychopharmacology (Berl) 156: 234-258. Braff DL, Swerdlow NR, Geyer M A (1999) Symptom correlates of prepulse inhibition deficits in male schizophrenic patients. Am J Psychiatry 156: 596-602. Braun AR, Jaskiw GE, Vladar K, Sexton R H , Kolachana BS, Weinberger DR (1993) Effects of ibotenic acid lesion of the medial prefrontal cortex on dopamine agonist-related behaviors in the rat. Pharmacol Biochem Behav 46: 51-60. Breier A , Su TP, Saunders R, Carson RE, Kolachana BS, de Bartolomeis A , Weinberger DR, Weisenfeld N , Malhotra A K , Eckelman WC, Pickar D (1997) Schizophrenia is associated with elevated amphetamine-induced synaptic dopamine concentrations: evidence from a novel positron emission tomography method. Proc Natl Acad Sci U S A 94: 2569-2574. Cadenhead KS, Swerdlow NR, Shafer K M , Diaz M , Braff D L (2000) Modulation of the startle response and startle laterality in relatives of schizophrenic patients and in subjects with schizotypal personality disorder: evidence of inhibitory deficits. A m J Psychiatry 157: 1660-1668. Cannon TD (1997) On the nature and mechanisms of obstetric influences in schizophrenia: a review and synthesis of epidemiologic studies. International Review of Psychiatry 9: 387-397. Cannon TD, Mednick SA, Parnas J, Schulsinger F, Praestholm J, Vestergaard A (1993) Developmental brain abnormalities in the offspring of schizophrenic mothers. I. Contributions of genetic and perinatal factors. Arch Gen Psychiatry 50: 551-564. Carlsson A, Waters N , Carlsson M L (1999) Neurotransmitter interactions in schizophrenia—therapeutic implications. Biol Psychiatry 46: 1388-1395. Castall B , Marsden CD, Naylor RJ, Pycock CJ (1977) Stereotyped behaviour patterns and hyperactivity induced by amphetamine and apomorphine after discrete 6-hydroxydopamine lesions of extrapyramidal and mesolimbic nuclei. Brain Res 123: 89-111. Church S M , Cotter D, Bramon E, Murray R M (2002) Does schizophrenia result from developmental or degenerative processes? Journal of Neural Transmission-Supplement 129-147. Cilia J, Hatcher PD, Reavill C, Jones D N (2005) Long-term evaluation of isolation-rearing induced prepulse inhibition deficits in rats: an update. Psychopharmacology (Berl) 180: 57-62. 25 Clinton S M , Meador-Woodruff JH (2004) Thalamic dysfunction in schizophrenia: neurochemical, neuropathological, and in vivo imaging abnormalities. Schizophrenia Research 69: 237-253. Costa E, Davis J, Pesold C, Tueting P, Guidotti A (2002) The heterozygote reeler mouse as a model for the development of a new generation of antipsychotics. Curr Opin Pharmacol 2: 56-62. Costa E, Davis J M , Dong E, Grayson DR, Guidotti A , Tremolizzo L , Veldic M (2004) A GABAergic cortical deficit dominates schizophrenia pathophysiology. Crit Rev Neurobiol 16: 1-23. Daenen EW, Van Der Heyden JA, Kruse CG, Wolterink G, Van Ree J M (2001) Adaptation and habituation to an open field and responses to various stressful events in animals with neonatal lesions in the amygdala or ventral hippocampus. Brain Res 918: 153-165. Daenen EW, Wolterink G, Gerrits M A , Van Ree J M (2002a) Amygdala or ventral hippocampal lesions at two early stages of life differentially affect open field behaviour later in life; an animal model of neurodevelopmental psychopathological disorders. Behav Brain Res 131: 67-78. Daenen EW, Wolterink G, Gerrits M A , Van Ree J M (2002b) The effects of neonatal lesions in the amygdala or ventral hippocampus on social behaviour later in life. Behav Brain Res 136: 571-582. Daenen EW, Wolterink G, Van Der Heyden JA, Kruse C G , Van Ree J M (2003a) Neonatal lesions in the amygdala or ventral hippocampus disrupt prepulse inhibition of the acoustic startle response; implications for an animal model of neurodevelopmental disorders like schizophrenia. Eur Neuropsychopharmacol 13: 187-197. Daenen EW, Wolterink G, Van Ree J M (2003b) Hyperresponsiveness to phencyclidine in animals lesioned in the amygdala on day 7 of life. Implications for an animal model of schizophrenia. Eur Neuropsychopharmacol 13: 273-279. Duncan E, Szilagyi S, Schwartz M , Kunzova A , Negi S, Efferen T, Peselow E, Chakravorty S, Stephanides M , Harmon J, Bugarski-Kirola D, Gonzenbach S, Rotrosen J (2003a) Prepulse inhibition of acoustic startle in subjects with schizophrenia treated with olanzapine or haloperidol. Psychiatry Res 120: 1-12. Duncan EJ, Szilagyi S, Efferen TR, Schwartz MP, Parwani A , Chakravorty S, Madonick SH, Kunzova A, Harmon JW, Angrist B , Gonzenbach S, Rotrosen JP (2003b) Effect of treatment status on prepulse inhibition of acoustic startle in schizophrenia. Psychopharmacology (Berl) 167: 63-71. Duncan GE, Moy SS, Perez A, Eddy D M , Zinzow W M , Lieberman JA, Snouwaert JN, Roller B H (2004) Deficits in sensorimotor gating and tests of social behavior in a genetic model of reduced N M D A receptor function. Behav Brain Res 153: 507-519. 26 Eastwood SL (2004) The synaptic pathology of schizophrenia: Is aberrant neurodevelopment and plasticity to blame? Disorders of Synaptic Plasticity and Schizophrenia 59: 47-72. Ellenbroek B A (2003) Animal models in the genomic era: possibilities and limitations with special emphasis on schizophrenia. Behav Pharmacol 14: 409-417. Ellenbroek B A , Cools A R (2000) The long-term effects of maternal deprivation depend on the genetic background. Neuropsychopharmacology 23: 99-106. Ellenbroek B A , van den Kroonenberg PT, Cools A R (1998) The effects of an early stressful life event on sensorimotor gating in adult rats. Schizophr Res 30: 251-260. Elvevag B, Goldberg TE (2000) Cognitive impairment in schizophrenia is the core of the disorder. Crit Rev Neurobiol 14: 1-21. Emsley R, Oosthuizen P (2003) The new and evolving pharmacotherapy of schizophrenia. Psychiatr Clin North Am 26: 141-163. Facchinetti F, Ciani E, Dallolio R, Virgili M , Contestabile A , Fonnum F (1993) Structural, Neurochemical and Behavioral Consequences of Neonatal Blockade of Nmda Receptor Through Chronic Treatment with Cgp-39551 Or Mk-801. Developmental Brain Research 74: 219-224. Famy C, Streissguth AP, Unis AS (1998) Mental illness in adults with fetal alcohol syndrome or fetal alcohol effects. Am J Psychiatry 155: 552-554. Flores G, Wood G K , Liang JJ, Quirion R, Srivastava L K (1996) Enhanced amphetamine sensitivity and increased expression of dopamine D2 receptors in postpubertal rats after neonatal excitotoxic lesions of the medial prefrontal cortex. J Neurosci 16: 7366-7375. Fox K, Schlaggar B L , Glazewski S, O'Leary DD (1996) Glutamate receptor blockade at cortical synapses disrupts development of thalamocortical and columnar organization in somatosensory cortex. Proc Nad Acad Sci U S A 93: 5584-5589. Fredriksson A, Archer T, Aim H , Gordh T, Eriksson P (2004) Neurofunctional deficits and potentiated apoptosis by neonatal N M D A antagonist administration. Behavioural Brain Research 153: 367-376. Gainetdinov RR, Mohn AR, Caron M G (2001) Genetic animal models: focus on schizophrenia. Trends Neurosci 24: 527-533. Geyer M A , Krebs-Thomson K, Braff DL, Swerdlow NR (2001) Pharmacological studies of prepulse inhibition models of sensorimotor gating deficits in schizophrenia: a decade in review. Psychopharmacology (Berl) 156: 117-154. 27 Geyer M A , Markou A (1995) Animal models of psychiatric disorders. In: Psychopharmacology: The Fourth Generation of Progress (Bloom FE, Kupfer DJ, eds), pp 787-798. New York: Raven Press, Ltd. Goff DC, Coyle JT (2001) The emerging role of glutamate in the pathophysiology and treatment of schizophrenia. Am J Psychiatry 158: 1367-1377. Gothelf D, Soreni N , Nachman RP, Tyano S, Hiss Y , Reiner O, Weizman A (2000) Evidence for the involvement of the hippocampus in the pathophysiology of schizophrenia. Eur Neuropsychopharmacol 10: 389-395. Gould E, Cameron HA, McEwen BS (1994) Blockade of N M D A receptors increases cell death and birth in the developing rat dentate gyrus. J Comp Neurol 340: 551-565. Grace A A (1991) Phasic versus tonic dopamine release and the modulation of dopamine system responsivity: a hypothesis for the etiology of schizophrenia. Neuroscience 41: 1-24. Hall FS (1998) Social deprivation of neonatal, adolescent, and adult rats has distinct neurochemical and behavioral consequences. Crit Rev Neurobiol 12: 129-162. Hamm A O , Weike A l , Schupp HT (2001) The effect of neuroleptic medication on prepulse inhibition in schizophrenia patients: current status and future issues. Psychopharmacology (Berl) 156: 259-265. Hanlon F M , Sutherland RJ (2000) Changes in adult brain and behavior caused by neonatal limbic damage: implications for the etiology of schizophrenia. Behav Brain Res 107:71-83. Harris L W , Sharp T, Gartlon J, Jones DNC, Harrison PJ (2003) Long-term behavioural, molecular and morphological effects of neonatal N M D A receptor antagonism. European Journal of Neuroscience 18: 1706-1710. Harrison PJ (1999) The neuropathology of schizophrenia. A critical review of the data and their interpretation. Brain 122 (Pt 4): 593-624. Heckers S (2001) Neuroimaging studies of the hippocampus in schizophrenia. Hippocampus 11: 520-528. ; Heckers S, Rauch SL, Goff D, Savage CR, Schacter DL , Fischman A J , Alpert N M (1998) Impaired recruitment of the hippocampus during conscious recollection in schizophrenia. Nat Neurosci 1: 318-323. Holt DJ, Weiss AP, Rauch SL, Wright CI, Zalesak M , Goff DC, Ditman T, Welsh RC, Heckers S (2005) Sustained activation of the hippocampus in response to fearful faces in schizophrenia. Biological Psychiatry 57: 1011-1019. 28 Honer WG, Bassett AS, Smith G N , Lapointe JS, Falkai P (1994) Temporal lobe abnormalities in multigenerational families with schizophrenia. Biol Psychiatry 36: 737-743. Hooks MS, Colvin A C , Juncos JL, Justice JB, Jr. (1992) Individual differences in basal and cocaine-stimulated extracellular dopamine in the nucleus accumbens using quantitative microdialysis. Brain Res 587: 306-312. Husum H , Termeer E, Mathe A A , Bolwig TG, Ellenbroek B A (2002) Early maternal deprivation alters hippocampal levels of neuropeptide Y and calcitonin-gene related peptide in adult rats. Neuropharmacology 42: 798-806. Ikonomidou C, Bosch F, Miksa M , Bittigau P, Vockler J, Dikranian K, Tenkova TI, Stefovska V , Turski L, Olney JW (1999) Blockade of N M D A receptors and apoptotic neurodegeneration in the developing brain. Science 283: 70-74. Impagnatiello F, Guidotti AR, Pesold C, Dwivedi Y , Caruncho H , Pisu M G , Uzunov DP, Smalheiser NR, Davis J M , Pandey G N , Pappas GD, Tueting P, Sharma RP, Costa E (1998) A decrease of reelin expression as a putative vulnerability factor in schizophrenia. Proc Natl Acad Sci U S A 95: 15718-15723. Jablensky A (2000) Epidemiology of schizophrenia: the global burden of disease and disability. Eur Arch Psychiatry Clin Neurosci 250: 274-285. Jaskiw GE, Karoum F, Freed WJ, Phillips I, Kleinman JE, Weinberger DR (1990a) Effect of ibotenic acid lesions of the medial prefrontal cortex on amphetamine-induced locomotion and regional brain catecholamine concentrations in the rat. Brain Res 534: 263-272. Jaskiw GE, Karoum FK, Weinberger DR (1990b) Persistent elevations in dopamine and its metabolites in the nucleus accumbens after mild subchronic stress in rats with ibotenic acid lesions of the medial prefrontal cortex. Brain Res 534: 321-323. Jentsch JD, Redmond DE, Jr., Elsworth JD, Taylor JR, Youngren K D , Roth R H (1997) Enduring cognitive deficits and cortical dopamine dysfunction in monkeys after long-term administration of phencyclidine. Science 277: 953-955. Jentsch JD, Roth R H (1999) The neuropsychopharmacology of phencyclidine: from N M D A receptor hypofunction to the dopamine hypothesis of schizophrenia. Neuropsychopharmacology 20: 201-225. Jones E G (1997) Cortical development and thalamic pathology in schizophrenia. Schizophr Bull 23: 483-501. Jones P, Rodgers B, Murray R, Marmot M (1994) Child development risk factors for adult schizophrenia in the British 1946 birth cohort. Lancet 344: 1398-1402. 29 Kanemoto K, Tsuji T, Kawasaki J (2001) Reexamination of interictal psychoses based on D S M IV psychosis classification and international epilepsy classification. Epilepsia 42: 98-103. Kapur S, Mamo D (2003) Half a century of antipsychotics and still a central role for dopamine D2 receptors. Prog Neuropsychopharmacol Biol Psychiatry 27: 1081-1090. Karper LP, Freeman GK, Grillon C, Morgan C A , UI, Charney DS, Krystal JH (1996) Preliminary evidence of an association between sensorimotor gating and distractibility in psychosis. J Neuropsychiatry Clin Neurosci 8: 60-66. Kelly PH, Seviour PW, Iversen SD (1975) Amphetamine and apomorphine responses in the rat following 6-OHDA lesions of the nucleus accumbens septi and corpus striatum. Brain Res 94: 507-522. Kennedy JL, Farrer L A , Andreasen NC, Mayeux R, George-Hyslop P (2003) The genetics of adult-onset neuropsychiatric disease: complexities and conundra? Science 302: 822-826. Kilts C D (2001) The changing roles and targets for animal models of schizophrenia. Biol Psychiatry 50: 845-855. Kumari V , Sharma T (2002) Effects of typical and atypical antipsychotics on prepulse inhibition in schizophrenia: a critical evaluation of current evidence and directions for future research. Psychopharmacology (Berl) 162: 97-101. Kumari V , Soni W, Sharma T (2002) Prepulse inhibition of the startle response in risperidone-treated patients: comparison with typical antipsychotics. Schizophr Res 55: 139-146. Lafleur, I, Marcotte, E. R., Quirion, R., and Srivastava, L. K. Increased adult locomotor activity following a single neonatal infusion of the N M D A antagonist AP-5 in the rat prefrontal cortex. Society for Neuroscience Abstracts , 876.10. 2001. Ref Type: Abstract Laruelle M , Abi-Dargham A, Gi l R, Kegeles L , Innis R (1999) Increased dopamine transmission in schizophrenia: relationship to illness phases. Biol Psychiatry 46: 56-72. Laruelle M , Kegeles LS, Abi-Dargham A (2003) Glutamate, dopamine, and schizophrenia: from pathophysiology to treatment. Ann N Y Acad Sci 1003: 138-158. Lawrie S M , Abukmeil SS (1998) Brain abnormality in schizophrenia. A systematic and quantitative review of volumetric magnetic resonance imaging studies. Br J Psychiatry 172: 110-120. Lawrie S M , Whalley H , Kestelman JN, Abukmeil SS, Byrne M , Hodges A , Rimmington JE, Best JJ, Owens DG, Johnstone EC (1999) Magnetic resonance imaging of brain in people at high risk of developing schizophrenia. Lancet 353: 30-33. 30 Le Pen G, Kew J, Alberati D, Borroni E, Heitz MP, Moreau JL (2003) Prepulse inhibition deficits of the startle reflex in neonatal ventral hippocampal-lesioned rats: reversal by glycine and a glycine transporter inhibitor. Biol Psychiatry 54: 1162-1170. Le Pen G, Moreau JL (2002) Disruption of prepulse inhibition of startle reflex in a neurodevelopmental model of schizophrenia: reversal by clozapine, olanzapine and risperidone but not by haloperidol. Neuropsychopharmacology 27: 1-11. Lehmann J, Pryce CR, Feldon J (2000) Lack of effect of an early stressful life event on sensorimotor gating in adult rats. Schizophr Res 41: 365-371. , Leumann L, Feldon J, Vollenweider F X , Ludewig K (2002) Effects of typical and atypical antipsychotics on prepulse inhibition and latent inhibition in chronic schizophrenia. Biol Psychiatry 52: 729-739. Lewis DA, Hashimoto T, Volk DW (2005) Cortical inhibitory neurons and schizophrenia. Nat Rev Neurosci 6: 312-324. Lewis D A , Levitt P (2002) Schizophrenia as a disorder of neurodevelopment. Annu Rev Neurosci 25: 409-432. Lewis R (2004) Should cognitive deficit be a diagnostic criterion for schizophrenia? Journal of Psychiatry & Neuroscience 29: 102-113. Lipska B K , Halim ND, Segal PN, Weinberger DR (2002) Effects of reversible inactivation of the neonatal ventral hippocampus on behavior in the adult rat. J Neurosci 22: 2835-2842. Lipska B K , Jaskiw GE, Chrapusta S, Karoum F, Weinberger DR (1992) Ibotenic acid lesion of the ventral hippocampus differentially affects dopamine and its metabolites in the nucleus accumbens and prefrontal cortex in the rat. Brain Res 585: 1-6. Lipska B K , Jaskiw GE, Karoum F, Phillips I, Kleinman JE, Weinberger DR (1991) Dorsal hippocampal lesion does not affect dopaminergic indices in the basal ganglia. Pharmacol Biochem Behav 40: 181-184. Lipska B K , Jaskiw GE, Weinberger DR (1993) Postpubertal emergence of hyperresponsiveness to stress and to amphetamine after neonatal excitotoxic hippocampal damage: a potential animal model of schizophrenia. Neuropsychopharmacology 9: 67-75. Lipska B K , Luu S, Halim ND, Weinberger DR (2003) Behavioral effects of neonatal and adult excitotoxic lesions of the mediodorsal thalamus in the adult rat. Behav Brain Res 141: 105-111. Lipska B K , Swerdlow NR, Geyer M A , Jaskiw GE, Braff DL, Weinberger DR (1995) Neonatal excitotoxic hippocampal damage in rats causes post-pubertal changes in prepulse inhibition of startle and its disruption by apomorphine. Psychopharmacology (Berl) 122: 35-43. 31 Lipska B K , Weinberger DR (1993) Delayed effects of neonatal hippocampal damage on haloperidol-induced catalepsy and apomorphine-induced stereotypic behaviors in the rat. Brain Res Dev Brain Res 75: 213-222. Lipska B K , Weinberger DR (1995) Genetic variation in vulnerability to the behavioral effects of neonatal hippocampal damage in rats. Proc Natl Acad Sci U S A 92: 8906-8910. Lipska B K , Weinberger DR (2000) To model a psychiatric disorder in animals: schizophrenia as a reality test. Neuropsychopharmacology 23: 223-239. Lomber SG (1999) The advantages and limitations of permanent or reversible deactivation techniques in the assessment of neural function. Journal of Neuroscience Methods 86: 109-117. Ludewig K, Geyer M A , Vollenweider F X (2003) Deficits in prepulse inhibition and habituation in never-medicated, first-episode schizophrenia. Biol Psychiatry 54: 121-128. Luthi A , Schwyzer L , Mateos J M , Gahwiler B H , McKinney R A (2001) N M D A receptor activation limits the number of synaptic connections during hippocampal development. Nat Neurosci 4: 1102-1107. Mackeprang T, Kristiansen KT, Glenthoj B Y (2002) Effects of antipsychotics on prepulse inhibition of the startle response in drug-naive schizophrenic patients. Biol Psychiatry 52: 863-873. Marcotte ER, Pearson D M , Srivastava L K (2001) Animal models of schizophrenia: a critical review. J Psychiatry Neurosci 26: 395-410. McCarley RW, Wible CG, Frumin M , Hirayasu Y , Levitt JJ, Fischer IA, Shenton M E (1999) MRI anatomy of schizophrenia. Biol Psychiatry 45: 1099-1119. McClure RK, Weinberger DR (2001) The neurodevelopmental hypothesis of schizophrenia: A review of the evidence. In: Current Issues in the Psychopharmacology of Schizophrenia. (Breier A , Tran PV, Herrea J M , Tollefson GD, Bymaster FP, eds), pp 27-56. New York: Lippincott Williams & Wilkins. McGowan S, Lawrence A D , Sales T, Quested D, Grasby P (2004) Presynaptic dopaminergic dysfunction in schizophrenia: a positron emission tomographic [18F]fluorodopa study. Arch Gen Psychiatry 61: 134-142. Meincke U , Morth D, Voss T, Thelen B , Geyer M A , Gouzoulis-Mayfrank E (2004) Prepulse inhibition of the acoustically evoked startle reflex in patients with an acute schizophrenic psychosis-a longitudinal study. Eur Arch Psychiatry Clin Neurosci 254: 415-421. Meltzer H Y , Lee M A , Ranjan R (1994) Recent advances in the pharmacotherapy of schizophrenia. Acta Psychiatr Scand Suppl 384: 95-101. 32 Meyer-Lindenberg A , Miletich RS, Kohn PD, Esposito G, Carson RE, Quarantelli M , Weinberger DR, Berman K F (2002) Reduced prefrontal activity predicts exaggerated striatal dopaminergic function in schizophrenia. Nat Neurosci 5: 267-271. Mitdeman G, Bratt A M , Chase R (1998) Heterogeneity of the hippocampus: effects of subfield lesions on locomotion elicited by dopaminergic agonists. Behav Brain Res 92: 31-45. Mohn AR, Gainetdinov RR, Caron M G , Roller B H (1999) Mice with reduced N M D A receptor expression display behaviors related to schizophrenia. Cell 98: 427-436. Morimoto T, Kida K, Nagao H , Yoshida K, Fukuda M , Takashima S (1995) The pathogenic role of the N M D A receptor in hyperthermia-induced seizures in developing rats. Brain Res Dev Brain Res 84: 204-207. Nelson M D , Saykin A J , Flashman L A , Riordan HJ (1998) Hippocampal volume reduction in schizophrenia as assessed by magnetic resonance imaging: a meta-analytic study. Arch Gen Psychiatry 55: 433-440. Noga JT, Bartley A J , Jones DW, Torrey EF, Weinberger DR (1996) Cortical gyral anatomy and gross brain dimensions in monozygotic twins discordant for schizophrenia. Schizophr Res 22: 27-40. Olney JW (2004) Fetal alcohol syndrome at the cellular level. Addict Biol 9: 137-149. Olney JW, Newcomer JW, Farber NB (1999) N M D A receptor hypofunction model of schizophrenia. J Psychiatr Res 33: 523-533. Owen MJ , Williams N M , O'Donovan M C (2004) The molecular genetics of schizophrenia: new findings promise new insights. Mo l Psychiatry 9: 14-27. Pakkenberg B (1990) Pronounced reduction of total neuron number in mediodorsal thalamic nucleus and nucleus accumbens in schizophrenics. Arch Gen Psychiatry 47: 1023-1028. Perry W, Braff D L (1994) Information-processing deficits and thought disorder in schizophrenia. Am J Psychiatry 151: 363-367. Perry W, Geyer M A , Braff D L (1999) Sensorimotor gating and thought disturbance measured in close temporal proximity in schizophrenic patients. Arch Gen Psychiatry 56: 277-281. Porrino LJ , Lucignani G, Dow-Edwards D, Sokoloff L (1984) Correlation of dose-dependent effects of acute amphetamine administration on behavior and local cerebral metabolism in rats. Brain Res 307: 311-320. 33 Pouzet B , Feldon J, Veenman CL, Yee B K , Richmond M , Nicholas J, Rawlins P, Weiner I (1999) The effects of hippocampal and fimbria-fornix lesions on prepulse inhibition. Behav Neurosci 113: 968-981. Rabacchi S, Bailly Y , Delhaye-Bouchaud N , Mariani J (1992) Involvement of the N -methyl D-aspartate (NMDA) receptor in synapse elimination during cerebellar development. Science 256: 1823-1825. Rapoport JL, Addington A M , Frangou S, Psych M (2005) The neurodevelopmental model of schizophrenia: update 2005. Molecular Psychiatry 10: 434-449. Rapoport JL, Giedd JN, Blumenthal J, Hamburger S, Jeffries N , Fernandez T, Nicolson R, Bedwell J, Lenane M , Zijdenbos A , Paus T, Evans A (1999) Progressive cortical change during adolescence in childhood-onset schizophrenia. A longitudinal magnetic resonance imaging study. Arch Gen Psychiatry 56: 649-654. Robbins TW (2004) Animal models of the psychoses. In: Neurobiology of Mental Illness, 2nd. Ed. (Charney DS, Nestler EJ, eds), pp 263-286. New York: Oxford University Press. Roberts GW, Colter N , Lofthouse R, Bogerts B , Zech M , Crow TJ (1986) Gliosis in schizophrenia: a survey. Biol Psychiatry 21: 1043-1050. Roth B L , Hanizavareh S M , Blum A E (2004) Serotonin receptors represent highly favorable molecular targets for cognitive enhancement in schizophrenia and other disorders. Psychopharmacology 174: 17-24. Sams-Dodd F, Lipska B K , Weinberger DR (1997) Neonatal lesions of the rat ventral hippocampus result in hyperlocomotion and deficits in social behaviour in adulthood. Psychopharmacology (Berl) 132: 303-310. Schneider M , Koch M (2005) Behavioral and morphological alterations following neonatal excitotoxic lesions of the medial prefrontal cortex in rats. Exp Neurol. Schoenfeld TA, Hamilton L W (1977) Secondary Brain Changes Following Lesions -New Paradigm for Lesion Experimentation. Physiology & Behavior 18: 951-967. Seidman LJ , Faraone SV, Goldstein J M , Kremen WS, Horton NJ, Makris N , Toomey R, Kennedy D, Caviness VS, Tsuang M T (2002) Left hippocampal volume as a vulnerability indicator for schizophrenia: a magnetic resonance imaging morphometric study of nonpsychotic first-degree relatives. Arch Gen Psychiatry 59: 839-849. Sharma T, Antonova L (2003) Cognitive function in schizophrenia - Deficits, functional consequences, and future treatment. Psychiatric Clinics of North America 26: 25-+. Snyder SH (1973) Amphetamine psychosis: a "model" schizophrenia mediated by catecholamines. A m J Psychiatry 130: 61-67. 34 Stein D G (1979) The Ghost in the Machine Is Still There. Behavioral and Brain Sciences 2: 346-348. Stevens JR (1997) Anatomy of schizophrenia revisited. Schizophr Bull 23: 373-383. Suddarh RL, Christison GW, Torrey EF, Casanova MF, Weinberger DR (1990) Anatomical abnormalities in the brains of monozygotic twins discordant for schizophrenia. N Engl J Med 322: 789-794. Swerdlow NR, Braff DL, Geyer M A (2000a) Animal models of deficient sensorimotor gating: what we know, what we think we know, and what we hope to know soon. Behav Pharmacol 11: 185-204. Swerdlow NR, Geyer M A , Braff D L (2001) Neural circuit regulation of prepulse inhibition of startle in the rat: current knowledge and future challenges. Psychopharmacology (Berl) 156: 194-215. Swerdlow NR, Lipska B K , Weinberger DR, Braff DL , Jaskiw GE, Geyer M A (1995) Increased sensitivity to the sensorimotor gating-disruptive effects of apomorphine after lesions of medial prefrontal cortex or ventral hippocampus in adult rats. Psychopharmacology (Berl) 122: 27-34. Swerdlow NR, Taaid N , Halim N , Randolph E, Kim Y K , Auerbach P (2000b) Hippocampal lesions enhance startle gating-disruptive effects of apomorphine in rats: a parametric assessment. Neuroscience 96: 523-536. Tenn CC, Kapur S, Fletcher PJ (2005) Sensitization to amphetamine, but not phencyclidine, disrupts prepulse inhibition and latent inhibition. Psychopharmacology (Berl) 180: 366-376. Vallano M L (1998) Developmental aspects of N M D A receptor function. Crit Rev Neurobiol 12: 177-204. Van den B M , Garner B , Koch M (2003) Neurodevelopmental animal models of schizophrenia: effects on prepulse inhibition. Curr Mol Med 3: 459-471. Varty G B , Braff DL, Geyer M A (1999) Is there a critical developmental 'window' for isolation rearing-induced changes in prepulse inhibition of the acoustic startle response? Behav Brain Res 100: 177-183. Varty G B , Higgins G A (1995) Examination of drug-induced and isolation-induced disruptions of prepulse inhibition as models to screen antipsychotic drugs. Psychopharmacology (Berl) 122: 15-26. Vestergaard M , Pedersen CB, Christensen J, Madsen K M , Olsen J, Mortensen PB (2005) Febrile seizures and risk of schizophrenia. Schizophr Res 73: 343-349. 35 Walker EF (1994) Developmentally moderated expressions of the neuropathology underlying schizophrenia. Schizophr Bull 20: 453-480. Walker EF, Savoie T, Davis D (1994) Neuromotor precursors of schizophrenia. Schizophr Bull 20: 441-451. Wang C, Mclnnis J, Ross-Sanchez M , Shinnick-Gallagher P, Wiley JL, Johnson K M (2001) Long-term behavioral and neurodegenerative effects of perinatal phencyclidine administration: Implications for schizophrenia. Neuroscience 107: 535-550. Weinberger DR (1987) Implications of normal brain development for the pathogenesis of schizophrenia. Arch Gen Psychiatry 44: 660-669. Weinberger DR (1996) On the plausibility of "the neurodevelopmental hypothesis" of schizophrenia. Neuropsychopharmacology 14: 1S-11S. Weiss IC, Feldon J (2001) Environmental animal models for sensorimotor gating deficiencies in schizophrenia: a review. Psychopharmacology (Berl) 156: 305-326. Wong A H , Van Tol H H (2003) Schizophrenia: from phenomenology to neurobiology. Neurosci Biobehav Rev 27: 269-306. Wood GK, Lipska B K , Weinberger DR (1997) Behavioral changes in rats with early ventral hippocampal damage vary with age at damage. Brain Res Dev Brain Res 101: 17-25. Wright IC, Ellison ZR, Sharma T, Friston KJ , Murray R M , McGuire P K (1999) Mapping of grey matter changes in schizophrenia. Schizophr Res 35: 1-14. Wright IC, Rabe-Hesketh S, Woodruff PWR, David AS, Murray R M , Bullmore ET (2000) Meta-analysis of regional brain volumes in schizophrenia. American Journal of Psychiatry 157: 16-25. Wynn JK, Sergi MJ , Dawson M E , Schell A M , Green M F (2005) Sensorimotor gating, orienting and social perception in schizophrenia. Schizophr Res 73: 319-325. Zhang W N , Bast T, Feldon J (2002) Prepulse inhibition in rats with temporary inhibition/inactivation of ventral or dorsal hippocampus. Pharmacol Biochem Behav 73: 929-940. Zornberg GL, Buka SL, Tsuang M T (2000) Hypoxic-ischemia-related fetal/neonatal complications and risk of schizophrenia and other nonaffective psychoses: A 19-year longitudinal study. American Journal of Psychiatry 157: 196-202. 36 CHAPTER TWO: ELECTRICAL STIMULATION OF THE HIPPOCAMPUS DISRUPTS PREPULSE INHIBITION IN RATS: FREQUENCY AND SITE DEPENDENT EFFECTS. 1 Introduction Prepulse inhibition (PPI) is the normal reduction in an acoustic startle response produced when a brief, quieter stimulus or prepulse is presented 30-500 ms prior to presentation of the starde-evoking stimulus (Koch, 1999;Swerdlow et al., 2000a). PPI provides a measure of sensorimotor gating, the inhibitory process by which responses to sensory stimuli are suppressed, thereby facilitating appropriate responding in complex, sensory rich environments (Swerdlow, 1996). Interest in the neural mechanisms underlying PPI has increased because it is often disrupted in humans diagnosed with a variety of psychiatric disorders including schizophrenia (Weike et al., 2000;Braff et al., 2001). As similar stimuli elicit PPI in rodents and humans, interventions in rodents that disrupt PPI may reveal aspects of neuronal dysfunction that underlie disrupted sensorimotor gating in schizophrenic patients (Swerdlow, 1996;Weike et al., 2000). Similarities exist in the neuroanatomical and neurochemical substrates related to both schizophrenia and PPI. A large body of research suggests that schizophrenic patients show abnormalities in cortico-limbic-striatal circuitry, in areas such as the frontal cortex, nucleus accumbens (NAc), and hippocampus (Pakkenberg, 1990;Bogerts, 1997;Harrison, 1999;Wright et al., 1999) likely involving alterations in dopamine (DA) and glutamate (Glu) transmission (Breier et al., 1997;Carlsson et al., 1999;Laruelle et al., 1999;Goff and Coyle, 2001;Meyer-Lindenberg et al., 2002). Consistent with these 1 A version of the chapter has been published: Howland JG, Mackenzie E M , Y i m TT, Taepavarapruk P, Phillips A G (2004). Electrical stimulation of the hippocampus disrupts prepulse inhibition in rats: frequency and site dependent effects. Behavioural Brain Research 152:187-197. 37 findings is evidence indicating that the regulation of PPI is also dependent on a complex array of structures commonly termed the cortico-striatal-pallidal-pontine (CSPP) circuitry (see Swerdlow et al., 2001a for review), including the medial prefrontal cortex (mPFC) (Koch and Bubser, 1994;Lacroix et al., 2000), amygdala (Wan and Swerdlow, 1997;Fendt et al., 2000), NAc (Swerdlow et al.,1986;1990;1994;Wan and Swerdlow, 1996), and hippocampus (Caine et al., 1992;Swerdlow et al., 1995;2001b;Wan et al., 1996;Bakshi and Geyer, 1998;Klarner et a l , 1998;Zhang et al., 1999;2002a;Bast et al., 2001;Caine et al., 2001). Importantly, the systemic administration of D A or D A agonists (Mansbach et a l , 1988;Swerdlow et al., 1990;Zhang et al., 2000a) and N-Methyl-D-Aspartate (NMDA) antagonists (Mansbach and Geyer, 1989;Bakshi and Geyer, 1998), likely acting in areas of the CSPP circuitry, also disrupt PPI in animals (Geyer et al., 2001). Recently, a complex role of NAc D A and Glu via D2, N M D A , and (±)-a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) receptors in the modulation of PPI has been described (Mansbach et al., 1988;Swerdlow et a l , 1990;Wan and Swerdlow, 1993;1996;Reijmers et al., 1995;Wan et al., 1995). Hippocampal dysfunction has been linked to symptoms of schizophrenia (Silbersweig et al., 1995;Heckers et al., 1998;Harrison, 1999;Dierks et al., 1999;Grace, 2000), and consequently the roles of distinct hippocampal subregions in the modulation of PPI have been examined extensively. Although permanent lesions of the ventral hippocampus (vHip) generally do not disrupt PPI (Swerdlow et al., 1995;2000b;Pouzet et al., 1999 but see also (Caine et al., 2001), temporary inactivation of vHip or dorsal hippocampal (dHip) with either the G A B A A agonist muscimol or the sodium channel blocker tetrodotoxin disrupts PPI (Zhang et al., 2002b). Debate also exists as to whether 38 infusion of glutamatergic antagonists, such as MK801, into the dHip, but not the vHip, disrupts PPI (Bakshi and Geyer, 1998;1999;Bast et al., 2000;Zhang et al., 2000b). Nevertheless, compelling evidence exists for the disruption of PPI following chemical stimulation of the vHip (Wan et al., 1996;Klarner et al., 1998;Zhang et al., 1999;Bast et al., 2001;Swerdlow et al., 2001b;Zhang et al., 2002a), but not dHip (Swerdlow et al., 2001b;Zhang et al., 2002a) with infusions of the ionotropic Glu agonist N M D A . As these studies suggest, various manipulations of the vHip and dHip can impair sensorimotor gating behavior, and regional differences clearly exist. Both the vHip and dHip are well positioned neuroanatomically to interact with the CSPP circuitry to modulate PPI. The vHip projects densely to the NAc shell, amygdala, and mPFC (Kelley and Domesick, 1982;Groenewegen et al., 1987;Brog et al., 1993;Conde et al., 1995) whereas the dHip projects to the NAc core (Swanson and Cowan, 1977;Whitter, 1986), and also communicates with the vHip via intra-hippocampal connections (Amaral and Whitter, 1995). At the level of the ventral striatum, projections from the vHip exert powerful control over D A efflux in the NAc shell and firing of NAc medium spiny projection neurons (O'Donnell and Grace, 1995;Taepavarapruk et al., 2000;Floresco et al., 2001). More specifically, stimulation of the vHip with N M D A (Brudzynski and Gibson, 1997;Legault et al., 2000) or brief trains of higher frequency electrical current (20 Hz, 10 s) (Blaha et al., 1997;Taepavarapruk et al., 2000) cause a long-lasting increase in NAc D A efflux and an increase in locomotor activity. Activation of ionotropic Glu receptors (iGluR's) in the NAc underlies the changes in NAc D A efflux observed following electrical stimulation of the vHip (Blaha et al., 1997;Taepavarapruk et al., 2000). In contrast, preliminary studies performed in our 39 laboratory demonstrate that lower frequency (2 Hz) stimulation of the vHip for 100 s can significantly reduce NAc D A efflux for at least 2 hr, an effect that may result from the activation of intra-NAc group 2 and 3 metabotropic Glu receptors (Taepavarapruk et al., 1998). The consequences of stimulating the dHip on NAc D A efflux have been poorly characterized, although stimulation of the previously kindled dHip results in a brief increase in NAc D A efflux (Strecker and Moneta, 1994). In the following experiments, the effects of electrical stimulation of the Hip on PPI were examined. Electrical stimulation was chosen because it has a number of distinct advantages over chemical stimulation, including precise control over the duration and intensity of the stimulation. Additionally, electrical stimulation parameters can approximate the physiological firing activity of the neurons that are stimulated. Given that intra-vHip N M D A infusions and 20 Hz electrical stimulation of the vHip produce similar increases in NAc D A efflux (Brudzynski and Gibson, 1997;Legault et al., 2000;Taepavarapruk et al., 2000), and that vHip N M D A infusions disrupt PPI (Wan et al., 1996;Klarner et a l , 1998;Zhang et a l , 1999;2002a;Bast et al., 2001;Swerdlow et al., 2001b), the first objective of the present study was to assess whether 20 Hz stimulation of the vHip would induce a reversible disruption of PPI. In a complementary experiment, we examined the effect of 20 Hz electrical stimulation to the dHip on PPI, hypothesizing that if dHip N M D A infusions fail to disrupt PPI (Swerdlow et al., 2001b;Zhang et a l , 2002a), then electrical stimulation at this frequency should also fail to reduce PPI. The final behavioral experiment examined the effect of 2 Hz vHip stimulation on PPI. Additional microdialysis experiments addressed two issues related to the effects of 20 Hz stimulation of the hippocampus. The first assessed whether unilateral vHip stimulation 40 would result in an increase in D A efflux bilaterally in the NAc. Secondly, as the effects of 20 Hz dHip stimulation on NAc D A are unknown, a similar microdialysis experiment assessed the effects of these stimulation parameters on NAc D A efflux. Male Long-Evans (LE) rats were used in all experiments in an effort to maintain consistency between the neurochemical data collected previously and the present behavioral experiments. Methods Subjects Male L E rats (Charles River Canada, St. Constant, Quebec, Canada) were pah-housed in Plexiglas cages and handled daily until surgery. The colony was kept on a 12/12 hour light/dark cycle (lights on at 0700), at a temperature of 22±1°C. Rats were given food (Purina Rat Chow) and tap water ad libitum. Experiments were conducted in accordance with the standards of the Canadian Council on Animal Care and the Committee on Animal Care at the University of British Columbia approved all procedures. Surgery At the time of surgery, subjects weighed 330 to 370 g. Rats were anaesthetized with ketamine hydrochloride (100 mg/kg, i.p., M T C Pharmaceuticals) and xylazine (10 mg/kg, i.p., Rompun), and placed in a stereotaxic frame. The dorsal surface of the skull was exposed, and holes were drilled. A bipolar stimulating electrode (Plastics One, Roanoke, V A ) was implanted into either the vHip (AP -5.8 mm from bregma, M L ±5.5 mm from midline, D V -6.0 mm from dura) or dHip (AP -3.0 mm, M L ±1.5 mm, D V -3.0 mm) of all rats. Guide cannulae were also implanted dorsal to the NAc in those rats used in the microdialysis experiments (AP +1.7 mm, M L ±1.1 mm, D V -1.0 mm). Cannulae and the electrode were secured to the skull with four jeweler's screws and dental acrylic. Wire obdurators were inserted into the cannula to keep them patent. Animals were allowed at least 7 days to recover from surgery before testing began, and were regularly handled beginning three days following surgery. Prepulse Inhibition Testing Testing was conducted in a single sound-attenuating startle chamber (ambient noise level 62 dB), containing a transparent Plexiglas tube (8.2 cm in diameter, 20 cm in length), mounted on a Plexiglas frame (SR-LAB, San Diego Instruments, San Diego, CA). Noise bursts were presented through a speaker mounted 24 cm above the tube. An accelerometer below the frame of the apparatus measured whole body startle amplitude, defined as the average of 100 1 ms accelerometer readings collected beginning at stimulus onset. Each PPI test session began with a 5 min acclimatization period during which a 70 dB background noise level was presented, which remained constant for the entire test session. Following the acclimatization period, six pulse alone trials (120 dB, 40 ms) were presented to achieve a relatively stable level of startle amplitude before presentation of the prepulse + pulse trials. The data from these pulse alone trials was not considered in the analysis of PPI. Immediately following the six initial pulse alone trials, presentation of the trials that were used in the calculation of PPI levels began. A total of 84 trials of five different types were presented. Trials presented were of three types: pulse alone (12 trials; 120 dB, 40 ms), prepulse + pulse (10 trials X 3 prepulse intensities - discussed below), or no stimulus (42 trials). Prepulse + pulse trials consisted of the presentation of a 20 ms prepulse of 73, 76, or 82 dB 80 ms before the presentation of the pulse. The pulse and prepulse + pulse trials were presented in a pseudorandom order. 42 One no stimulus trial was presented between each pair of pulse and prepulse + pulse trials. The inter-trial interval varied randomly from 3 to 12 s (average 7.5 s). The total length of each PPI session was approximately 18 min. Calibration of the apparatus was performed using a RadioShack Digital Sound Level Meter and adjustments were made as necessary. A within-subjects design was used in all PPI experiments. Experiments were conducted in two successive weeks; all animals were tested four times during these two weeks. The four PPI sessions were referred to as follows. The 'stim' PPI session occurred 2 min following electrical stimulation of either the vHip or dHip, 'stim+48' was the session 48 hr following stimulation, 'control' was the PPI session 2 min following exposure to the stimulation box without stimulation, and 'control+48' followed the 'control' session by 48 hrs. Animals were randomly assigned to one of two sequences to control for potential order effects. Rats in sequence one received their PPI sessions in the following order: stim, stim+48, control, and control+48. Rats in sequence two received their PPI sessions in the control, control+48, stim, and stim+48 order. Following recovery from surgery, animals were habituated to the stimulation environment on the two days immediately preceding their first PPI test. Each rat was removed from the colony and taken to the stimulation room. This small room was immediately adjacent to the room used for PPI testing, and contained two transparent Plexiglas chambers (32 cm X 32 cm X 41 cm) used for stimulation. In the stimulation room, each rat was connected to a stimulation lead and placed in a Plexiglas chamber for 10 min, following which they were returned to the colony. On the day of their first PPI test, the animals were treated exactly as on the two previous days except that after 10 min 43 in the stimulation box, electrical stimulation (20 Hz: 200 cathodal, constant current pulses, 300 uA, 20 Hz, pulse width 0.5 ms; 2 Hz: identical except frequency of pulses was 2 Hz) was applied to half the rats, while the other half were given no current. Stimulation was delivered through an isolator (Iso-flex, A.M.P.I , Israel) via a Master-8 stimulator (A.M.P.I.). The experimenter observed the animals for 90 sec following stimulation, disconnected them, and quickly placed them in the PPI chamber. This procedure took a total of 2 min. Immediately following the PPI session, the animals were returned to the colony. A l l rats were then tested for PPI 48 hours later. For this test, the rats were removed from the colony and immediately placed in the PPI apparatus. Following the second PPI session, the animals were handled daily for five days. On the sixth day following the first PPI test session, all rats were given one habituation session in the stimulation chamber for 10 min. The following day, the PPI testing procedure described above was repeated, with control animals receiving electrical stimulation and previously stimulated animals receiving no current. Forty-eight hours later, all rats received their last PPI session. Neurochemical Experiments The microdialysis procedure commonly employed in our laboratory has been described in detail elsewhere (Taepavarapruk et a l , 2000;Howland et al., 2002). Briefly, concentric-style microdialysis probes (2 mm of exposed membrane) were constructed in our laboratory. Probes were attached to gas-tight syringes via a liquid swivel containing perfusion medium (147 mM NaCl, 3.0 mM KC1,1.3 mM CaCl 2 .H 2 0, 1.0 mM MgCl 2 .6H 2 0, 0.01 sodium phosphate buffer; pH 7.3-7.4) and flushed for 10 to 20 min using a syringe pump. Probes were then secured in a copper collar and inserted into the 44 NAc (7.8 mm ventral to dura) and the rats were allowed to move freely in a Plexiglas box (32 cm x 32 cm x 41 cm high) with access to food and water for 12 to 18 hours before experimental testing began the following morning. The probes were continuously perfused at 1 pL/min overnight. Probes were inserted bilaterally for the vHip stimulation experiment and unilaterally ipsilateral to the electrode for the dHip stimulation experiments. Two high-performance liquid chromatography systems with electrochemical detection (HPLC-ED) consisting of an ESA 582 pump (ESA Inc., Bedford, M A ) , Rheodyne Inert manual injector (Rheodyne, Rohnert Park, CA), an Ultrasphere column (Beckmann, Fullerton, CA. ; ODS 5 pm, 15 cm x 4.6 mm), an ESA 5011 analytical cell, and a Coulochem II EC detector (ESA Inc.) were used to quantify D A levels in all experiments. The working potentials were: +450 mV (electrode 1), -300 mV (electrode 2), and +450 mV (guard cell). The mobile phase (pH 3.5) consisted of 6 g/L sodium acetate, 10 mg/L ethylenediaminetetra-acetic acid (EDTA), 150 mg/L octyl sulfate (adjustable), 35 ml/L glacial acetic acid and 865 mL M i l l i Q purified water. Chromatograms were registered on a dual-pen chart recorder (Kipp and Zonen, Bohemia, NY) . A l l samples were injected immediately after collection and D A peak heights were measured manually. Dialysate samples were collected at 10 min intervals throughout the experiment. Once four baseline samples were collected that did not differ by more than ± 10%, the rats received 20 Hz electrical stimulation of either the vHip or dHip (same parameters described for the PPI experiments). Stimulation was timed to ensure that the next dialysis sample reflected only "stimulation-evoked" changes in D A efflux. The 45 experimenter remained in the testing room following the stimulation to record any behavioral effects of the stimulation. Nine samples were collected following stimulation, after which the animals were disconnected and sacrificed. Histology Upon completion of each experiment, animals were sacrificed with an overdose of pentobarbital, and perfused transcardially with 0.9% saline followed by 10% formaldehyde. Brains were stored in 10% sucrose in 10% formaldehyde for at least 1 week, after which they were sectioned (50 pm) using a cryostat and stained with cresyl violet. Placements of the electrodes and probes were confirmed under a light microscope with the assistance of a rat brain atlas (Paxinos and Watson, 1997). Data Analysis Data were analysed using repeated measures analyses of variance with the aid of SPSS (version 10.0). A l l post-hoc tests were performed separately. Variance is indicated on all graphs with the standard error of the mean (SEM). Significance levels for all statistical tests were set at 0.05. For the PPI experiments, two measures were calculated for each animal. The startle amplitude represented the mean startle amplitude of the 12 pulse alone trials presented after the six habituation trials. For calculation of PPI, startle amplitudes were averaged for each trial type. The percent PPI for each prepulse intensity was calculated using the formula: [100 - (100 X startle amplitude on prepulse + pulse trials) (startle amplitude on pulse alone trials)]. Two-way (prepulse intensity, test session as factors) repeated measures A N O V A s were performed on the data obtained from each stimulation site. As the prepulse intensity factor did not significantly interact with any of the other 46 factors, percent PPI data was averaged across the three prepulse intensities for each rat, thereby creating a global PPI score (Wan et al., 1995;Bakshi and Geyer, 1998;1999). Both startle amplitude and percent PPI were then analyzed using separate one-way A N O V A s with condition as a repeated measures factor. The repeated measures assumption of sphericity was met in all cases. Post-hoc analyses were performed using the Neuman-Keul's test where appropriate. Following the microdialysis experiments, baseline D A levels for each rat were calculated by averaging the four baseline samples. In all figures, D A levels are expressed as a percentage change from baseline (baseline is expressed as 0%). One-way repeated measures analyses of variance with time as a within-subjects factor were performed on all data and Dunnett's post-hoc tests were performed where appropriate. The baseline sample taken immediately before the first stimulation was used as the critical value during computation of the Dunnett's post-hoc tests. Results Immediate Behavioral Effects of Stimulation During the 2 min that the rats remained in the stimulation apparatus after stimulation was applied, a variety of behavioral effects were noted. The rats were generally awake and alert before administration of stimulation. At the onset of stimulation, behavioral arrest was observed in all animals. Higher frequency (20 Hz) stimulation of the vHip resulted in 5 to 20 wet dog shakes (WDS) that were restricted to 45 s following initiation of the stimulation in 20 of the 21 animals (rats used in the dialysis studies are included in this description). Additionally, all rats engaged in vigorous forward locomotor behavior following cessation of the 20 Hz stimulation, as has 47 been previously quantified (Taepavarapruk et al., 2000). Stimulation of the dHip resulted in an initial period of WDS activity in 4 of the 28 animals. A l l rats displayed a period of increased activity and rearing within 30 sec of the cessation of stimulation. Between 70 and 100 s following the cessation of stimulation, 18 of the 28 rats had a second bout of WDS activity. One to 20 WDS were displayed during this period. Two Hz stimulation of the vHip caused an initial period of behavioral arrest followed by behavioral activation manifested by grooming and some rearing. Wet dog shakes were observed in 3 of 15 animals during application of the 2 Hz stimulation. Differential Effects of 20 Hz Stimulation of the vHip or dHip on PPI and Startle Amplitude Twenty Hz stimulation of the vHip (n=14) resulted in a significant and reversible disruption of PPI (Fig. 2-1 A). A one-way repeated measures A N O V A performed on the PPI data for the vHip stimulation group revealed a significant main effect for the test condition factor (F(3, 39) = 9.32, p< 0.001). Post-hoc analyses revealed that percent PPI levels were significantly lower in the stim session (20.19 ± 6 %) than in the stim+48 (41.73 ± 4%), control (42.39 ± 4 %), or control+48 (44.47 ± 4 %) sessions. This effect was replicated with rats tested previously with 2 Hz stimulation (data discussed in section 3.3 below). In contrast, 20 Hz stimulation of the dHip (n=13) failed to induce any significant changes in percent PPI (Fig. 2-1C; F(3, 36) = 2.01, N.S.). Percent PPI levels were similar in the stim (39.40 ± 3%), control (46.94 ± 5 %), stim+48 (51.40 ± 4 %), and control+48 (48.67 ± 5 %) sessions. 48 startle stim control stim control +48 +48 Condition stim control stim control +48 +48 Condition dHip - startle stim control stim: control +48 +48 Condition stim control stim control +48 +48 Condition Figure 2-1. Effects of 20 Hz stimulation of the hippocampus on PPI and acoustic startle amplitude. A, Percent PPI scores 2 min (stim) or 48 hr (stim+48) following 20 Hz stimulation (10 s) of the vHip (n=14), and respective control PPI scores (control and control+48). The asterisk denotes a significant reduction in PPI in the stim group when compared to all other groups (p<0.05). B, Average startle amplitudes for those rats included in A. C, The effects of 20 Hz stimulation of the dHip (n=13) on percent PPI. Identification of the groups is the same as that described for A. D, Average startle amplitudes following 20 Hz dHip stimulation (n=13). The cross denotes a significant difference between the stim+48 and control groups. In all panels, error bars represent SEM. 49 Presentation of the auditory pulse alone induced robust startle in all rats (Fig. 2-1B, D). A one-way repeated measure A N O V A revealed that average startle amplitudes for the 4 PPI test sessions of the vHip 20 Hz stimulation group did not differ significantly (F(3, 39) = 1.40, N.S.). Thus, stimulation of the vHip did not significantly affect startle amplitude (average startle amplitude 116.61 ± 20 arbitrary startle units). Although stimulation of the dHip did not alter percent PPI levels (Fig. 2-1C), a significant main effect of test session was observed when startle amplitude was examined (Fig. 2-1D, F(3, 36) = 3.37, p < 0.03). Post-hoc analyses revealed that when tested in the stim+48 condition, rats had significantly lower startle amplitudes (74.24 ± 10) than when tested in the control condition (101.30 ± 13). Although a similar trend was noted between the stim and stim+48 test sessions, it was not significant. Effects of 2 Hz Stimulation of the vHip on PPI and Startle Amplitude Two hertz stimulation of the vHip (n=15) did not significantly alter PPI levels (Fig. 2-2A, F(3, 42) = 0.34, N.S.). When tested in the stim (46.93 ± 4 %), control (42.35 ± 4 %), stim+48 (43.03 ± 4 %), and control+48 (43.52 ± 3 %) conditions, the 2 Hz group demonstrated similar levels of PPI. In order to verify that 20 Hz stimulation would disrupt PPI in this group of rats, one week following their last PPI session, nine of the rats received 20 Hz stimulation of the vHip, and were tested for PPI 2 min and 24 hr later. Results revealed that 2 min following stimulation, PPI scores were significantly lower (30.01 ± 6 %) than 24 hr following stimulation (46.96 ± 4 %, F ( l , 8) = 5.88, p < 0.05, data not shown). These data confirmed the short-term disruptive effect of 20 Hz stimulation of the vHip on PPI previously described. Stimulation (2 Hz) of the vHip produced an unexpected significant increase in 50 A v H i p - P P I B stim control stim control +48 +48 Condition 300, vHip - startle stim control stim control: +48 +48 Condition Figure 2-2. Effects of 2 Hz stimulation of the vHip on PPI and acoustic startle amplitude. A, Percent PPI scores resulting from 2 Hz stimulation (100 s) of the vHip (n=15) either 2 min (stim) or 48 hr (stim+48) prior to testing, and respective control PPI scores (control and control+48). Control testing sessions were run exactly the same as 'stim' sessions; however, no stimulation was applied to the vHip. B, Amplitude of the startle response during the same test conditions depicted in panel A. The cross denotes a significant difference between the stim condition and the stim+48 and control+48 groups (p<0.05). Error bars represent SEM. 51 startle amplitude (Fig. 2-2B, F(3, 42) = 9.04, p < 0.001). Post-hoc analyses revealed that following 2 Hz stimulation, startle amplitudes were significantly higher (213.68 ± 29) than during the stim+48 (137.04 ± 15) or control+48 (134.71 ± 19) test sessions. Effects of Stimulation of the vHip and dHip on NAc DA Efflux As depicted in Fig. 2-3, baseline levels of D A in the NAc were relatively stable in all groups prior to stimulation. Stimulation of the vHip (20 Hz, n=7) induced a robust, long-lasting 35.41 ±4% increase in D A efflux in the NAc that was restricted to the hemisphere ipsilateral to the stimulating electrode. Separate one-way repeated measures A N O V A ' s confirmed that the increase in D A efflux in the ipsilateral hemisphere was significantly greater than baseline for 20 min (F(12, 72) = 4.04, p < 0.001, Dunnett's p < 0.05), whereas no significant changes in D A efflux were observed in the contralateral hemisphere following vHip stimulation (F(12, 72) = 1.23, N.S.). Stimulation of the dHip (n=7) failed to evoke an increase in D A efflux in the NAc (F(12, 72) = 1.52, N.S.). Histology The locations of representative stimulation electrodes and the active tips of microdialysis probes are depicted in Fig. 2-4A-C. Ventral hippocampal placements (Fig. 4A) were similar to those previously reported in the CAl/ventral subiculum (Wan et al., 1996;Blaha et al., 1997;Klarner et a l , 1998;Zhang et al., 1999;2002a;Taepavarapruk et a l , 2000;Bast et al., 2001;Swerdlow et al., 2001b). Electrodes aimed at the dHip (Fig. 4B) were similarly positioned to dHip cannulae reported by a number of different groups (Swerdlow et al., 2001b;Zhang et al., 2002a) in the CA3/dentate gyrus region of the dHip. The placements of the active tips of the microdialysis probes aimed at the NAc did 52 ^ -20 -I 1 1 1 1 1 1 1 1 1 1 1 1 1 2 3 4 5 6 7 8 9 10 11 12 13 Time (X 10 min) Figure 2-3. Unilateral stimulation (20 Hz, 10 s) of either the vHip (n=7, stim) or dHip (n=7), and its effect on NAc D A efflux in the ipsilateral (vHip - black diamonds; dHip -white triangles) or contralateral (vHip - white squares) hemisphere. Data points represent the mean level of D A obtained over a 10 min sampling period, and error bars represent S E M . Asterisks denote a significant difference from baseline (p<0.05). 53 Figure 2-4. Schematic diagram of the placements of stimulating electrodes and microdialysis probes in all experiments. A, Representative placements of electrode tips (black circles) aimed at the vHip. B, Representative placements of electrode tips (black circles) aimed at the dHip. C, Representative placements of microdialysis probes aimed at the NAc. The black bars illustrate the position of the probes and are scaled to 2 mm in length to accurately reflect only the area of the brain from which each probe sampled. Numbers correspond to the anterior or posterior distance (in mm) of each plate from bregma. Plates adapted from Paxinos and Watson (Paxinos and Watson, 1997). 54 not substantially differ between the vHip and dHip stimulation experiments and are depicted on the same panels (Fig. 2-4C). Discussion In the present study, the effects of electrical stimulation of the hippocampus on PPI were site and frequency specific. Unilateral 20 Hz stimulation of the vHip, but not the dHip, caused a significant reduction in PPI at all prepulse intensities when testing began 2 min, but not 24 or 48 hr, after stimulation. Two Hz stimulation of the vHip failed to produce any changes in PPI. Changes in startie amplitude were unpredictable and difficult to interpret. Higher frequency stimulation of the vHip did not change startle amplitude significantly, whereas 2 Hz stimulation significantly increased startle amplitude 2 min following stimulation. When tested in the control condition, rats in the dHip group showed significantly higher startle amplitudes than when tested in the stim+48 condition. Experiments performed with microdialysis in freely moving rats confirmed our previous report that 20 Hz stimulation of the vHip results in a significant increase in NAc D A efflux (Taepavarapruk et al., 2000;Bast et al., 2001), and showed further that this increase was restricted to the hemisphere ipsilateral to the stimulating electrode. Finally, 20 Hz stimulation of the dHip failed to significantly increase NAc D A efflux. Potent Disruption of Sensorimotor Gating Induced by 20 Hz Stimulation of the vHip, but not dHip Numerous studies have reported that prolonged activation of the vHip, but not dHip, induced by bilateral infusion of N M D A , disrupts PPI in both Sprauge-Dawley (SD) and Wistar (W) rat strains (Wan et al., 1996;Klarner et al., 1998;Zhang et a l , 55 1999;2002a;Bast et al., 2001;Swerdlow et a l , 2001b). The present study demonstrated a similar disruption of PPI following 20 Hz electrical stimulation of the vHip in the L E strain. Although hooded rats, such as the L E strain, are less commonly used in PPI experiments than the SD and W strains, they have been employed in studies of the genetic determinants (Swerdlow et al., 2001c;2003) and other pharmacological aspects of PPI (Rasmussen et al., 1997;Faraday et al., 1999;Binder et al., 2001). Recent experiments suggest that the three strains are differentially sensitive to the disruptive effects of D A agonists (Swerdlow et al., 2001c;2003), which is interesting given the role of the vHip in modulating NAc D A efflux discussed below. The degree (~ 50 %) and duration (< 24 hr) of PPI disruption following the brief 20 Hz electrical stimulation employed in these studies were similar to that observed following N M D A infusion (Wan et a l , 1996;Zhang et al., 1999;2002a;Bast et al., 2001;Swerdlow et al., 2001b), although some inconsistencies have been reported (Klarner et al., 1998). By using electrical stimulation of the vHip, we have demonstrated the brevity (10 s) and specificity (20 Hz vs. 2 Hz) by which aberrant activity in the vHip results in disrupted PPI. Clearly, the mechanism by which vHip stimulation disrupts PPI must significantly outlast the duration of stimulation. Analysis of the startle amplitude data revealed that no significant changes in startle occurred following 20 Hz stimulation of the vHip. Inspection of the startle amplitude data following N M D A infusion into the vHip suggests that startle amplitude is typically either unchanged (Wan et al., 1996;Klarner et al., 1998;Bast et al., 2001) or reduced (Zhang et al., 1999;2002a;Swerdlow et al., 2001b) following chemical stimulation of the vHip. Given the inconsistent changes in startie amplitude relative to consistent disruption of PPI 56 following stimulation of the vHip, it is unlikely that the reduction in PPI observed here is due to changes in startle amplitude. The role of the hippocampus in the modulation of PPI has also been assessed with a variety of other manipulations. Permanent lesions of either the vHip or dHip do not affect PPI (Swerdlow et al., 1995;2000b;Pouzet et al., 1999 but see also Caine et al., 2001), although lesions of the vHip increase the sensitivity of rats to apomorphine-induced disruption of PPI (Swerdlow et al., 1995;2000b). Interestingly, temporary inactivation of either the vHip or dHip reversibly disrupts PPI (Zhang et al., 2002b). Data regarding modulation of PPI by the dHip are inconsistent and difficult to interpret. Infusion of the noncompetitive N M D A antagonist MK-801 into the vHip does not affect PPI (Bakshi and Geyer, 1998), whereas MK-801 infusions into the dHip may (Bakshi and Geyer, 1998;1999) or may not (Bast et al., 2000;Zhang et al., 2000b) alter PPI responding. In summary, these previous data demonstrate a subtle role for the vHip in the modulation of PPI under normal conditions. However, the present 20 Hz stimulation data, and those of others groups using N M D A (Wan et al., 1996;Zhang et al., 1999;2002a;Bast et al., 2001;Swerdlow et al., 2001b), indicate that overactivity in the vHip can cause a potent reduction in PPI. Although the dHip may also modulate PPI under certain conditions, data from this study and others (Swerdlow et al., 2001b;Zhang et al., 2002a) clearly indicate that induced overactivity of this structure does not significantly disrupt PPI. Frequency Dependence of the vHip Stimulation-induced Disruption of PPI Two Hz stimulation of the vHip, which causes a significant reduction in NAc D A efflux that may be mediated by the activation of group 2/3 mGluR's (Taepavarapruk et 57 al., 1998), failed to significantly affect PPI in this study (Fig. 2-2). Available pharmacological evidence is consistent with the observed lack of effect on PPI following 2 Hz vHip stimulation. For instance, blockade of D A receptors with D A antagonists, such as haloperidol, does not alter levels of PPI in rats (Mansbach et al., 1988;Depoortere et al., 1997;Hart et al., 1998;Feifel and Priebe, 1999). Additionally, systemic administration of the group 2/3 mGluR agonist LY354740 has no effect on PPI levels (Schreiber et al., 2000). Thus, pharmacological treatments that mimic some of the effects of 2 Hz vHip stimulation are also without effect on PPI responding. Unexpectedly, 2 Hz stimulation of the vHip resulted in a significant increase in startle amplitude immediately following the stimulation when compared to the stim+48 and control+48 conditions. Treatment with group 2/3 mGluR agonists or haloperidol decreased or had no effect on startle magnitude respectively (Mansbach et al., 1988;Depoortere et al., 1997;Hart et al., 1998;Feifel and Priebe, 1999;Schreiber et al., 2000). Given that these drugs were administered systemically, it is possible that their effects may result from actions in brain areas other than those affected by 2 Hz stimulation. However, it is interesting that stimulation of the vHip with two different frequencies had different effects on both startle and PPI. Given the inconsistencies in startle changes observed following N M D A stimulation (Wan et al., 1996;Zhang et al., 1999;2002a;Legault et al., 2000;Taepavarapruk et al., 2000;Bast et al., 2001;Swerdlow et al., 2001b), it will be important to replicate these findings in separate groups and/or strains of rats to ensure their reliability. 58 Increases in NAc DA Efflux following Hip Stimulation are Site and Hemisphere Specific Stimulation of the vHip with either N M D A or brief periods of 20 Hz electrical stimulation produces a long-lasting increase in NAc D A efflux (Legault et al., 2000;Taepavarapruk et al., 2000). The effect of 20 Hz stimulation is mediated by iGluR's in the NAc, thereby implicating the direct glutamatergic projection from the vHip to NAc in this effect (Blaha et a l , 1997;Taepavarapruk et al., 2000). The present neurochemical experiments (Fig. 3) extend our previous findings, as the increase in NAc D A efflux following 20 Hz electrical stimulation of the vHip was restricted to the hemisphere ipsilateral to the stimulating electrode. This finding is consistent with the fact that the vHip projections to forebrain areas such as the NAc are primarily unilateral (Kelley and Domesick, 1982;Groenewegen et a l , 1987;Brog et al., 1993). In contrast to stimulation of the vHip, 20 Hz stimulation of the dHip failed to increase NAc D A efflux significantly. Although the dHip sends glutamatergic efferents to the NAc, it is important to note that the vHip projects primarily to the NAc shell whereas the dHip projects primarily to the NAc core (Swanson and Cowan, 1977;Kelley and Domesick, 1982;Whitter, 1986;Groenewegen et al., 1987;Brog et al., 1993). Probe placements adjacent to both the shell and core subregions of the NAc were employed in studies in which stimulation of the vHip increased NAc D A efflux (Fig. 2-4C); therefore, we monitored D A efflux in a similar region of the NAc following stimulation of the dHip. This raises the possibility that any effect of the dHip stimulation on NAc D A efflux was restricted to the NAc core. However, unpublished observations failed to confirm an increase in NAc D A efflux with dialysis probes located in the core of the NAc (Howland, Taepavararpuk, and Phillips). 59 Microdialysis experiments have examined dynamic changes in NAc D A efflux during PPI test sessions. Zhang and colleagues (Zhang et al., 2000a) demonstrated that systemic administration of amphetamine disrupted PPI, whereas treatment with cocaine had no effect (but also see Martinez et al., 1999). Interestingly, both drugs increased NAc D A efflux by at least 100 % of baseline during the PPI test sessions. Consequently, these authors suggest that the behavioral effects of DA agonists on PPI are critically dependent on their mechanism of action. Other experiments have shown that presentation of startle pulses alone cause a significant decrease in NAc D A efflux that is not observed when prepulses of a moderate intensity are presented prior to the startle pulses (Humby et al., 1996). These data suggest that during PPI, the presentation of the prepulses prevents perturbation of the mesoaccumbens D A system in response to the startling stimuli. Together, these data imply that certain drugs, such as amphetamine, can interfere with the effect of a prepulse on D A efflux in the NAc, thereby disrupting PPI. Given these data, it is intriguing that two different methods of stimulating the vHip, both of which increase D A efflux in the NAc, can disrupt PPI. The fact that 20 Hz stimulation of the dHip or 2 Hz stimulation of the vHip failed to disrupt PPI and did not increase NAc D A efflux is consistent with the hypothesis that increased NAc D A efflux following 20 Hz vHip stimulation may be a factor in the disruption of PPI. However, systemic administration of the D 2 antagonist haloperidol (Wan et al., 1996;Zhang et al., 1999;Bast et al., 2001) or intra-NAc infusions of the non-NMDA receptor antagonist C N Q X (Wan et al., 1996) fail to block the disruptive effects of intra-vHip N M D A infusion on PPI responding. Therefore, it is unlikely that the disruptive effects of 60 stimulation of the vHip on PPI are mediated solely by activation of the mesoaccumbens D A system or non-NMDA receptors in the NAc (Wan et al., 1996;Bast et al., 2001). Potential Mechanisms Underlying vHip Stimulation-induced Disruption in PPI Normal D A and Glu transmission appears to be important for PPI (Zhang et al., 2000a;Geyer et al., 2001). For example, infusions of D A or D A agonists into the NAc disrupt PPI (Mansbach et al., 1988;Swerdlow et al., 1990;Wan et al., 1995), and infusions of Glu agonists and antagonists such as A M P A , A P V , CNQX, and MK-801 into the NAc have complex and regionally specific effects (Reijmers et al., 1995;Wan et al., 1995;Wan and Swerdlow, 1996). Recent physiological experiments raise the possibility that the effects of vHip stimulation on PPI may involve interactions between multiple receptor subtypes in the NAc. Stimulation of the vHip shifts NAc neurons to an activated or 'up' state (O'Donnell and Grace, 1995) and recent experiments from our laboratory indicate that 20 Hz stimulation (10 s) of the vHip alters the responses of medium spiny neurons in the NAc shell to afferent input from the vHip and B L A (Floresco et al., 2001). These effects are dependent on the 20 Hz stimulation-induced increase in NAc D A and a complex pharmacology involving activation of both D t and N M D A receptors, but not D 2 and non-NMDA receptors (Floresco et al., 2001). Importantly, the potentiating effect of 20 Hz stimulation of the vHip on NAc neurons is maintained for at least 20 min, which corresponds to the duration of PPI trials in the present study. Stimulation of the vHip may also disrupt PPI via the widespread connections of the vHip to a number of areas in the CSPP circuitry critical for normal PPI (Klarner et al., 1998). Data obtained in the present study do not refute this hypothesis. The vHip is reciprocally connected to a number of areas other than the NAc, such as the mPFC, 61 amygdala, thalamus, septum, and ventral pallidum (Groenewegen et a l , 1987;Amaral and Whitter, 1995;Conde et al., 1995), all of which are important for the regulation of PPI (Swerdlow et al., 2001a). Stimulation of the vHip with doses of N M D A that disrupt PPI cause increased fos-protein expression in a number of limbic and cortical areas such as the NAc, lateral septum, mPFC, and dorsomedial striatum (Klarner et al., 1998). Stimulation of the vHip also induces various types of plasticity in the mPFC such as long-term potentiation and depression (Laroche et al., 2000). Therefore, subtle changes in multiple areas induced by vHip stimulation may disrupt PPI (Klarner et al., 1998). Although the dHip projects to the NAc core, it is not directly connected to other areas implicated in PPI (Swanson and Cowan, 1977;Kelley and Domesick, 1982;Whitter, 1986;Amaral and Whitter, 1995), and therefore aberrant activity generated in the dHip may not have access to the neural substrates critical to disrupt PPI. This conjecture is consistent with the failure of 20 Hz stimulation of the dHip to increase NAc D A efflux in the NAc or to disrupt PPI in the present study. Conclusion Our data clearly demonstrate that brief, higher frequency stimulation of vHip, but not dHip, disrupts PPI and increases D A efflux in the NAc. Given the anatomical and functional abnormalities frequently observed in the hippocampal formation of patients with schizophrenia, these data support the assertion that abnormalities in limbic-cortico-striatal interactions may underlie deficits in sensorimotor gating in schizophrenia. Identification of the mechanism underlying the disruptive effects of vHip stimulation on PPI may provide insight into potential targets for pharmacological treatment strategies for schizophrenia. 62 \ References Amaral DG, Whitter M P (1995) Hippocampal formation. In: The Rat Nervous System (Paxinos G, ed), pp 443-493. San Diego: Academic Press. Bakshi VP, Geyer M A (1998) Multiple limbic regions mediate the disruption of prepulse inhibition produced in rats by the noncompetitive N M D A antagonist dizocilpine. J Neurosci 18: 8394-8401. Bakshi VP, Geyer M A (1999) Alpha-1-adrenergic receptors mediate sensorimotor gating deficits produced by intracerebral dizocilpine administration in rats. Neuroscience 92: 113-121. Bast T, Zhang W, Feldon J, White JJVI (2000) Effects of MK801 and neuroleptics on prepulse inhibition: re-examination in two strains of rats. Pharmacol Biochem Behav 67: 647-658. Bast T, Zhang W N , Heidbreder C, Feldon J (2001) Hyperactivity and disruption of prepulse inhibition induced by N-methyl-D-aspartate stimulation of the ventral hippocampus and the effects of pretreatment with haloperidol and clozapine. Neuroscience 103: 325-335. Binder EB, Kinkead B, Owens MJ , Kilts CD, Nemeroff CB (2001) Enhanced neurotensin neurotransmission is involved in the clinically relevant behavioral effects of antipsychotic drugs: evidence from animal models of sensorimotor gating. J Neurosci 21: 601-608. Blaha CD, Yang CR, Floresco SB, Barr A M , Phillips A G (1997) Stimulation of the ventral subiculum of the hippocampus evokes glutamate receptor-mediated changes in dopamine efflux in the rat nucleus accumbens. Eur J Neurosci 9: 902-911. Bogerts B (1997) The temporolimbic system theory of positive schizophrenic symptoms. Schizophr Bull 23: 423-435. Braff DL, Geyer M A , Swerdlow NR (2001) Human studies of prepulse inhibition of starde: normal subjects, patient groups, and pharmacological studies. Psychopharmacology (Berl) 156: 234-258. Breier A , Su TP, Saunders R, Carson RE, Kolachana BS, de Bartolomeis A , Weinberger DR, Weisenfeld N , Malhotra A K , Eckelman WC, Pickar D (1997) Schizophrenia is associated with elevated amphetamine-induced synaptic dopamine concentrations: evidence from a novel positron emission tomography method. Proc Natl Acad Sci U S A 94: 2569-2574. Brog JS, Salyapongse A, Deutch A Y , Zahm DS (1993) The patterns of afferent innervation of the core and shell in the "accumbens" part of the rat ventral striatum: immunohistochemical detection of retrogradely transported fluoro-gold. J Comp Neurol 338: 255-278. 63 Bradzynski S M , Gibson CJ (1997) Release of dopamine in the nucleus accumbens caused by stimulation of the subiculum in freely moving rats. Brain Res Bull 42: SOS-SOS. Caine SB, Geyer M A , Swerdlow NR (1992) Hippocampal modulation of acoustic startle and prepulse inhibition in the rat. Pharmacol Biochem Behav 43: 1201-1208. Caine SB, Humby T, Robbins TW, Everitt BJ (2001) Behavioral effects of psychomotor stimulants in rats with dorsal or ventral subiculum lesions: locomotion, cocaine self-administration, and prepulse inhibition of startle. Behav Neurosci 115: 880-894. Carlsson A , Waters N , Carlsson M L (1999) Neurotransmitter interactions in schizophrenia—therapeutic implications. Biol Psychiatry 46: 1388-1395. Conde F, Maire-Lepoivre E, Audinat E, Crepel F (1995) Afferent connections of the medial frontal cortex of the rat. II. Cortical and subcortical afferents. J Comp Neurol 352: 567-593. Depoortere R, Perrault G, Sanger DJ (1997) Potentiation of prepulse inhibition of the startle reflex in rats: pharmacological evaluation of the procedure as a model for detecting antipsychotic activity. Psychopharmacology (Berl) 132: 366-374. Dierks T, Linden DE, Jandl M , Formisano E, Goebel R, Lanfermann H , Singer W (1999) Activation of Heschl's gyrus during auditory hallucinations. Neuron 22: 615-621. Faraday M M , O'Donoghue V A , Grunberg N E (1999) Effects of nicotine and stress on startle amplitude and sensory gating depend on rat strain and sex. Pharmacol Biochem Behav 62: 273-284. Feifel D, Priebe K (1999) The effects of subchronic haloperidol on intact and dizocilpine-disrupted sensorimotor gating. Psychopharmacology (Berl) 146: 175-179. Fendt M , Schwienbacher I, Koch M (2000) Amygdaloid N-methyl-D-aspartate and gamma-aminobutyric acid(A) receptors regulate sensorimotor gating in a dopamine-dependent way in rats. Neuroscience 98: 55-60. Floresco SB, Blaha CD, Yang CR, Phillips A G (2001) Modulation of hippocampal and amygdalar-evoked activity of nucleus accumbens neurons by dopamine: cellular mechanisms of input selection. J Neurosci 21: 2851-2860. Geyer M A , Krebs-Thomson K, Braff DL, Swerdlow NR (2001) Pharmacological studies of prepulse inhibition models of sensorimotor gating deficits in schizophrenia: a decade in review. Psychopharmacology (Berl) 156: 117-154. Goff DC, Coyle JT (2001) The emerging role of glutamate in the pathophysiology and treatment of schizophrenia. Am J Psychiatry 158: 1367-1377. 64 Grace A A (2000) Gating of information flow within the limbic system and the pathophysiology of schizophrenia. Brain Res Brain Res Rev 31: 330-341. Groenewegen HJ, Vermeulen-Van der Zee E, te K A , Witter M P (1987) Organization of the projections from the subiculum to the ventral striatum in the rat. A study using anterograde transport of Phaseolus vulgaris leucoagglutinin. Neuroscience 23: 103-120. Harrison PJ (1999) The neuropathology of schizophrenia. A critical review of the data and their interpretation. Brain 122 (Pt 4): 593-624. Hart S, Zreik M , Carper R, Swerdlow NR (1998) Localizing haloperidol effects on sensorimotor gating in a predictive model of antipsychotic potency. Pharmacol Biochem Behav 61: 113-119. Heckers S, Rauch SL, Goff D, Savage CR, Schacter DL, Fischman A J , Alpert N M (1998) Impaired recruitment of the hippocampus during conscious recollection in schizophrenia. Nat Neurosci 1: 318-323. Howland JG, Taepavarapruk P, Phillips A G (2002) Glutamate receptor-dependent modulation of dopamine efflux in the nucleus accumbens by basolateral, but not central, nucleus of the amygdala in rats. J Neurosci 22: 1137-1145. Humby T, Wilkinson LS, Robbins TW, Geyer M A (1996) Prepulses inhibit startle-induced reductions of extracellular dopamine in the nucleus accumbens of rat. J Neurosci 16: 2149-2156. Kelley A E , Domesick V B (1982) The distribution of the projection from the hippocampal formation to the nucleus accumbens in the rat: an anterograde- and retrograde-horseradish peroxidase study. Neuroscience 7: 2321-2335. Klarner A , Koch M , Schnitzler H U (1998) Induction of Fos-protein in the forebrain and disruption of sensorimotor gating following N-methyl-D-aspartate infusion into the ventral hippocampus of the rat. Neuroscience 84: 443-452. Koch M (1999) The neurobiology of startle. Prog Neurobiol 59: 107-128. Koch M , Bubser M (1994) Deficient sensorimotor gating after 6-hydroxydopamine lesion of the rat medial prefrontal cortex is reversed by haloperidol. Eur J Neurosci 6: 1837-1845. Lacroix L , Spinelli S, White W, Feldon J (2000) The effects of ibotenic acid lesions of the medial and lateral prefrontal cortex on latent inhibition, prepulse inhibition and amphetamine-induced hyperlocomotion. Neuroscience 97: 459-468. Laroche S, Davis S, Jay T M (2000) Plasticity at hippocampal to prefrontal cortex synapses: dual roles in working memory and consolidation. Hippocampus 10: 438-446. 65 Laruelle M , Abi-Dargham A, Gi l R, Kegeles L , Innis R (1999) Increased dopamine transmission in schizophrenia: relationship to illness phases. Biol Psychiatry 46: 56-72. Legault M , Rompre PP, Wise R A (2000) Chemical stimulation of the ventral hippocampus elevates nucleus accumbens dopamine by activating dopaminergic neurons of the ventral tegmental area. J Neurosci 20: 1635-1642. Mansbach RS, Geyer M A (1989) Effects of phencyclidine and phencyclidine biologs on sensorimotor gating in the rat. Neuropsychopharmacology 2: 299-308. Mansbach RS, Geyer M A , Braff D L (1988) Dopaminergic stimulation disrupts sensorimotor gating in the rat. Psychopharmacology (Berl) 94: 507-514. Martinez ZA, Ellison GD, Geyer M A , Swerdlow NR (1999) Effects of sustained cocaine exposure on sensorimotor gating of startle in rats. Psychopharmacology (Berl) 142: 253-260. Meyer-Lindenberg A , Miletich RS, Kohn PD, Esposito G, Carson RE, Quarantelli M , Weinberger DR, Berman K F (2002) Reduced prefrontal activity predicts exaggerated striatal dopaminergic function in schizophrenia. Nat Neurosci 5: 267-271. O'Donnell P, Grace A A (1995) Synaptic interactions among excitatory afferents to nucleus accumbens neurons: hippocampal gating of prefrontal cortical input. J Neurosci 15: 3622-3639. Pakkenberg B (1990) Pronounced reduction of total neuron number in mediodorsal thalamic nucleus and nucleus accumbens in schizophrenics. Arch Gen Psychiatry 47: 1023-1028. Paxinos G, Watson C (1997) The rat brain in stereotaxic co-ordinates. San Diego: Academic Press. Pouzet B , Feldon J, Veenman CL, Yee B K , Richmond M , Nicholas J, Rawlins P, Weiner I (1999) The effects of hippocampal and fimbria-fornix lesions on prepulse inhibition. Behav Neurosci 113: 968-981. Rasmussen K, Gates MR, Burger JE, Czachura JF (1997) The novel atypical antipsychotic olanzapine, but not the C C K - B antagonist LY288513, blocks apomorphine-induced disruption of pre-pulse inhibition. Neurosci Lett 222: 61-64. Reijmers L G , Vanderheyden P M , Peeters B W (1995) Changes in prepulse inhibition after local administration of N M D A receptor ligands in the core region of the rat nucleus accumbens. Eur J Pharmacol 272: 131-138. Schreiber R, Lowe D, Voerste A , De Vry J (2000) LY354740 affects startle responding but not sensorimotor gating or discriminative effects of phencyclidine. Eur J Pharmacol 388: R3-R4. 66 Silbersweig D A , Stern E, Frith C, Cahill C, Holmes A , Grootoonk S, Seaward J, McKenna P, Chua SE, Schnorr L , . (1995) A functional neuroanatomy of hallucinations in schizophrenia. Nature 378: 176-179. Strecker RE, Moneta M E (1994) Electrical stimulation of the kindled hippocampus briefly increases extracellular dopamine in the nucleus accumbens. Neurosci Lett 176: 173-177. Swanson L W , Cowan W M (1977) An autoradiographic study of the organization of the efferent connections of the hippocampal formation in the rat. J Comp Neurol 172: 49-84. Swerdlow NR (1996) Cortico-striatal substrates of cognitive, motor, and sensory gating: speculations and implications for psychological dysfunction. Advances in Biological Psychiatry 2: 179-207. Swerdlow NR, Braff DL , Geyer M A (2000a) Animal models of deficient sensorimotor gating: what we know, what we think we know, and what we hope to know soon. Behav Pharmacol 11: 185-204. Swerdlow NR, Braff DL , Geyer M A , Koob GF (1986) Central dopamine hyperactivity in rats mimics abnormal acoustic startle response in schizophrenics. Biol Psychiatry 21: 23-33. Swerdlow NR, Braff DL , Masten V L , Geyer M A (1990) Schizophrenic-like sensorimotor gating abnormalities in rats following dopamine infusion into the nucleus accumbens. Psychopharmacology (Berl) 101: 414-420. Swerdlow NR, Braff DL, Taaid N , Geyer M A (1994) Assessing the validity of an animal model of deficient sensorimotor gating in schizophrenic patients. Arch Gen Psychiatry 51: 139-154. Swerdlow NR, Geyer M A , Braff D L (2001a) Neural circuit regulation of prepulse inhibition of startle in the rat: current knowledge and future challenges. Psychopharmacology (Berl) 156: 194-215. Swerdlow NR, Hanlon F M , Henning L, Kim Y K , Gaudet I, Halim N D (2001b) Regulation of sensorimotor gating in rats by hippocampal N M D A : anatomical localization. Brain Res 898: 195-203. Swerdlow NR, Lipska B K , Weinberger DR, Braff DL , Jaskiw GE, Geyer M A (1995) Increased sensitivity to the sensorimotor gating-disruptive effects of apomorphine after lesions of medial prefrontal cortex or ventral hippocampus in adult rats. Psychopharmacology (Berl) 122: 27-34. Swerdlow NR, Platten A , Kim Y K , Gaudet I, Shoemaker J, Pitcher L , Auerbach P (2001c) Sensitivity to the dopaminergic regulation of prepulse inhibition in rats: evidence for genetic, but not environmental determinants. Pharmacol Biochem Behav 70: 219-226. 67 Swerdlow NR, Shoemaker J M , Platten A, Pitcher L , Goins J, Crain S (2003) Heritable differences in the effects of amphetamine but not DOI on startle gating in albino and hooded outbred rat strains. Pharmacol Biochem Behav 75: 191-197. Swerdlow NR, Taaid N , Halim N , Randolph E, Kim Y K , Auerbach P (2000b) Hippocampal lesions enhance startle gating-disruptive effects of apomorphine in rats: a parametric assessment. Neuroscience 96: 523-536. Taepavarapruk P, Blaha CD, Phillips A G (1998) Decrease in extracellular dopamine levels in the nucleus accumbens induced by low frequency stimulation of the ventral subiculum/CAl region of the rat hippocampus. SfN Abstracts 24, 823.2. Taepavarapruk P, Floresco SB, Phillips A G (2000) Hyperlocomotion and increased dopamine efflux in the rat nucleus accumbens evoked by electrical stimulation of the ventral subiculum: role of ionotropic glutamate and dopamine D l receptors. Psychopharmacology (Berl) 151: 242-251. Wan FJ, Caine SB, Swerdlow NR (1996) The ventral subiculum modulation of prepulse inhibition is not mediated via dopamine D2 or nucleus accumbens non-NMDA glutamate receptor activity. Eur J Pharmacol 314: 9-18. Wan FJ, Geyer M A , Swerdlow NR (1995) Presynaptic dopamine-glutamate interactions in the nucleus accumbens regulate sensorimotor gating. Psychopharmacology (Berl) 120: 433-441. Wan FJ, Swerdlow NR (1993) Intra-accumbens infusion of quinpirole impairs sensorimotor gating of acoustic startle in rats. Psychopharmacology (Berl) 113: 103-109. Wan FJ, Swerdlow NR (1996) Sensorimotor gating in rats is regulated by different dopamine-glutamate interactions in the nucleus accumbens core and shell subregions. Brain Res 722: 168-176. Wan FJ, Swerdlow NR (1997) The basolateral amygdala regulates sensorimotor gating of acoustic startle in the rat. Neuroscience 76: 715-724. Weike A l , Bauer U , Hamm A O (2000) Effective neuroleptic medication removes prepulse inhibition deficits in schizophrenia patients. Biol Psychiatry 47: 61-70. Whitter M P (1986) A survey of the anatomy of the hippocampal formation, with emphasis on the septotemporal organization of its intrinsic and extrinsic connections. In: Advances in experimental and medical biology: Vol . 203. Excitatory amino acids and epilepsy. (Schwarcz R, Ben-Ari Y , eds), pp 67-82. New York: Plenum. Wright IC, Ellison ZR, Sharma T, Friston KJ , Murray R M , McGuire P K (1999) Mapping of grey matter changes in schizophrenia. Schizophr Res 35: 1-14. Zhang J, Forkstam C, Engel JA, Svensson L (2000a) Role of dopamine in prepulse inhibition of acoustic startle. Psychopharmacology (Berl) 149: 181-188. 68 Zhang W, Pouzet B , Jongen-Relo A L , Weiner I, Feldon J (1999) Disruption of prepulse inhibition following N-methyl-D-aspartate infusion into the ventral hippocampus is antagonized by clozapine but not by haloperidol: a possible model for the screening of atypical antipsychotics. Neuroreport 10: 2533-2538. Zhang W N , Bast T, Feldon J (2000b) Microinfusion of the non-competitive N-methyl-D-aspartate receptor antagonist MK-801 (dizocilpine) into the dorsal hippocampus of wistar rats does not affect latent inhibition and prepulse inhibition, but increases startle reaction and locomotor activity. Neuroscience 101: 589-599. Zhang W N , Bast T, Feldon J (2002a) Effects of hippocampal N-methyl-D-aspartate infusion on locomotor activity and prepulse inhibition: differences between the dorsal and ventral hippocampus. Behav Neurosci 116: 72-84. Zhang W N , Bast T, Feldon J (2002b) Prepulse inhibition in rats with temporary inhibition/inactivation of ventral or dorsal hippocampus. Pharmacol Biochem Behav 73: 929-940. 69 CHAPTER THREE: DELAYED ONSET OF PREPULSE INHIBITION DEFICITS FOLLOWING KAINIC ACID TREATMENT ON POSTNATAL DAY SEVEN IN RATS. 2 Introduction Given the heterogeneity and complexity of schizophrenia, its etiology has been difficult to define. The neurodevelopmental hypothesis of schizophrenia proposes that adverse events or genetic abnormalities may disrupt early brain development, and interact with environmental factors or influence subsequent brain development to cause the disorder (Weinberger, 1995;Benes et al., 2000;McClure and Weinberger, 2001). Once the illness is expressed, a diverse array of subde changes are found in temporal and frontal brain areas such as the hippocampus, striatum, thalamus, and frontal cortex (Pakkenberg, 1990;Suddath et al., 1990;Bogerts, 1997;Jones, 1997;Heckers etal., 1998;Harrison, 1999;Gothelf et al., 2000;McClure and Weinberger, 2001;Meyer-Lindenberg et al., 2002). Neurodegeneration in these areas is rarely observed in the brains of patients with schizophrenia (but see Stevens, 1994), therefore alterations in normal patterns of connectivity between affected brain areas may underlie the expression of some symptoms of schizophrenia (Harrison, 1999;Friston, 1999;Benes, 2000;Penn, 2001). Accordingly, the consequences of disrupting the normal development of limbic and cortical areas on behaviors in rodents related to schizophrenia have been examined (for reviews see Lipska and Weinberger, 2000;Van den et al., 2003). Specific behavioral abnormalities have been observed in adult, but not prepubescent rats, that received 2 A version of this chapter has been published: Howland JG, Hannesson D K , Phillips A G (2004). Delayed onset of prepulse inhibition deficits following kainic acid treatment on postnatal day seven in rats. European Journal of Neuroscience 20:2639-2648. 70 ventral hippocampal (vHip) lesions on postnatal day (PND) 7 (Lipska and Weinberger, 2000). Behavioral changes in prepulse inhibition (PPI) and locomotor responses to novelty, which resemble certain positive symptoms in schizophrenia, are robust and can be exacerbated by acute administration of dopamine (DA) agonists immediately before testing in adulthood (Lipska et al., 1993; 1995;Le Pen and Moreau, 2002). Importantly, temporary reduction of neural activity within the vHip on PND7, with the sodium channel blocker tetrodotoxin (TTX) increased locomotor activity in response to novelty, injection stress, and amphetamine after puberty (Lipska et al., 2002). Similar results have been observed following neonatal lesions of the amygdala (Daenen et al., 2001; 2002; 2003), but not the medial prefrontal cortex (Lipska et al., 1998;Brake et al., 2000;Van den et al., 2003 but see Flores et al., 1996). Additionally, systemic administration of the non-competitive N M D A receptor antagonist dizocilpine (MK-801) on PND7 can reduce PPI levels and increase locomotor activity in female rats (Harris et al., 2003; but see Beninger et al., 2002). The apparent sensitivity of limbic-cortical circuits on PND7 to functional disruption raises the possibility that increased neural activity induced by the ionotropic glutamate agonist kainic acid (KA) at this developmental age would result in behavioral changes in rats similar to those discussed above. Kainic acid has been used extensively to study limbic-motor seizures and excitotoxicity in adult rats (for reviews see Sperk, 1994;Ben Ari and Cossart, 2000); however, its effects during early development are not as well characterized. During the first two postnatal weeks, systemic administration of K A results in tonic-clonic seizures and the selective activation of the hippocampus and lateral septum without the high levels of cell death normally observed in limbic and 71 cortical areas following administration during or after the 3 r postnatal week (Nitecka et al., 1984;Tremblay et al., 1984;Stafstrom et al., 1992;Khalilov et al., 1999;Koh et al., 1999;Lynch et al., 2000;Silveira et al., 2002). Although few studies have analyzed behavioral changes in adulthood following a single administration of K A during the first week of life, there are reports of deficits in conditioned avoidance (de Feo et al., 1986) and spatial learning using the radial arm maze (Lynch et al., 2000), but not the water maze (Stafstrom et al., 1993;Koh et al., 1999). The present study sought to examine whether systemic K A administration on PND7 would alter the behavior of rats in a pattern similar to that observed in other animal models of schizophrenia (Lipska et al., 1993;Harris et al., 2003). Kainic acid or saline was administered to rat pups on PND7 and their PPI levels and locomotor responses to a novel environment were measured before and after puberty. To assess potential alterations of the D A system following K A administration, we tested the potential interactive effects of K A treatment and apomorphine, a direct D A agonist, on PPI and also with amphetamine-induced locomotion. Subgroups of K A - and saline-treated rats were also tested on a standard hippocampal-dependent Morris Water Maze task in adulthood to examine aspects of spatial learning and memory that are dependent on normal hippocampal function (Morris et al., 1990). Methods Subjects Two independent groups of animals (i.e. Group 1 and Group 2) were tested in this study. Testing protocols for both groups were similar with small procedural differences noted in the methods section where appropriate. Pregnant Long-Evans rats were obtained 72 from Charles River (Quebec, Canada) at 13 to 15 days of gestation. They were singly housed and left undisturbed until giving birth. The colony was maintained on a 12/12 hour light/dark cycle (lights on at 0700), at a temperature of 22+1 °C. A l l rats were given food (Purina Rat Chow) and tap water ad libitum. Experiments were conducted in accordance with the standards of the Canadian Council on Animal Care and were approved by the Committee on Animal Care at the University of British Columbia. Kainic Acid Administration The day of birth of the pups was designated post-natal day (PND) 0. On PND3, the litters were sexed and culled to include only males (6-9 rats per litter). On PND7, all but one of the pups were removed from the nest, weighed and individually placed in small compartments of a cardboard box for K A administration. They were then removed from the colony and taken to a small heated room. K A (1.5 mg/kg, Tocris) or saline was injected (i.p.) with a 30-gauge needle (10 ml/kg). Care was taken to ensure that both K A and saline was administered to members from each litter. Previous reports indicated that K A administration of 1-2 mg/kg on PND7 causes behavioral seizures in most pups without high mortality rates (Stafstrom et al., 1993;Lynch et al., 2000). The behavior of all rats was recorded with an overhead video camera for 180 minutes. Before being returned to their mothers, the pups were earmarked according to treatment condition. The litters were then left undisturbed (except for normal cage changing) until weaning on PND25. Weanling rats were housed in cages of 2 or 3 with members of their litter. A l l rats were handled for at least 5 days before behavioral testing. Prepulse Inhibition On PND35 and 56, rats were removed individually from the colony and taken immediately to the PPI apparatus. Testing was conducted in a single sound-attenuating startle chamber (ambient noise level 64 dB), containing a transparent Plexiglas tube (8.2 cm in diameter, 20 cm in length), mounted on a Plexiglas frame (SR-LAB, San Diego Instruments, San Diego). Noise bursts were presented through a speaker mounted 24-cm above the tube. An accelerometer below the frame of the apparatus measured whole body startle amplitude, defined as the average of 100 1-ms accelerometer readings collected from stimulus onset. Each PPI test session began with a 5-min acclimatization period during which a 70-dB background noise level was presented, which remained constant for the entire test session. Following the acclimatization period, six pulse alone trials (120 dB, 40 ms) were presented to achieve a relatively stable startle amplitude before PPI testing. Data from these pulse-alone trials was not considered in the analysis of PPI. Immediately following the six initial pulse-alone trials, presentation of the trials that were used in the calculation of PPI levels were initiated. Trials presented were of three types: pulse alone (12 trials, 120 dB, 40 ms), prepulse + pulse (10 trials X 3 prepulse intensities - discussed below), or no stimulus (42 trials). Prepulse + pulse trials consisted of the presentation of a 20 ms prepulse of 73, 76, or 82 dB 80 ms before the presentation of the pulse. The pulse and prepulse + pulse trials were presented in a pseudorandom order. One no-stimulus trial was presented between each pair of pulse and prepulse + pulse trials. The inter-trial interval varied randomly from 3 to 12 s (average 7.5 s). Calibration of the apparatus was performed using a RadioShack Digital Sound Level Meter and adjustments were made as necessary. 74 Between PND98 and 105, a randomly selected subset of the rats from Group 2 were re-tested on PPI following injection with vehicle (0.1% ascorbic acid) and apomorphine (Sigma; 0.2 mg/kg; s.e.) 72 hr later to assess: 1) the consistency of the PPI reduction in KA-treated animals and 2) the potential role of dopaminergic mechanisms in the observed PPI decreases in the KA-treated animals. The PPI sessions were conducted in a manner identical to that described above, except that immediately before the PPI session, all rats were weighed and injected with the appropriate volume of vehicle or drug. Locomotor Activity On PND36 and 57, rats were weighed, removed from the colony, and immediately placed in locomotor boxes. For Group 1, the locomotor boxes were in two similar, small adjacent rooms (4 boxes in one, 3 in the other) constructed from clear Plexiglas (32 cm X 32 cm X height 41 cm) and fitted with four pairs of infra-red beams positioned 10 cm apart and 2.5 cm above a metal grid floor. A l l sensors were interfaced to a computer-controlled system (MANX) . Motor activity counts were collected in 10-min bins during testing. Care was taken to ensure that rats from each group were tested in both rooms and in all boxes. Spontaneous locomotor activity levels in a novel environment were measured for 60 min. Immediately following the spontaneous locomotor activity test, all rats were injected with D-amphetamine (1.5 mg/kg, i.p.) and returned to the locomotor boxes. Locomotor activity was then monitored for an additional 90 min before the rats were returned to the colony room. For Group 2, testing procedures were identical, except that a Med Associates System was used to collect the data. Eight Med Associates Test Chambers (ENV-008; 30.5 cm X 24.1cm X height 21.0 75 cm) were fitted with 4 pairs of infrared photocells 3.5 cm from the floor evenly spaced on the long walls. The chambers had metal grid floors and two operant levers (which were retracted during locomotor activity testing). Each chamber was contained within a Med Associates sound attenuating cubicle with a house light that was illuminated during testing. Water Maze Testing A subgroup of the animals from Group 1 was tested between PND65 and 75 in a water maze. Testing was performed in a circular, white swimming pool 180 cm in diameter, 54 cm in height. The escape platform was 34 cm in height and 14 cm in diameter. The pool was filled with water such that the top of the platform was 3 cm below the surface of the water. For visible platform trials, an additional piece of wood and wire mesh was secured to the top of the invisible platform such that it extended 3 cm out of the water. The water was rendered opaque by the addition of white Tempura Powder paint. Data were recorded using a computerized tracking system (HVS Image, Hampton, England). Rats were transported to the pool room in groups and testing was conducted over 3 consecutive days. Rats were released facing the wall of the pool from one of the geographic poles in a pseudorandom order. Each rat was allowed 60 s to swim to the platform, after which they were left on the platform for 10 s. They were then removed from the water maze, and the next rat was tested. The intertrial interval for each rat was approximately 7 min. On the first day, rats were trained to swim to the visible platform over 5 trials to allow habituation to the testing environment and swimming. The location of the platform was varied for each trial, and was never in the location of the hidden platform used on day 2. Hidden platform training was performed on day 2. Rats 76 were given 10 trials to learn to find the constant location of the hidden platform. Finally, on day 3, the rats were tested in a 30-s probe trial without the platform present. The percent time spent in the quadrant that contained the hidden platform on day 2 was taken as a measure of spatial memory. Histology Following behavioral testing, a subgroup of animals (n=5 from each group) was randomly selected, sacrificed with an overdose of pentobarbital, and perfused transcardially with 0.9% saline followed by 10% formalin. Brains were stored in 10% sucrose/10% formalin for at least 1 week, after which coronal sections (40 u.m) containing the hippocampus were taken using a vibratome. Every fourth section was saved and stained with cresyl violet. An experimenter blind to the treatment group examined each section using a light microscope for gross cell loss in the hippocampus with particular attention paid to the CA3 region due to its established sensitivity to the excitotoxic effects of K A in adult rats (Ben Ar i and Cossart, 2000). Data Analysis For the PPI experiments, two measures were calculated for each animal. The startle amplitude represented the mean startle amplitude of the 12 pulse-alone trials presented after the 6 habituation trials. Startle amplitude data were compared with an independent samples t-test for each age. PPI was calculated by averaging startle amplitudes for each trial type. The percent PPI for each prepulse intensity was calculated using the formula: [100 - (100 X startle amplitude on prepulse + pulse trials) -s- (startle amplitude on pulse alone trials)]. A repeated measures A N O V A was performed on the data obtained from each age with prepulse intensity as a within subjects factor and 77 treatment on PND7 as a between subjects factor. Results from the apomorphine experiment were also analyzed with a mixed A N O V A (prepulse intensity and test as within subjects factors, and treatment at PND7 as a between subjects factor). Locomotor activity data were compared using repeated measures A N O V A at each age with test type as a within subjects factor and treatment on PND7 as a between subjects factor. For the water maze task, trials were blocked into pairs to reduce variance; latencies and path lengths to find the hidden platform were analyzed using a repeated measures A N O V A (trial block as a within subjects factor, treatment on PND7 as a between subjects factor). Additionally, swim speeds were measured for all rats and compared using a repeated measures A N O V A . After the probe trials, the percent time spent in each quadrant was calculated by dividing the time spent in that quadrant by the total length of the probe trial (30 s). A repeated measures A N O V A was performed on the data with quadrant as a within subjects factor and treatment on PND7 as a between subjects factor. For all A N O V A ' s , post-hoc analyses were performed using the Neuman-Keuls test where appropriate. The significance level for all statistical tests was p < 0.05. Results Immediate Behavioral Effects ofPND7 KA Administration Ten to 20 min following K A administration, the majority of pups began to show behavioral signs of seizure activity as previously reported (Tremblay et al., 1984;Stafstrom et a l , 1992;Lynch et al., 2000;Silveira et a l , 2002). Initially, the majority of the pups showed evidence of a hunched posture and heavy breathing followed by the appearance of a continuous, clonic 'scratching' activity of the rear limbs. Many of the rats developed swimming seizures during which they rolled onto their sides during 78 bouts of forelimb and hindlimb clonus, which continued for 30 to 120 min. Some of the pups developed more severe tonic seizures in the second hour after K A administration, which lasted up to 60 min. Only data from pups that displayed behavioral signs of seizure activity for at least 30 minutes were analyzed in these experiments, and no differences in seizure severity between pups from Group 1 and Group 2 could be readily quantified. Six pups (11%) died as a result of the K A administration, while 10 (18%) were not used for the behavioral experiments due to mild, intermittent seizures. None of the saline-treated animals displayed seizure-related behaviors. Effects ofPND7 KA Administration on Body Weight On PND7, pups randomly selected for K A administration weighed 18.13 ± 0.37 g while those administered saline weighed 17.86 ± 0.46 g. On PND8, the pups were weighed again to ensure that suckling behavior was not altered between the groups after treatment on PND7. The K A - and saline-treated rats weighted 20.45 ± 0.45 g and 20.77 ± 0.55 g on PND8 respectively. An independent samples t-test revealed that both groups of animals gained a similar amount of weight during the 24 hr after treatment (t(50) = -1.28, N.S.). On PND36, the rats treated with K A on PND7 weighed slightly more than those treated with saline (KA: 181.90 ± 2 g; saline: 175.90 ± 2 g); however, when tested on PND57, the rats treated with K A weighed significantly more than those treated with saline (KA: 395.33 ± 5 g; saline: 379.66 ± 5 g) as revealed by a significant age by weight interaction (F(l, 68) = 3.98, p = 0.05). 79 A PND35 7 0 - , PP3 PP6 PP12 average Prepulse Intensity PP3 PP6 PP12 average Prepulse Intensity B PND35 140 i •g 120 H 100 -80 -Q. E •<; a> 60 -i OJ 40 CO CO 20 0 T i i saline kainic Treatment D PND56 140 -o 120-1 100 80 Q. E < a) 60 H oq 40 20 A 1 saline kainic Treatment Figure 3-1. Prepulse inhibition (PPI) scores from rats in Group 1 treated on postnatal day (PND) 7 with saline (white bars, n=17) or kainic acid (KA; black bars, n=20). A, Effects of.PND 7 K A treatment on PPI at PND35. B, Average starde amplitudes of the rats on PND35. C, Percent PPI scores for those rats tested at PND56. D, Average startle amplitudes of the rats on PND56. Asterisks indicate a significant difference between groups (p<0.05) and a cross indicates a difference between groups that approached significance (p<0.06). 80 Prepulse Inhibition is Reduced in Post-Pubescent, but not Pre-pubescent Rats, Following PND7 KA Administration Group 1: As shown in Fig. 3-1 A , rats administered either K A (n=20) or saline (n=17) on PND7 had similar levels of PPI on PND35. Percent PPI scores were similar in response to trials with 3 and 6 dB prepulses, whereas PPI was substantially increased for trials during which a 12 dB prepulse was presented.' A repeated measures A N O V A revealed a significant effect of prepulse (F(2, 70) = 78.92, p< 0.001), but no significant prepulse by treatment interaction (F(2, 70) = 0.26, N.S.) or main effect of treatment (F(l , 35) = 2.04, N.S.). On PND35, rats in the KA-treated group demonstrated slightly lower starde amplitudes than those in the saline treated group to the pulse alone trials (55.27 ± 5 vs. 67.51 ± 7 arbitrary startle units) groups (Fig. 3-1B). However, this difference was not significant (t(35) = -1.41, N.S.). In contrast to PND35, PPI levels obtained from K A - and saline-treated rats in young adulthood (PND56) were significantly different (Fig. 3-1C). A repeated measures A N O V A revealed a significant effect of prepulse intensity (F(2, 70) = 33.05, p < 0.001), as well as a significant prepulse by treatment interaction (F(2, 70) = 5.57, p < 0.01). Rats in the saline-treated condition showed a typical pattern of increasing percent PPI in response to prepulses of increasing intensity (24.80 ± 5% for PP3, 41.77 ± 4% for PP6, and 54.38 ± 4% for PP12 trials). When compared to the saline-treated animals, K A -treated rats responded with significantly lower percent PPI scores for the PP6 trials (19.17 ± 6% PPI, p < 0.05), and attenuated PPI in response to PP12 trials (47.15 ± 4%). As a result, animals in the KA-treated group demonstrated a 25% reduction in average percent PPI scores when compared with the saline-treated group (KA-treated = 30.18 ± 81 3%; saline-treated = 40.32 ± 4%), which approached significance (F(l, 35) = 3.93, p = 0.055). The average startle amplitude of the KA-treated rats (77.20 ± 12 arbitrary startle units) was lower than that of the saline-treated group (102.11 ± 18), although this difference was not significant (t(35) = -1.17, N.S.). Group 2: As in Group 1, rats in both the K A - (n=19) and saline- (n=14) treated groups tested on PND35 had similar levels of PPI (Fig. 3-2A). A repeated measures A N O V A revealed a significant main effect of prepulse intensity (F(2, 62) = 28.62, p <0.001), but no treatment effect (F(l, 31) = 0.15, N.S.) or prepulse by treatment interaction (F(2, 62) = 0.46, N.S.). Additionally, there was no significant difference in startle amplitude between the two groups (Fig. 3-2B; t(31) = 0.94, N.S.). When tested on PND56 rats in the KA-treated group again showed significantly reduced levels of PPI compared to saline-treated controls (KA-treated average PPI = 31.71 ± 2%, saline-treated average PPI = 39.95 ± 3%, Fig. 3-2C). A repeated measure A N O V A revealed the expected main effect of prepulse intensity (F(2, 62) = 47.28, p < 0.001) and a significant main effect of treatment (F(l, 31) = 4.17, p < 0.05), but no significant prepulse by treatment interaction (F(2, 62) = 0.393, N.S.). As with group 1, K A treatment on PND7 had no significant effect on startle amplitude at PND56 (Fig. 3-2D;t(31) = 0.85,N.S.). To further explore the effect of KA-treatment on PPI, we re-tested a subgroup of rats from group 2 following an injection of either vehicle (0.1% ascorbic acid) or apomorphine (0.2 mg/kg, s.e.) in separate test sessions 72 hr apart. Analysis of these data revealed a significant main effect of prepulse (F(2, 32) = 43.74, p < 0.001), thus indicating that PPI levels increased with increased intensity of the prepulse (data not 82 A PND35 PP3 PP6 PP12 average Prepulse Intensity C PND56 £-•30 CD OL 20 A PP3 PP6 PP 12 average Prepulse Intensity B PND35 140 . 120 "§ 100 CO co 80 60 40 20 saline kainic Treatment D PND56 140 „ 1 2 0 "§ 100 | - 80 60 tr. •a-C O 40 20 0-saline kainic Treatment Figure 3-2. Prepulse inhibition (PPI) scores from rats in Group 2 treated on postnatal day (PND) 7 with saline (white bars, n=14) or kainic acid (black bars, n=19). A, Effects of postnatal day (PND) 7 K A treatment on PPI at PND35. B, Average startle amplitudes of the rats on PND35. C, Percent PPI scores for those rats tested at PND56. D, Average startle amplitudes of the rats on PND56. The asterisk indicates a significant difference between groups (p<0.05). 83 shown). Additionally, a significant main effect of test (F(l, 16) = 16.94, p < 0.005) and a test by prepulse interaction (F(2,32) = 4.82, p < 0.02) were observed. Inspection of the data confirmed that apomorphine treatment significantly disrupted PPI in both groups of rats (Fig. 3-3A) while post-hoc analyses revealed that apomorphine disrupted PPI significantly more during the 6 dB prepulse trials than during either the 3 or 12 dB trials (p < 0.05, data not shown). Importantly, the absence of a significant test by treatment interaction (F(l,16) = 0.09, N.S.) does not support the hypothesis that animals treated with K A on PND7 are more sensitive to the disruptive effects of low doses of apomorphine on PPI. A separate analysis of the data obtained following the vehicle injection revealed that those animals treated with K A (n=10) on PND7 had lower PPI scores (38.36 ± 4%) than those treated with saline (n=8; 50.00 ± 6%, Fig. 3-3 A). Although this difference was of a similar magnitude to the differences in PPI reported at PND56 for groups 1 and 2 above, it failed to reach significance perhaps due to insufficient power (t(16) = -1.65, p = 0.12). Apomorphine treatment significantly decreased startle amplitude in both groups (Fig. 3-3B). This is reflected by a significant main effect of test (F(l,16) = 6.15, p < 0.05), but not group (F(l, 16) = 0.13, N.S.) or a group by test interaction (F(l, 16) = 0.07, N.S.). Locomotor Activity in Response to Novelty and Amphetamine in Rats Following PND7 KA Administration Group 1: As depicted in Fig. 3-4A and B, K A - (n=20) and saline- (n=17) treated rats in this group showed similar levels of spontaneous locomotor activity in a novel environment (60 min) or following amphetamine administration (1.5 mg/kg, 90 min) on 84 S-V K-V S-A K-A Treatment S-V K-V S-A K-A V T r e a t m e n t Figure 3-3. A, Effects of pretreatment with vehicle (0.1% ascorbic acid, A) or apomorphine (0.2 mg/kg, B) on average percent PPI scores in adulthood. Animals were administered either saline (n=8, white bars) or kainic acid (n=10, black bars) on postnatal day 7. B, Average startle amplitudes of those rats depicted in panel A. In both panels, asterisks indicate a significant difference between groups. The cross indicates a between group difference that approached significance (p<0.12). S-V= saline-vehicle treatment, S-A = saline-apomorphine treatment, K-V = kainic acid-vehicle treatment, K-A - kainic acid-apomorphine treatment. 85 A Nov PND36 o 1 4 o 12 X 10 £ 8 < 4 • TO 2 , S-1 K-1 S-2 K-2 Treatment o 1 4 0 12 >< 10 1 8 £ 6 4 < 4 -iS 2 S-1 K-1 S-2 K-2 Treatment B A m p h PND36 o 3 5 -° 3 0 -X;25-^20-•^ •'1.5-o < 10-I 5 0 S-1 K-1 S-2 K-2 Treatment C Nov PND57 D A m p h PND57 S-1 K-1 S-2 K-2 : Treatment Figure 3-4. Locomotor activity levels in response to novelty (A, C) or amphetamine (1.5 mg/kg; B, D) of all rats tested on either postnatal day (PND) 36 (A, B) or PND57 (C, D). Group labels on the x-axis of each panel reflect the treatment of the rats on PND7 (5 = saline, K - kainic acid) and their group (1 = group 1,2 = group 2). The asterisk indicates a significant difference between the denoted groups (p<0.05). Note the units for activity levels (y-axis) for each panel. 86 PND36. A repeated measures A N O V A failed to confirm a significant treatment effect (F(l, 35) = 0.16, N.S.) or treatment by test interaction (F(l , 35) = 0.12, N.S.). At PND57, a repeated measures A N O V A did not reveal a main effect of treatment (F(l, 35) = 2.01, N.S.), but did reveal a significant test by treatment interaction (F(l, 35) = 4.10, p = 0.05). Post-hoc analyses revealed that although both groups showed similar locomotor responses in the novelty condition (Fig. 3-4C), rats treated with K A on PND7 had significantly higher locomotor responses (2640.25 ± 225 beam crosses) following injection with amphetamine than rats treated with saline (2145.06 ± 143; Fig. 3-4D). Group 2: Similar to the data presented for Group 1, the locomotor responses of rats treated with either K A (n=19) or saline (n=14) on PND7 did not differ in either the spontaneous activity or amphetamine conditions on PND36 (Fig. 3-4A and B). This is reflected in an insignificant main effect of treatment (F(l, 31) = 1.30, N.S.) and treatment by test interaction (F(l , 31) = 2.29, N.S.). In contrast to the data obtained from Group 1, when tested on PND57, rats in Group 2 failed to show significant differences in locomotor activity in either condition (main effect of treatment: F ( l , 31) = 1.70, N.S.; treatment by test interaction: F ( l , 31) = 1.66, N.S.). Spatial Learning and Memory in the Morris Water Maze is not Altered in Rats Following PND7 KA Administration On the first test day, all rats learned to escape by swimming to the visible platform (data not shown). On the second day of testing, rats in both groups learnt to swim to the hidden platform (Fig. 3-5A). A repeated measures A N O V A performed on the latency data revealed a significant main effect of trial block (F(4, 52) = 17.43, p < 0.001), but not a significant effect of group (F(l, 13) = 0.279, N.S.) or a significant block 87 A E F 50 40 30 20 10 0 -n———r———i •'•.- i-B Trial Block 1 • ' / .2 . . 3 ; Quadrant Figure 3-5. A, Average latencies (s) to locate the hidden platform in the water maze of rats treated with saline (white diamonds, n=7) or kainic acid (black squares, n=8) on postnatal day 7. Trial blocks represent the average search time of 2 trials for each rat. The asterisk indicates a significant difference between trial blocks (p<0.05). B, Percent time spent by the rats in each quadrant of the water maze 24 hr after the training trials depicted in A. On the training day, the platform was located in quadrant 2. The asterisk denotes a significant difference in percent time between quadrant 2 and quadrant 3 (p<0.05), while the # denotes a significant difference in percent time between quadrant 4 and quadrant 1 and 2 (p<0.05). 88 by group interaction (F(4, 52) = 0.487, N.S.). Post-hoc analyses revealed that both the K A - (n=8) and saline-treated (n=7) rats were significantly slower at finding the platform during block 1 (i.e. trials 1 and 2) than the subsequent 4 blocks (average search time -block 1: 38.66 ± 6 s; blocks 2 to 5: 14.38 ± 3 s; p < 0.05). Path lengths were significantly reduced across trial blocks in both groups (F(4, 52) = 14.33, p < 0.01; data not shown); however, neither total path length (KA-treated = 4,029.10 ± 483 cm, saline-treated = 4597.04 ± 609 cm; F ( l , 13) = 1.56, N.S.) nor swim speed (KA-treated = 22.33 ± 1 cm/s, saline-treated = 25.14 ± 1 cm/s; F ( l , 13) = 3.21, N.S.) differed significandy between the groups. When retention of the platform location was tested 24 hr later using a 30 s probe trial, both groups spent most of their time searching in the quadrant which had contained the platform the previous day (Fig. 3-5B). A repeated measures A N O V A revealed a significant main effect of quadrant (F(3, 39) = 20.88, p < 0.001), but no significant group by quadrant interaction (F(3, 39) = 0.581, N.S.). Post-hoc analyses revealed that both groups spent significandy more time in quadrant 2 (41.65 ± 5 %), which contained the platform, than in either quadrants 3 (19.22 ± 4 %) or 4 (8.24 ± 3 %). Inspection of the data presented in Fig. 3-5B reveals that the animals also spent more time in quadrant 2 than quadrant 1 (30.89 ± 3 %), although this difference failed to reach significance. Histology In agreement with previous studies (Nitecka et al., 1984;Stafstrom et al., 1992;Koh et al., 1999;Lynch et al., 2000), histological examination of the hippocampi of rats which had received either K A or saline on PND7 revealed no signs of gross cell loss or hippocampal damage (Fig. 3-6). 89 Figure 3-6. A representative cresyl violet stained section of the dorsal hippocampus of a KA-treated (A) and saline-treated rat (B). 90 Discussion The present study demonstrates that treatment with K A on PND7 results in the delayed emergence of behavioral changes in adulthood not seen before puberty. Following treatment with K A on PND7, rats in two independent groups showed disrupted PPI on PND56, but not PND35. When re-tested 6 weeks later, a subgroup of the K A -treated animals continued to exhibit relatively lower PPI than saline-treated controls. Additionally, administration of a low dose of apomorphine (0.2 mg/kg) disrupted PPI in both K A - and saline-treated rats; however, KA-treated rats failed to show enhanced sensitivity to the disruptive effects of apomorphine. When the locomotor responses of these animals in response to a novel environment and amphetamine challenge were analyzed, an inconsistent pattern emerged. K A - and saline-treated rats had similar locomotor responses to both novelty and amphetamine on PND36, as well as novelty on PND57. Following amphetamine administration (1.5 mg/kg) on PND57, locomotor activity was significantly increased in the KA-treated rats from Group 1, but not from Group 2. Furthermore, neither spatial learning nor memory were affected by the administration of K A on PND7 as assessed in the Morris water-maze, confirming previous studies (Stafstrom et al., 1993;Koh et al., 1999). Effects of Neonatal KA Administration on PPI In two separate groups of rats, we observed that K A treatment on PND7 significantly disrupted PPI in postpubescence, but not before puberty. It is important to note that PPI has been reliably measured in rat pups as young as 16 days of age (Martinez et al., 2000;Swerdlow et al., 2000), and that the rats tested in the present experiments on PND35 showed consistent levels of PPI with relatively low variance. Post-pubescent rats 91 treated with K A on PND7 had mean PPI levels that were 75 to 80% of those shown by saline-treated animals. This effect was observed consistently in two groups of animals tested independendy (Fig. 3-1C and 3-2C) and was evident without changes in startle amplitude (Fig. 3-1D and 3-2D). Previous studies have reported the effects of neonatal manipulations on PPI in adult rats. Data presented by Lipska and colleagues (Lipska et a l , 1995; Fig. 4) from rats that had sustained permanent lesions of the vHip on PND7, indicate PPI levels that were approximately 70% of their control littermates, although a significant difference between groups was noted only at the lowest prepulse intensity (4 dB above baseline). Other studies report slightiy larger effects over a wider range of prepulse intensities following neonatal lesions of the vHip (Le Pen and Moreau, 2002;Daenen et al., 2003) and the amygdala (Daenen et al., 2003) or treatment with M K -801 on PND7 (Harris et al., 2003). Given that these treatments result in relatively subtle disruptions in PPI under baseline testing conditions, it is perhaps not surprising that K A treatment on PND7, which does not cause gross brain damage (Nitecka et al., 1984;Stafstrom et al., 1992;Koh et al., 1999;Lynch et a l , 2000; present data), causes relatively small disruptions in PPI. Rats with neonatal vHip lesions also show heightened sensitivity to the disruptive effects of apomorphine (0.1 mg/kg), thereby supporting the hypothesis that mesolimbic D A transmission is altered in the lesioned rats (Lipska et al., 1995). In the present study, K A treatment on PND7 failed to enhance the disruptive effects of apomorphine treatment on PPI. However, administration of 0.2 mg/kg of apomorphine immediately before testing disrupted PPI in both the K A - and saline-treated animals (Fig. 3-3A). This result is surprising because Long-Evans rats are reported to be less sensitive than Sprague 92 Dawley rats (the strain used by Lipska et al., 1995) to the disruptive effects of apomorphine on PPI. Indeed, it has been demonstrated that Long-Evans fats fail to show disrupted PPI following administration of doses of apomorphine as high as 0.5 mg/kg (Swerdlow et al., 2001). Although no clear explanation exists for these discrepant data, complex interactions between rat strain and PPI have been studied extensively (Varty and Geyer, 1998;Ellenbroek and Cools, 2000;Swerdlow et al., 2000). In this context, further examination of the effect of early KA-treatment's potentially interactive effects with apomorphine may be profitable in other rat strains. Using C57BL6 mice, Yee and colleagues (Yee et al., 2004a; 2004b) elegandy demonstrated that pretreatment with dopamine agonists such as apomorphine or non-competitive A/-methyl-D-aspartate antagonists, such as dizocilpine and phencyclidine, disrupts PPI, a finding that has previously been well documented (Geyer et al., 2001). However, when prepulse-elicited reactivity was examined, a dissociation between the two classes of drugs was observed whereby apomorphine increases and dizocilpine and phencyclidine decrease prepulse-elicited reactivity (Yee et al., 2004a; 2004b). Incorporation of a prepulse alone condition in future experiments may allow for parallels to be drawn between these pharmacological studies and K A treatment. For example, if K A administration on PND7 decreased prepulse-elicited startle, it could be argued that K A administration has effects similar to dizocilpine or phencyclidine pretreatment, but not apomorphine pretreatment. Effects of Neonatal KA on Locomotor Activity Increased locomotor activity in response to treatments that activate the mesolimbic D A system is a cardinal feature of many animal models of schizophrenia 93 (Lipska et al., 1993;Hall, 1998;Weiss et al., 2000;Lipska et al., 2002;Harris et a l , 2003). Neonatal K A treatment in the present study failed to increase locomotor behavior consistently following exposure to a novel environment or an amphetamine injection, although Group 1 did show a significant increase in locomotor activity following treatment with amphetamine (Fig. 3-4D). It is unclear why KA-treated rats in Group 2 failed to show elevated locomotor responses following amphetamine treatment on PND57 given that both groups displayed similar seizure-related behaviors following K A administration on PND7 and similar deficits in PPI when tested at PND56. These results, considered in conjunction with the failure of apomorphine to affect PPI, question the role of postsynaptic alterations in the mesolimbic D A system in the observed effects of K A treatment on PPI. Furthermore, the absence of consistent changes in locomotor behavior in early adulthood following KA-induced aberrant activity in developing cortico-limbic circuits on PND7, underscores important differences between these data and the decreased activity induced by the T T X administration into the vHip (Lipska et al., 2002) and systemic MK-801 administration (Harris et al., 2003). Potential Mechanisms Underlying the Observed PPI Changes TTX infusions into the vHip on PND7 potentiate locomotor activity in early adulthood (Lipska et al., 2002) and systemic administration of the N M D A antagonist MK-801 on PND7 may also increase locomotor activity and disrupt PPI (Harris et al., 2003, but see Beninger et al., 2002). Lipska and colleagues failed to detect any gross morphological changes in the adult brains of animals injected with T T X on PND7 (Lipska et al., 2002). In contrast, administration of MK-801 on PND7 increases apoptosis in limbic and cortical brain areas such as the hippocampus and thalamus 94 (Harris et al., 2003), and this effect may underlie the observed behavioral changes in adult animals. Taken together, these data suggest that neural circuits subserving locomotor behavior and PPI are particularly sensitive to alterations in neural activity during the first postnatal week. A l l rats included in this study displayed generalized tonic-clonic seizures for 1 to 2 hr following K A treatment on PND7, therefore it is reasonable to assume that aberrant activity would have occurred in neural circuits that regulate PPI and locomotor activity in adulthood (i.e. the hippocampus and septum; (Tremblay et al., 1984;Khalilov et al., 1999;Silveira et al., 2002). This raises the question of whether the behavioral effects observed in this study are the result of the activation of glutamate receptors and/or the consequence of seizures early in postnatal development. Obviously, these possibilities cannot be resolved based on the present experiments. However, it is well known that ionotropic glutamate receptor activity is critical for the normal formation of cortico-limbic circuits (Ben Ar i et al., 1997;Feldman and Knudsen, 1998;Luthi et al., 2001). Although most of this research has focused on N M D A and A M P A receptor mediated events, some recent studies suggest that kainate receptors are dynamically regulated during critical periods of experience-dependent plasticity in thalamocortical synapses (Kidd and Isaac, 1999;Kidd et al., 2002). As a result, it is conceivable that hyperstimulation of kainate receptors could result in developmental alterations of the neural circuitry known to regulate PPI. Given that K A was administered systemically in the present study and genes encoding kainate receptor subunits are widely expressed in the brain during early postnatal development (Bahn et al., 1994), K A administration 95 could have subtly altered a number of brain regions, thereby producing the observed behavioral effects. The dose of K A (1.5 mg/kg) used in the present study induced seizures in the neonatal rats, therefore the effects of seizures, and not ionotropic glutamate receptor hyperstimulation per se, may also underlie the behavioral effects observed. Seizures induced by systemic administration of K A on PND7 increase glucose metabolism primarily in the dorsal hippocampus and lateral septum (Tremblay et al., 1984) and transiendy increase c-fos expression in the hippocampus (Silveira et al., 2002). Significant changes in the morphology of the hippocampus observed routinely following K A administration post-weaning are not evident after acute systemic treatment with K A during postnatal weeks 1 and 2 (Nitecka et al., 1984;Stafstrom et al., 1992;Koh et al., 1999;Lynch et al., 2000). More subtle alterations in the excitability of limbic circuits have been noted following administration of K A on PND7 including a reduction in long-term potentiation in the dentate gyrus, slower kindling rates, and increased paired pulse inhibition in the perforant path in adult rats, accompanied by impairments of spatial learning as assessed on a radial arm maze (Lynch et al., 2000). Exposure to K A on PND15 may also exacerbate cognitive impairment and cell loss in the hippocampus induced by seizures in adulthood (Koh et al., 1999). Recently, i.c.v. administration of K A (10-50 nmol) on PND7 has been demonstrated to cause a relatively subtle loss of hippocampal neurons, particularly in the CA3 and CA1 subregions of the hippocampus (Montgomery et al., 1999;Dong et al., 2003a). Although some cell loss is observed immediately following K A administration on PND7, greater cell loss occurs in adulthood (Humphrey et al., 96 2002). Interestingly, dynamic alterations in neurogenesis are correlated with this cell loss; when more hippocampal cells are lost, the rate of neurogenesis in the dentate gyrus is increased (Dong et al., 2003b). These results obtained with i.e.v. administration suggest that hippocampal circuits may be particularly vulnerable to alterations caused by K A during the end of the first postnatal week. It is important to note that the histological methods used in the present study preclude a detailed analysis of subtle changes in hippocampal circuits such as those discussed above following i.e.v. administration. However, given the findings of Lynch and colleagues (2000) and those using i.e.v. administration of K A , the changes in PPI observed in the present study may be attributed to alterations in the development of the hippocampus, particularly the dentate gyrus. This hypothesis receives further support from studies in adult rats that confirm a role for the hippocampus and dentate gyrus in the neural circuitry that regulates PPI. For example, stimulation or deactivation of the hippocampus disrupts PPI (for review see Bast and Feldon, 2003), and infusion of the cholinergic agonist carbachol into the dentate gyrus disrupts PPI (Caine et al., 1991; 1992), an effect that is not reversed by pretreatment with the D2 antagonist spiperone (Caine et al., 1991). Additionally, reduced synaptophysin immunoreactivity (Varty et al., 1999) and alterations in specific interneurons (Greene et al., 2001) are observed in the dentate gyri of socially-isolated rats that exhibit disrupted PPI. Conclusion Attempts to model the etiologies of psychiatric disorders such as schizophrenia in animals are challenging. A l l current models have drawbacks, mainly attributable to the specific brain manipulations performed (Lipska and Weinberger, 2000;Penn, 2001 ;Van 97 den Buuse et al., 2003). The data reported here demonstrate that a single period of intense cortico-limbic activity induced by the systemic administration of K A on PND7 can cause a selective disruption of PPI that is only observed in adult rats. This provides further support for the hypothesis that in rats, the end of the first postnatal week is a critical period for the development of cortico-limbic circuits that mediate behaviors such as PPI and locomotor activity (Lipska et al., 1993; 1995; Wood et al., 1997;Daenen et al., 2002; 2003;Harris et al., 2003). However, the present apomorphine-PPI experiment and locomotor activity experiments do not support the notion that acute administration of K A on PND7 results in the expression of an array of behavioral changes consistent with symptoms of schizophrenia. 98 References Bahn S, Volk B , Wisden W (1994) Kainate Receptor Gene-Expression in the Developing Rat-Brain. J Neurosci 14: 5525-5547. Bast T, Feldon J (2003) Hippocampal modulation of sensorimotor processes. Prog Neurobiol 70: 319-345. Ben Ari Y , Cossart R (2000) Kainate, a double agent that generates seizures: two decades of progress. Trends Neurosci 23: 580-587. Ben Ar i Y , Khazipov R, Leinekugel X , Caillard O, Gaiarsa JL (1997) G A B A A , N M D A and A M P A receptors: a developmentally regulated 'menage a trois'. Trends Neurosci 20: 523-529. Benes F M (2000) Emerging principles of altered neural circuitry in schizophrenia. Brain Res Brain Res Rev 31: 251-269. Benes F M , Taylor JB, Cunningham M C (2000) Convergence and plasticity of monoaminergic systems in the medial prefrontal cortex during the postnatal period: implications for the development of psychopathology. Cereb Cortex 10: 1014-1027. Beninger RJ, Jhamandas A , Aujla H , Xue L , Dagnone RV, Boegman RJ, Jhamandas K (2002) Neonatal exposure to the glutamate receptor antagonist MK-801: effects on locomotor activity and pre-pulse inhibition before and after sexual maturity in rats. Neurotox Res 4: 477-488. Bogerts B (1997) The temporolimbic system theory of positive schizophrenic symptoms. Schizophr Bull 23: 423-435. Brake WG, Flores G, Francis D, Meaney M J , Srivastava L K , Gratton A (2000) Enhanced nucleus accumbens dopamine and plasma corticosterone stress responses in adult rats with neonatal excitotoxic lesions to the medial prefrontal cortex. Neuroscience 96: 687-695. Caine SB, Geyer M A , Swerdlow NR (1991) Carbachol infusion into the dentate gyrus disrupts sensorimotor gating of startle in the rat. Psychopharmacology (Berl) 105: 347-354. Caine SB, Geyer M A , Swerdlow NR (1992) Hippocampal modulation of acoustic startle and prepulse inhibition in the rat. Pharmacol Biochem Behav 43: 1201-1208. Daenen EW, Van Der Heyden JA, Kruse CG, Wolterink G, Van Ree J M (2001) Adaptation and habituation to an open field and responses to various stressful events in animals with neonatal lesions in the amygdala or ventral hippocampus. Brain Res 918: 153-165. 99 Daenen EW, Wolterink G, Gerrits M A , Van Ree J M (2002) Amygdala or ventral hippocampal lesions at two early stages of life differentially affect open field behaviour later in life; an animal model of neurodevelopmental psychopathological disorders. Behav Brain Res 131: 67-78. Daenen EW, Wolterink G, Van Der Heyden JA, Kruse C G , Van Ree J M (2003) Neonatal lesions in the amygdala or ventral hippocampus disrupt prepulse inhibition of the acoustic startle response; implications for an animal model of neurodevelopmental disorders like schizophrenia. Eur Neuropsychopharmacol 13: 187-197. de Feo MR, Mecarelli O, Palladini G, Ricci GF (1986) Long-term effects of early status epilepticus on the acquisition of conditioned avoidance behavior in rats. Epilepsia 27: 476-482. Dong H , Csernansky C A , Chu Y , Csernansky JG (2003a) Intracerebroventricular kainic acid administration to neonatal rats alters interneuron development in the hippocampus. Brain Res Dev Brain Res 145: 81-92. Dong H , Csernansky C A , Goico B , Csernansky JG (2003b) Hippocampal neurogenesis follows kainic acid-induced apoptosis in neonatal rats. J Neurosci 23: 1742-1749. Ellenbroek B A , Cools A R (2000) The long-term effects of maternal deprivation depend on the genetic background. Neuropsychopharmacology 23: 99-106. Feldman DE, Knudsen EI (1998) Experience-dependent plasticity and the maturation of glutamatergic synapses. Neuron 20: 1067-1071. Flores G, Wood GK, Liang JJ, Quirion R, Srivastava L K (1996) Enhanced amphetamine sensitivity and increased expression of dopamine D2 receptors in postpubertal rats after neonatal excitotoxic lesions of the medial prefrontal cortex. J Neurosci 16: 7366-7375. Friston KJ (1999) Schizophrenia and the disconnection hypothesis. Acta Psychiatr Scand Suppl 395: 68-79. Geyer M A , Krebs-Thomson K, Braff DL, Swerdlow NR (2001) Pharmacological studies of prepulse inhibition models of sensorimotor gating deficits in schizophrenia: a decade in review. Psychopharmacology (Berl) 156: 117-154. Gothelf D, Soreni N , Nachman RP, Tyano S, Hiss Y , Reiner O, Weizman A (2000) Evidence for the involvement of the hippocampus in the pathophysiology of schizophrenia. Eur Neuropsychopharmacol 10: 389-395. Greene JR, Kerkhoff JE, Guiver L , Totterdell S (2001) Structural and functional abnormalities of the hippocampal formation in rats with environmentally induced reductions in prepulse inhibition of acoustic startle. Neuroscience 103: 315-323. Hall FS (1998) Social deprivation of neonatal, adolescent, and adult rats has distinct neurochemical and behavioral consequences. CritRev Neurobiol 12: 129-162. 100 Harris L W , Sharp T, Gartlon J, Jones D N , Harrison PJ (2003) Long-term behavioural, molecular and morphological effects of neonatal N M D A receptor antagonism. Eur J Neurosci 18: 1706-1710. Harrison PJ (1999) The neuropathology of schizophrenia. A critical review of the data and their interpretation. Brain 122 (Pt 4): 593-624. Heckers S, Rauch SL, Goff D, Savage CR, Schacter DL , Fischman A J , Alpert N M (1998) Impaired recruitment of the hippocampus during conscious recollection in schizophrenia. Nat Neurosci 1: 318-323. Humphrey W M , Dong H , Csernansky C A , Csernansky JG (2002) Immediate and delayed hippocampal neuronal loss induced by kainic acid during early postnatal development in the rat. Brain Res Dev Brain Res 137: 1-12. Jones E G (1997) Cortical development and thalamic pathology in schizophrenia. Schizophr Bull 23: 483-501. Khalilov I, Dzhala V , Medina I, Leinekugel X , Melyan Z, Lamsa K, Khazipov R, Ben Ar i Y (1999) Maturation of kainate-induced epileptiform activities in interconnected intact neonatal limbic structures in vitro. Eur J Neurosci 11: 3468-3480. Kidd FL, Coumis U , Collingridge GL, Crabtree JW, Isaac JT (2002) A presynaptic kainate receptor is involved in regulating the dynamic properties of thalamocortical synapses during development. Neuron 34: 635-646. Kidd FL, Isaac JT (1999) Developmental and activity-dependent regulation of kainate receptors at thalamocortical synapses. Nature 400: 569-573. Koh S, Storey TW, Santos TC, Mian A Y , Cole AJ (1999) Early-life seizures in rats increase susceptibility to seizure-induced brain injury in adulthood. Neurology 53: 915-921. Le Pen G, Moreau JL (2002) Disruption of prepulse inhibition of startle reflex in a neurodevelopmental model of schizophrenia: reversal by clozapine, olanzapine and risperidone but not by haloperidol. Neuropsychopharmacology 27: 1-11. Lipska B K , al Amin H A , Weinberger DR (1998) Excitotoxic lesions of the rat medial prefrontal cortex. Effects on abnormal behaviors associated with neonatal hippocampal damage. Neuropsychopharmacology 19: 451-464. Lipska B K , Halim ND, Segal PN, Weinberger DR (2002) Effects of reversible inactivation of the neonatal ventral hippocampus on behavior in the adult rat. J Neurosci 22: 2835-2842. Lipska B K , Jaskiw GE, Weinberger DR (1993) Postpubertal emergence of hyperresponsiveness to stress and to amphetamine after neonatal excitotoxic hippocampal damage: a potential animal model of schizophrenia. Neuropsychopharmacology 9: 67-75. 101 Lipska B K , Swerdlow NR, Geyer M A , Jaskiw GE, Braff DL, Weinberger DR (1995) Neonatal excitotoxic hippocampal damage in rats causes post-pubertal changes in prepulse inhibition of startle and its disruption by apomorphine. Psychopharmacology (Berl) 122: 35-43. Lipska B K , Weinberger DR (2000) To model a psychiatric disorder in animals: schizophrenia as a reality test. Neuropsychopharmacology 23: 223-239. Luthi A , Schwyzer L , Mateos J M , Gahwiler B H , McKinney R A (2001) N M D A receptor activation limits the number of synaptic connections during hippocampal development. Nat Neurosci 4: 1102-1107. Lynch M , Sayin U , Bownds J, Janumpalli S, Sutula T (2000) Long-term consequences of early postnatal seizures on hippocampal learning and plasticity. Eur J Neurosci 12: 2252-2264. Martinez ZA, Halim ND, Oostwegel JL, Geyer M A , Swerdlow NR (2000) Ontogeny of phencyclidine and apomorphine-induced startle gating deficits in rats. Pharmacol Biochem Behav 65: 449-457. McClure RK, Weinberger DR (2001) The neurodevelopmental hypothesis of schizophrenia: A review of the evidence. In: Current Issues in the Psychopharmacology of Schizophrenia. (Breier A , Tran PV, Herrea J M , Tollefson GD, Bymaster FP, eds), pp 27-56. New York: Lippincott Williams & Wilkins. Meyer-Lindenberg A, Miletich RS, Kohn PD, Esposito G, Carson RE, Quarantelli M , Weinberger DR, Berman K F (2002) Reduced prefrontal activity predicts exaggerated striatal dopaminergic function in schizophrenia. Nat Neurosci 5: 267-271. Montgomery E M , Bardgett M E , Lall B , Csernansky C A , Csernansky JG (1999) Delayed neuronal loss after administration of intracerebroventricular kainic acid to preweanling rats. Brain Res Dev Brain Res 112: 107-116. Morris RG, Schenk F, Tweedie F, Jarrard L E (1990) Ibotenate Lesions of Hippocampus and/or Subiculum: Dissociating Components of Allocentric Spatial Learning. Eur J Neurosci 2: 1016-1028. Nitecka L , Tremblay E, Charton G, Bouillot JP, Berger M L , Ben Ari Y (1984) Maturation of kainic acid seizure-brain damage syndrome in the rat. II. Histopathological sequelae. Neuroscience 13: 1073-1094. Pakkenberg B (1990) Pronounced reduction of total neuron number in mediodorsal thalamic nucleus and nucleus accumbens in schizophrenics. Arch Gen Psychiatry 47: 1023-1028. Penn A A (2001) Early brain wiring: activity-dependent processes. Schizophr Bull 27: 337-347. 102 Silveira DC, Sogawa Y , Holmes G L (2002) The expression of Fos following kainic acid-induced seizures is age-dependent. Eur J Neurosci 15: 329-344. Sperk G (1994) Kainic acid seizures in the rat. Prog Neurobiol 42: 1-32. Stafstrom CE, Chronopoulos A, Thurber S, Thompson JL, Holmes G L (1993) Age-dependent cognitive and behavioral deficits after kainic acid seizures. Epilepsia 34: 420-432. Staf strom CE, Thompson JL, Holmes G L (1992) Kainic acid seizures in the developing brain: status epilepticus and spontaneous recurrent seizures. Brain Res Dev Brain Res 65: 227-236. Stevens JR (1994) Brain atrophy or dystrophy in schizophrenia: when did it happen? Arch Gen Psychiatry 51: 927. Suddath RL, Christison GW, Torrey EF, Casanova MF, Weinberger DR (1990) Anatomical abnormalities in the brains of monozygotic twins discordant for schizophrenia. N Engl J Med 322: 789-794. Swerdlow NR, Martinez ZA, Hanlon F M , Platten A, Farid M , Auerbach P, Braff DL, Geyer M A (2000) Toward understanding the biology of a complex phenotype: rat strain and substrain differences in the sensorimotor gating-disruptive effects of dopamine agonists. J Neurosci 20: 4325-4336. Swerdlow NR, Platten A , Kim Y K , Gaudet I, Shoemaker J, Pitcher L , Auerbach P (2001) Sensitivity to the dopaminergic regulation of prepulse inhibition in rats: evidence for genetic, but not environmental determinants. Pharmacol Biochem Behav 70: 219-226. Tremblay E, Nitecka L, Berger M L , Ben Ar i Y (1984) Maturation of kainic acid seizure-brain damage syndrome in the rat. I. Clinical, electrographic and metabolic observations. Neuroscience 13: 1051-1072. Van den B M , Garner B , Koch M (2003) Neurodevelopmental animal models of schizophrenia: effects on prepulse inhibition. Curr Mol Med 3: 459-471. Varty G B , Geyer M A (1998) Effects of isolation rearing on startle reactivity, habituation, and prepulse inhibition in male Lewis, Sprague-Dawley, and Fischer F344 rats. Behav Neurosci 112: 1450-1457. Varty G B , Marsden C A , Higgins G A (1999) Reduced synaptophysin immunoreactivity in the dentate gyrus of prepulse inhibition-impaired isolation-reared rats. Brain Res 824: 197-203. Weinberger DR (1995) Schizophrenia as a neurodevelopmental disorder: A review of the concept. In: Schizophrenia (Hirsch SR, Weinberger DR, eds), pp 293-323. London: Blackwood. 103 Weiss IC, D i Iorio L , Feldon J, Domeney A M (2000) Strain differences in the isolation-induced effects on prepulse inhibition of the acoustic startle response and on locomotor activity. Behav Neurosci 114: 364-373. Wood GK, Lipska B K , Weinberger DR (1997) Behavioral changes in rats with early ventral hippocampal damage vary with age at damage. Brain Res Dev Brain Res 101: 17-25. Yee B K , Chang DL, Feldon J (2004a) The Effects of dizocilpine and phencyclidine on prepulse inhibition of the acoustic startle reflex and on prepulse-elicited reactivity in C57BL6 mice. Neuropsychopharmacology 29: 1865-1877. Yee B K , Russig H, Feldon J (2004b) Apomorphine-induced prepulse inhibition disruption is associated with a paradoxical enhancement of prepulse stimulus reactivity. Neuropsychopharmacology 29: 240-248. 104 CHAPTER FOUR: BEHAVIORAL CONVULSIONS INDUCED BY E A R L Y POSTNATAL ADMINISTRATION OF THE NR2B ANTAGONIST R025-6981 FAIL TO AFFECT SENSORIMOTOR GATING OR LOCOMOTOR BEHAVIOR IN PRE-AND POST-PUBESCENT RATS. 3 Introduction Schizophrenia is a complex disorder resulting from multiple etiological risk factors. Adverse events during early development have been proposed to contribute to the etiology of schizophrenia (Weinberger, 1987). Supporting evidence includes increased risk of developing schizophrenia following perinatal events such as maternal infection and obstetrical complications (for review see Lewis and Levitt, 2002). Additionally, subtle signs of cognitive and behavioral impairment are present during childhood and early adolescence in those who go on to develop schizophrenia in adulthood (Lewis and Levitt, 2002). These observations, among others, led to the proposal that the primary pathology of schizophrenia occurs during early development and its effects somehow remain largely silent until adulthood (Weinberger, 1987;Lewis and Levitt, 2002). The effects of disturbing the early neural development of rats on behavioral measures relevant to schizophrenia are being examined to test this hypothesis. During early postnatal life, glutamate neurotransmission, particularly via N M D A receptors, is critical for the formation of neural circuits due to its established role in processes such as synaptogenesis (Rabacchi et al., 1992;Fox et al., 1996;Luthi et al., 2001), cell birth (Gould et al., 1994), and cell survival (Vallano, 1998;Ikonomidou et a l , 1999). Abnormalities of the glutamate system, with an emphasis On N M D A hypofunction, have been linked to patients with schizophrenia (Jentsch and Roth, 3 A version of this Chapter will be submitted for publication: Howland JG, Choi F Y , Phillips A G (2005) Behavioral convulsions induced by early postnatal administration of the NR2B antagonist Ro25-6981 fail to affect sensorimotor gating or locomotor behavior in pre- and post-pubescent rats. 105 1999;01ney et al., 1999;Goff and Coyle, 2001;Coyle and Tsai, 2004). Accordingly, previous studies have administered non-competitive N M D A antagonists such as phencyclidine, ketamine, and MK-801 to neonatal rats and demonstrated alterations in several behavioral paradigms including prepulse inhibition (PPI; (Wang et al., 2001;Harris et al., 2003), locomotor activity (Facchinetti et a l , 1993;Wang et al., 2001;Harris et al., 2003;Fredriksson et al., 2004), stereotypy (Semba et al., 2001), set-shifting (Stefani and Moghaddam, 2005), and spatial memory (Gorter and de Bruin, 1992;Wang et al., 2001;Fredriksson et al., 2004;Stefani and Moghaddam, 2005). Importandy, blocking N M D A receptor function at the end of the first postnatal week causes extensive apoptosis throughout forebrain areas implicated in schizophrenia such as the hippocampus, prefrontal cortex, striatum, and thalamus (Ikonomidou et al., 1999;Wang et al., 2001;Harris et al., 2003;Fredriksson et a l , 2004) thereby providing a potential mechanism that may underlie the behavioral changes observed in adulthood. Recently, an explosion in knowledge surrounding the composition of N M D A receptors and their subunit composition has emerged (Cull-Candy et a l , 2001). This provides a unique opportunity to examine the potential role of various subclasses of the receptors in disorders such as schizophrenia. N M D A receptors are heterodimers composed of a variety of subunits (Fig. 4-1) and over the course of development, the expression levels of some subunits undergo dynamic change. At birth, NR2B subunits are expressed at high levels, whereas expression of NR2A subunit levels is low. Over the first two to three weeks of development, this pattern reverses, and during late adolescence and into adulthood, N M D A receptors containing NR2A subunits predominate (Monyer et a l , 1994;Guilarte and McGlothan, 1998;Ritter et al., 2002;Zhang et al., 2002). As the 106 Figure 4-1. Cartoon illustrating an N M D A receptor within the cell membrane. N M D A receptors are assemblies of various subunits including NR1, NR2A to NR2D, and NR3A and NR3B. Functional receptors contain two NR1 subunits and some combination of two additional subunits. As is depicted, the binding site for non-competitive N M D A antagonists such as MK-801 and phencyclidine is located within the pore of the receptor. Thus, the receptor must be open in order for these antagonists to bind. In the present experiments, the NR2B-specific antagonists Ro25-6981 and ifenprodil were used, which have been reported to bind in the region of the polyamine site. Interestingly, these drugs only bind to receptors termed diheteromers that contain two NR1 subunits and two NR2B subunits. In this manner, their high degree of specificity is achieved. 107 subunit composition of N M D A receptors determine their precise kinetics, the unique subunit composition of N M D A receptors during the neonatal period is likely to be an important factor in normal development (Cull-Candy et al., 2001). Interestingly, numerous lines of evidence suggest that subunit-specific changes of the N M D A receptor may exist in schizophrenia: (1) significant increases in the expression of NR2B subunits have been noted in the hippocampus (Gao et al., 2000), thalamus (Clinton and Meador-Woodruff, 2004), and temporal cortex (Grimwood et al., 1999) of schizophrenic patients, (2) increased expression of NR2D in the prefrontal cortex (Akbarian et al., 1996) and decreases in NR1 expression in the hippocampus (Gao et al., 2000) have been demonstrated, (3) novel polymorphisms and variants in the promoter regions of the NR2A (Itokawa et a l , 2003) and NR2B genes (Miyatake et al., 2002; Qin et al., 2005) may also exist in schizophrenia, (4) behavioral and neurochemical abnormalities consistent with the disorder are observed in mice with genetically altered NR1 (Mohn et al., 1999;Duncan et al., 2004) and NR2A (Miyamoto et al., 2001) subunits. Therefore, in an effort to further understand the effects of transiently disrupting glutamate receptors containing specifically NR2B subunits, we administered the subunit-selective N M D A antagonist [(+/-)-(R*,S*)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-l-piperidine propanol] (Ro25-6981) to neonatal rats on postnatal days (PND) 6 and 7. Ro25-6981 is a high affinity non-competitive antagonist of receptors containing the NR2B subunit (Fischer et al., 1997). As NR2B-containing N M D A receptors are overexpressed in the neonatal period, administration of Ro25-6981 provides a unique opportunity to assess the importance of these specific receptors in the maturation of the 108 neural circuits that mediate behaviors with potential relevance to schizophrenia. Importantly, Ro25-6981 has low affinities for a-adrenergic and serotoninergic receptors, a problem recognized for other NR2B antagonists such as ifenprodil (Mutel et al., 1998). Following neonatal treatment, rats were tested for PPI and locomotor responses to a novel environment and amphetamine challenge both before and after puberty. These behavioral tests are extensively used to model the information processing deficits and striatal dopamine elevations observed in patients with schizophrenia (Lipska and Weinberger, 2000;Braff et al., 2001 ;van den et al., 2003;Bast and Feldon, 2003). Finally, the disruptive effect of the dopamine agonist apomorphine on PPI was also assessed in a sub-group of adult rats. Unexpectedly, the dose of the Ro25-6981 used (15 mg/kg) caused behavioral convulsions when administered on PND 6. As is detailed in the methods section, this dose has been used extensively in adult rats without reports of convulsive activity, and was chosen based on "affinity estimates for NR2B-containing receptors. Due to this observation, we also performed a series of parametric studies to further examine the convulsive effects of a number of N M D A antagonists during early development. Additionally, previous work performed in our laboratory demonstrated that seizures induced by kainic acid on PND 7 resulted in the delayed expression of PPI deficits in rats (Howland et al., 2004). From this standpoint, the long-term behavioral effects of convulsions following Ro25-6981 were of considerable interest. 109 Methods Experiment 1 - Incidence and Characteristics of Behavioral Convulsions in Postnatal Rats Following Antagonism of NMDA Receptors Containing the NR2B Subunit Subjects Long-Evans or Sprague-Dawley rat pups were used in all experiments. Pregnant Long-Evans rats were obtained from Charles River (Quebec, Canada) at 13 to 15 days of gestation. They were singly housed and left undisturbed until giving birth. The colony was maintained on a 12/12 hour light/dark cycle (lights on at 0700), at a temperature of 22±1°C. A l l rats were given food (Purina Rat Chow) and tap water ad libitum. Sprague-Dawley rat pups were obtained from the University of British Columbia Animal Care Center. The pups were delivered to our animal colony on the desired postnatal day without their mother. A l l experiments were initiated within 60 min of the arrival of the rat pups in the laboratory. Experiments were conducted in accordance with the standards of the Canadian Council on Animal Care and were approved by the Committee on Animal Care at the University of British Columbia. Experimental Procedures The day of birth of the pups was designated PND 0. Rat pups of the appropriate age (PND 6, 9, 12,15) were weighed and separated into individual compartments of a cardboard box for drug administration. The pups were removed from the colony and taken to a room where the behavioral effects of the administered drugs were videotaped and scored by an observer. The subunit-selective NMDA-NR2B antagonists Ro25-6981 (7.5, 15, and 30 mg/kg) and ifenprodil (15 and 30 mg/kg) were injected (10 ml/kg; i.p.) and the behavior of the animals was videotaped for 60 minutes. Reports indicate that 15 110 mg/kg of Ro25-6981 is in the dose range necessary to block most NR2B-containing receptors in the adult rodent brain (Murray et al., 2000;Lee and Rajakumar, 2003), and doses of 20 to 30 mg/kg have been used in vivo (Chaperon et al., 2003;Higgins et al., 2005). Similar data exists for ifenprodil (Murray et al., 2000). Following the experiment, the pups were sacrificed with carbon dioxide. A l l drugs were obtained from Sigma. Experiment 2 - Long-term Behavioral Effects of Convulsions Resulting from Antagonism of NMDA Receptors Containing the NR2B Subunit Subjects Two independent groups of Long-Evans rats (i.e. Group 1 and Group 2) were tested in this study. The animals were obtained, housed and cared for in a manner identical to that described in Experiment 1. Testing protocols for both groups were similar with small procedural differences noted in the methods section where appropriate. Ro25-6981 Administration On PND3, the litters were sexed and culled to include only males (4-9 rats per litter). At 4:00 p.m. on the afternoon of PND 6 and 9:00 a.m. and 4:00 p.m. of PND 7, all pups were removed from the nest, weighed and placed individually in small compartments of a cardboard box. They were then removed from the colony and taken to a small heated room. Ro25-6981 (15 mg/kg) or saline was injected (i.p.) with a 30-gauge needle (10 ml/kg). Care was taken to ensure that both treatments were administered to members of each litter. The behavior of all rats was recorded with an overhead video camera for 60 min. Before being returned to their mothers, the pups were earmarked according to treatment condition. The litters were then left undisturbed (except for 111 normal cage changing) until weaning on PND 25. Weanling rats were housed in cages of 2 or 3 with members of their litter and these groups remained together for the duration of the experiment. A l l rats were handled before behavioral testing. Prepulse Inhibition On PND 35 and 56, rats were removed individually from the colony and taken immediately to the PPI apparatus. Testing was conducted in a single sound-attenuating startle chamber (ambient noise level 64 dB), containing a transparent Plexiglas tube (8.2 cm in diameter, 20 cm in length), mounted on a Plexiglas frame (SR-LAB, San Diego Instruments, San Diego). Noise bursts were presented through a speaker mounted 24-cm above the tube. An accelerometer below the frame of the apparatus measured whole body startle amplitude, defined as the average of 100 1-ms accelerometer readings collected from stimulus onset. Each PPI test session began with a 5-min acclimatization period during which a 70-dB background noise level was presented, which remained constant for the entire test session. Following the acclimatization period, six pulse alone trials (120 dB, 40 ms) were presented to achieve a relatively stable startle amplitude before PPI testing. Data from these pulse-alone trials was not considered in the analysis of PPI. Immediately following the six initial pulse-alone trials, presentation of the trials that were used in the calculation of PPI levels were initiated. Trials presented were of four types: pulse alone (12 trials, 120 dB, 40 ms), prepulse + pulse (12 trials X 3 prepulse intensities - discussed below), prepulse alone (12 trials X 3 prepulse intensities) or no stimulus (12 trials). Prepulse + pulse trials consisted of the presentation of a 20 ms prepulse of 73, 76, or 82 dB 80 ms before the presentation of the pulse. Prepulse alone trials consisted only of a 20 ms prepulse (73, 76, 82 dB). A l l trials were presented in a 112 pseudorandom order. After the trials used for calculation of PPI were presented, 6 additional pulse-alone trials were presented. These, along with the first 6 pulse-alone trials, were used to calculate the level of habituation over the testing period. As no significant differences were observed between the groups in habituation and or for the prepulse-alone trials, these data are not presented. The inter-trial interval varied randomly from 3 to 12 s (average 7.5 s). Calibration of the apparatus was performed using a RadioShack Digital Sound Level Meter and adjustments were made as necessary. Spontaneous Locomotor Activity On PND 36, rats were weighed, removed from the colony, and immediately placed in 1 of 8 Med Associates Test Chambers (ENV-008; 30.5 cm X 24.1cm X height 21.0 cm) to measure spontaneous locomotor activity for 60 min. The chambers were fitted with 4 pairs of infrared photocells 3.5 cm from the floor evenly spaced on the long walls and had metal grid floors and two operant levers (which were retracted during locomotor activity testing). Each chamber was contained within a Med Associates sound attenuating cubicle with a house light illuminated during testing. Dopaminergic Challenges of PPI and Locomotor Activity For those rats in Group 1, 7 to 14 days after PPI testing in early adulthood, the responses of the animals to administration of dopamine agonists were measured in both the PPI and locomotor activity tests. Some rats were tested for PPI first followed by locomotor activity while others were tested in the reverse order. A minimum of 7 days separated locomotor activity and PPI testing. The PPI sessions were conducted in a manner identical to that described above, except that immediately before the PPI session, all rats were weighed and injected with the appropriate volume of vehicle (ascorbic acid, 113 0.1%) or drug (apomorphine, Sigma, 0.2 mg/kg; s.e.). Each rat was tested twice (i.e. following either vehicle or apomorphine injection), and the PPI tests were at least 5 days apart. For locomotor activity testing, the rats were weighed, removed from the colony, and placed in the locomotor activity boxes for 60 min (as described above for PND 36). A l l rats were then immediately injected with D-amphetamine (1.5 mg/kg, i.p.) and returned to the locomotor boxes. Locomotor activity was monitored for an additional 90 min before the rats were returned to the colony room. For rats in Group 2, PPI responses following apomorphine challenge were not measured. However, locomotor responses to D-amphetamine were examined at both PND 36 and PND 57. Rats were weighed and placed in the locomotor boxes as previously described, however, at both ages, they were injected with D-amphetamine (1.5 mg/kg) immediately following the spontaneous locomotor activity test. Locomotor activity following D-amphetamine was measured for 90 min in all cases. Data Analysis For the PPI experiments, two measures were calculated for each animal. The startle amplitude represented the mean startle amplitude of the 12 pulse-alone trials presented after the 6 habituation trials. Startle amplitude data were compared using an independent samples t-test for each age. PPI was calculated by averaging startle amplitudes for each trial type. The percent PPI for each prepulse intensity was calculated using the formula: [100 - (100 X startle amplitude on prepulse + pulse trials) (startle amplitude on pulse alone trials)]. A repeated measures A N O V A was performed on the data obtained from each age with prepulse intensity as a within-subjects factor and treatment on PND 6 and 7 as a between-subjects factor. Results from the apomorphine 114 experiment were also analyzed using a repeated measures A N O V A (prepulse intensity and test as within-subjects factors, and treatment at PND 7 as a between-subjects factor). Locomotor activity data were compared using repeated measures A N O V A at each age with test type as a within-subjects factor and treatment on PND 7 as a between-subjects factor. For all A N O V A ' s , post-hoc analyses were performed using the Neuman-Keuls test where appropriate. The significance level for all statistical tests was p < 0.05. Results Experiment 1 - Behavioral Effects of Ro25-6981 Administration in the Early Postnatal Period Approximately 5 min following injection with Ro25-6981, the majority of PND 6 pups displayed behavioral convulsions. The convulsions were similar those typically described following administration of kainic acid (Howland et al., 2004) or bicuculline (Lai et al., 2002) in neonatal rats. Generally, they consisted of loss of posture, vocalizations, and rapid bursts of motor activity (especially with the hind legs). The majority of the animals showed bouts of bilateral forelimb and hindlimb clonus and/or tonus on their backs, in what has been called a 'swimming' posture by other authors (Stafstrom et al., 1993). In most cases, the behavioral convulsions subsided within 30 to 60 min following injection. Interestingly, following their initial bout of convulsions, some animals would have another period of convulsive activity if they were handled by the experimenter. Those pups injected repeatedly with Ro25-6981 (see Experiment 2) showed no evidence of tolerance to the convulsive effects of the drug. As depicted in Table 4-1, the convulsive effects of Ro25-6981 were age and dose dependent. Whereas all pups treated with 15 mg/kg of the drug on PND 6 had 115 convulsions, only 25% of PND 9 pups, and 0% of PND 12 or 15 (using either 15 or 30 mg/kg) pups displayed evidence of convulsions. However, pups injected with Ro25-6981 were more active than saline-injected controls. Additionally, none of the rats treated with 7.5 mg/kg of Ro25-6981 displayed behavioral convulsions. In an effort to rule out the possibility that the convulsions observed in the PND 6 pups were caused by an unknown property of Ro25-6981 unrelated to NR2B antagonism, ifenprodil was also administered to some animals on PND 6. Similarly to the results obtained with Ro25-6981, almost all pups (89%) treated with ifenprodil had behavioral convulsions. Experiment 2 - Effects of Neonatal Ro25-6981 Administration on Body Weight As described in detail for experiment 1, administration of Ro25-6981 (15 mg/kg; Group 1: n=25; Group 2: n=16) resulted in behavioral convulsions for 30 to 60 minutes. Some individual differences in the severity of the convulsions were noted; although these did not correlate with performance on the behavioral tests performed. As a result, data from all rats is included. A l l rats were weighed on PND 6, twice on PND 7, and once on PND 9, 36 and 57. Repeated measures A N O V A ' s performed on these data indicate that administration of Ro25-6981 to the pups had no significant effect on body weight at any age (Group 1: F ( l , 41)=0.90, N.S.-; Group 2: F ( l , 30)=0.49, N.S.). However, comparing the average weights of rats in Group 1 and Group 2 revealed that the animals in Group 1 were significandy lighter than those in Group 2 (F(l, 73)=48.97, p < 0.001). On PND 6 and 7, rats in Group 1 weighed approximately 70% of those in Group 2 (PND 6: Group 1 mean = 12.16 g; Group 2 = 17.00 g). During puberty and early adulthood, the difference in weights 116 Drug Postnatal Dose (i.p., Total Number Total Number Day mg/kg) Injected Exhibiting Convulsions Ro25-6981 6 7.5 8 0 Ro25-6981 6 15 8 8 Ro25-6981 9 15 4 1 Ro25-6981 12 15 5 0(A) Ro25-6981 15 15 4 0(A) Ro25-6981 15 30 3 0(A) Ifenprodil 6 15 4 4 Ifenprodil 6 30 5 4 Table 4-1. The incidence of behavioral convulsions in rats administered NR2B-selective N M D A antagonists at various postnatal ages. i.p. = intraperitoneal. 117 narrowed to a 10 to 15% difference (PND 36: Group 1 = 159.77 g; Group 2 = 173.58 g; PND 57: Group 1 = 339.26 g; Group 2 = 398.96 g). Neonatal Ro25-6981 Administration Does not Alter PPI Responding Either before Puberty or in Early Adulthood Before Puberty (PND 35): As is shown in Figure 4-2A and 4-3A, comparable levels of PPI was elicited in both saline- and Ro25-6981-treated rats for Groups 1 (saline: n=19, average PPI: 27.86±2%; Ro25-6981: n=25, average PPI: 26.53±3%) and 2 (saline n=16, average PPI: 32.24±3%; Ro25-6981: n=16, average PPI: 26.38±4%). Repeated measures A N O V A ' s performed separately for each Group revealed a significant effect of prepulse (Group 1: F(2, 84)=96.62, p < 0.001; Group 2: F(2, 60)=49.92, p < 0.001), but no significant treatment effects (Group 1: F ( l , 43)=0.13, N.S.; Group 2: F ( l , 30)=1.45, N.S.) or treatment by prepulse interactions (Group 1: F(2, 84)=0.08, N.S.; Group 2: F(2, 60)=0.14, N.S.). Figures 4-2B and 4-3B depict the average startie amplitudes for all animals tested in Groups 1 and 2. Independent samples t-tests revealed no significant differences between saline- and Ro25-6981-treated rats in either Group (Group 1: t(42)=1.35, N.S.; Group 2: t(30)=0.03, N.S.). Early Adulthood (PND 56): Similar to before puberty, all groups tested demonstrated robust PPI in early adulthood (Fig. 4-2C and 4-3C), and no significant differences were observed between animals treated with either saline (average PPI, Group 1: 30.60±3%; Group 2: 31.74±3%) or Ro25-6981 (average PPI, Group 1: 28.33±3%; Group 2: 37.00±5%). Repeated measures A N O V A ' s confirmed these observations with no significant treatment (Group 1: F ( l , 42)=0.29, N.S.; Group 2: F ( l , 30)=0.68, N.S.) or treatment by prepulse interactions (Group 1: F(2, 84)=0.63, N.S.; Group 2: F(2, 60)=0.69, 118 A B Q_ Q. :<D.' <u.: CO 180n 160 140-120 100 80 60 40 20 pP6 pp12 Prepulse Intensity average saline Ro Group PP3 pp6 pp12 average Prepulse Intensity saline Ro Group Figure 4-2. Prepulse inhibition (PPI) scores of rats in Group 1 treated on postnatal day (PND) 6 and 7 with saline (white bars, n=19) or Ro25-6981 (Ro, 15 mg/kg; black bars, n=25). A, Effects of postnatal Ro treatment on PPI at PND35. B, Average startle amplitudes of the rats on PND 35. C, Percent PPI scores for those rats tested at PND 56 D, Average startle amplitudes of the rats on PND 56. 119 pp3 pp6 pp12 average Prepulse Intensity : pp3 pp6 pp12 Prepulse Intensity average B 160i saline Ro Group D 160! 1120 I 80] I 40] saline Ro Group Figure 4-3. Prepulse inhibition (PPI) scores from rats in Group 2 treated on postnatal day (PND) 6 and 7 with saline (white bars, n=16) or Ro25-6981 (Ro, 15mg/kg; black bars, n=16). A, Effects of postnatal day (PND) 7 K A treatment on PPI at PND 35. B, Average starde amplitudes of the rats on PND 35. C, Percent PPI scores for those rats tested at PND 56. D, Average startle amplitudes of the rats on PND 56. 120 N.S.)- As expected, significant main effects for prepulse intensity were found for both groups (Group 1: F(2, 84)=70.90, p < 0.001; Group 2: F(2, 60)=32.71, p < 0.001). Additionally, startle amplitudes did not differ as a result of treatment in either Group 1 (Fig. 4-2D; t(42)=l .05, N.S.) or Group 2 (Fig. 4-3D; t(30)=0.73, N.S.) Apomorphine Challenge in Adulthood has Similar Effects on PPI in Neonatally Saline- or Ro25-6981 -treated Rats To examine the potential effects of neonatal Ro25-6981 treatment on the dopaminergic regulation of PPI, 16 saline-treated and 18 Ro25-6981 -treated rats from Group 1 were retested for PPI following injection with either vehicle (0.1% ascorbic acid in saline) or apomorphine (0.2 mg/kg). Figure 4-4A summarizes the results of these tests. Following vehicle injection, PPI levels in both saline- and Ro25-6981-treated rats were similar to those observed when PPI was tested on PND 56. Apomorphine (0.2 mg/kg) significantly disrupted PPI in both the saline- and Ro25-6981-treated rats, as reflected by a significant main effect of test (vehicle versus apomorphine; F ( l , 32)=31.18, p < 0.01) and a significant test by prepulse interaction (F(2, 64)=10.75, p < 0.001, post-hoc, p < 0.05). Additional analyses revealed that significant effects only existed for trials with 6 and 12 dB prepulses in both groups (p < 0.05). As shown in Fig. 4-4B, startle amplitude was also significantly reduced by apomorphine administration in both groups (F(l, 32)=8.59, p < 0.01). Neonatal Treatment with Ro25-6981 and Amphetamine-Induced Locomotor Activity in Adult Rats Group 1: Spontaneous locomotor activity in response to a novel environment was assessed for 60 min in the saline- and Ro25-6981-treated rats. Both before puberty (Fig. 121 G L D-: a> A 70-60-50-40-30-20 \ lO-CI asal+veh •Ro+veh •sal+apol oRo+apo Condition average sal+ Ro+ sal+ Ro+ veh veh apo apo Group Figure 4-4. A, Effects of pretreatment with vehicle (0.1% ascorbic acid) or apomorphine (0.2 mg/kg) on percent PPI scores in adulthood. Animals were administered either saline (n=16) or Ro25-6981 (15 mg/kg; n=18) on postnatal day 6 and 7. B, Average starde amplitudes of those rats depicted in panel A. In both panels, asterisks indicate a significant difference between groups. 122 Figure 4-5. Locomotor activity levels (photobeam breaks) in response to novelty (A, B left side) or amphetamine (1.5 mg/kg; B right side) of rats tested on either postnatal day (PND) 36 (A) or PND 57 (B) in Group 1. Rats were treated with either saline (white bars) or Ro25-6981 (black bars, 15 mg/kg) on PND 6 and 7. The asterisk denotes a significant difference between amphetamine-treated groups (p<0.05). Note the difference in scale for the units of activity levels (y-axis) for each panel. PND 36: saline n = 19, Ro25-6981 n = 25; PND 57: saline n = 12, Ro25-6981 n = 12. • 123 Figure 4-6. Locomotor activity levels (photobeam breaks) in response to novelty or amphetamine (1.5 mg/kg) of rats tested on either postnatal day (PND) 36 (A) or PND 57 (B) in Group 2. Rats were treated with either saline (white bars, n = 12) or Ro25-6981 (black bars, 15 mg/kg, n = 12) on PND 6 and 7. Note the difference in scale for the units of activity levels (y-axis) for each panel. 124 4-5A) and in early adulthood (Fig. 4-5B), exploration levels were similar in both groups (before puberty: t(42)=0.45, N.S.; early adulthood: see A N O V A below). Interestingly, administration of the dopamine agonist D-amphetamine (1.5 mg/kg) further increased locomotor activity by approximately 28% in rats neonatally treated with Ro25-6981 (2193.75 ± 143 beam breaks) than saline (1712.67 ± 133 beam breaks) during the 90 min test (Fig. 4-5B). This effect was confirmed by a significant treatment by test interaction (F(l, 22)=7.83, p < 0.05), and post-hoc analyses (p < 0.05). Group 2: Spontaneous and amphetamine-induced locomotor activity was compared between treatment groups both before puberty (Fig. 4-6A) and in early adulthood (Fig. 4-6B) for those rats in Group 2. Unexpectedly, the groups did not significandy differ on either test at either age. Statistical analyses revealed insignificant main effects of treatment (PND 36: F ( l , 22)= 0.27, N.S.; PND 57: 0.18, N.S.) and treatment by test interactions (PND 36: F ( l , 22)=0.12, N.S.; PND 57: F ( l , 22)=0.18, N.S.). Discussion The present experiments assessed the acute and delayed behavioral effects of the administration of Ro25-6981, an antagonist of NR2B-containing N M D A receptors, in rats. During PND 6 or 7, administration of Ro25-6981 resulted in behavioral convulsions that typically lasted 30 to 60 minutes. The convulsions were dose-dependent and could be elicited with a second NR2B antagonist, ifenprodil. Parametric experiments demonstrated that convulsions could not be reliably elicited in rats older than PND 7. The delayed behavioral effects of administering Ro25-6981 on PND 6 and 7 were inconsistent. Ro25-6981-treated rats in Group 1 performed similarly to saline-treated animals on PPI and spontaneous locomotion tests both before and after puberty. 125 However, the Ro25-6981 -treated animals were significantly more active when treated with amphetamine in early adulthood, but failed to show increased sensitivity to the disruptive effects of apomorphine on PPI. In contrast, Ro25-6981-treated rats tested in Group 2 failed to show significant differences from saline-treated animals in any of the behavioral tests conducted. Antagonism of NR2B-containing NMDA Receptors Induces Behavioral Convulsions The convulsive effects of Ro25-6981 were unexpected in the present experiments; however, they appear to be reliable as they were dose-dependently elicited in two strains of rats by both Ro25-6981 and ifenprodil. Interestingly, some groups (Fischer et al., 1997;Mutel et al., 1998;Mallon et al., 2005) have described agonist-like properties of Ro25-6981 when the drug is applied in conjunction with low concentrations of N M D A . In Xenopus oocytes, Ro25-6981 (1 uM) profoundly inhibits the current elicited by 100 u.M of N M D A , but surprisingly potentiates the current elicited by 1 u M of N M D A (Fischer et al., 1997). In another study using hippocampal slices from pubescent rats, Ro25-6981 (3 uM) potentiated the effects of N M D A (4 to 10 uM) on CA1 field potentials and paired-pulse interactions (Mallon et al., 2005). As also reported by Fisher et al. (1997), these effects were more significant with lower concentrations of N M D A (Mallon et al., 2005). Interestingly, the NR2A specific antagonist NVP-AAM077 (Auberson et al., 2002) effectively blocked the effects of Ro25-6981 in the hippocampal slice suggesting that the NR2B subunit may reduce N M D A receptor activity by exerting tonic inhibitory effects on receptors containing the NR2A subunit (Mallon et al., 2005). A number of issues make integrating the present data with other reports in the literature challenging. Most importantly, the subunit composition of N M D A receptors 126 dynamically changes over development; therefore, care must be taken when comparing data sets gathered from animals of different ages (for example, comparing the present data to those of Mallon et al., 2005). During the first postnatal week, NR2A subunits are expressed at very low levels in the rodent brain, whereas in adulthood NR2A subunits predominate (Monyer et al., 1994;Guilarte and McGlothan, 1998;Cull-Candy et al., 2001;Ritter et al., 2002;Zhang et al., 2002). Given these expression differences, it is unlikely that the observations made by Mallon and colleagues (2005) apply during the early neonatal period since very few NR2A subunits are expressed at that age. Additionally, numerous reports indicate that in vivo Ro25-6981 antagonizes NR2B containing N M D A receptors at doses within the range used in the present study (Lee and Rajakumar, 2003;Chaperon et al., 2003;De Vry and Jentzsch, 2003;Boyce-Rustay and Holmes, 2005). Therefore, the most likely explanation of the present data is that the behavioral convulsions observed resulted from the antagonist action of Ro25-6981 on NR2B-containing N M D A receptors. Although convulsions resulting from N M D A receptor antagonism are counterintuitive, they are not without precedence in the literature. For example, systemic administration of the non-competitive N M D A antagonist MK801 (0.1, 0.5, 1.0 mg/kg) to neonatal rat pups induces behavioral and electrographic seizures and exacerbates seizures caused by kainic acid (Stafstrom et al., 1997). Given that MK-801 would primarily act on N M D A receptors containing NR2B subunits at this developmental age, these results support our assertion that the neonatal brain may be prone to convulsions following the blockade of receptors containing NR2B subunits. 127 Further experiments are necessary to understand the mechanism underlying the convulsive effects of neonatal N M D A receptor antagonism. Seizures are more common neonatally than later in development, an effect that may be related to differences in the net ratio of excitation and inhibition early in development (Ben Ar i et al., 1997;Holmes et a l , 2002). As previously described, intricate developmental regulation of N M D A receptor expression exists during the first few weeks of life. The expression patterns and function of other receptors, including G A B A A and A M P A receptors, are also developmentally regulated. For instance, G A B A A receptors are expressed early in the embryonic period and provide much of the excitatory drive in the developing brain until the end of the first postnatal week when N M D A and A M P A currents begin to predominate (Ben Ar i et al., 1997;Holmes et al., 2002;Khazipov et al., 2004). The excitatory action of G A B A is primarily due to delayed expression of the K + / C f co-transporter KCC2, resulting in increased intracellular C f concentrations. Thus, when G A B A A channels open under these conditions, Cl" ions exit the neuron resulting in depolarization (Holmes et al., 2002). However, under some conditions, G A B A A activation may also increase the resting membrane conductance sufficiently that the net effect of opening these channels is inhibitory (Leinekugel et al., 1999;Khalilov et al., 1999a;Holmes et al., 2002). As resting membrane conductance at this age is also critically dependent on glutamate receptor conductances (Khalilov et al., 1999a), one possible explanation of our observations is that antagonism of N M D A receptors with Ro25-6981 may 'tip the balance' in favor of excitation, thereby triggering a convulsion. 128 The Long-Term Behavioral Effects of Ro25-6981 on PPI and Locomotor Activity The levels of PPI and locomotor activity observed before and after puberty in the present study are similar to previous experiments from our group (Howland et al., 2004). Accordingly, percent PPI increased with higher prepulse intensities (Fig. 4-2 and 4-3). Average PPI levels were also lower in prepubescence than in early adulthood. Interestingly, a relatively low dose of apomorphine (0.2 mg/kg) disrupted PPI in the present study (Fig. 4-4A). This effect is consistent with previous results using the Long-Evans strain in our laboratory (Howland et al., 2004), but not others (Swerdlow et al., 2001). Although the significantly potentiated locomotor activity following amphetamine administration in the Ro25-6981-treated rats of Group 1 (Fig. 4-5B) is intriguing, the failure to replicate this effect in Group 2 (Fig. 4-6B) questions the reliability of these data. However, an interaction between Ro25-6981 administration and the factors that led to significantly reduced body weight in Group 1 may explain these behavioral changes. When the present body weights are compared to previous studies from our laboratory (Howland et al., 2004) in which Long-Evans pups were reared under identical conditions to those described herein, it is clear that the pups tested in Group 1 had abnormally low body weights. The cause of these differences cannot be confirmed, although some renovations to our facility occurred during the period which the pregnant females arrived and gave birth to the pups in Group 1. Thus, the mothers of the rats tested in Group 1 may have been abnormally stressed during the pre- and postnatal period. Indeed, stress during these developmental periods has been shown to regulate dopamine-dependent behaviors such as locomotor activity in adult rats (Kofman, 2002;Brake et al., 2004). 129 The present experiments fail to provide convincing evidence that neonatal treatment with Ro25-6981 results in specific behavioral changes related to schizophrenia. However, these data stand in contrast to reports of significant behavioral effects of neonatal N M D A antagonism in adulthood using non-competitive antagonists. For example, neonatal treatment with phencyclidine (PND 7, 9, and 11) reduces PPI and significantly increases locomotor activity in response to a phencyclidine challenge, both of which were reversed following antipsychotic treatment (Wang et al., 2001). Increased spontaneous locomotor activity is also observed following ketamine administration to mice on PND 10 (Fredriksson et al., 2004). Finally, neonatal administration of MK-801 alters PPI (Harris et al., 2003), locomotor activity (Harris et al., 2003), and set-shifting (Stefani and Moghaddam, 2005), although the PPI and locomotor effects were not independently replicated (Beninger et al., 2002) and were only found in female animals (Harris et a l , 2003). A number of differences between non-competitive N M D A antagonists and Ro25-6981 may underlie the different behavioral effects observed following their administration. For example, non-competitive N M D A antagonists have actions at dopamine D 2 and serotonin 5-HT 2 receptors, in addition to their effects of N M D A receptors (Kapur and Seeman, 2002). Importantly, the neonatal administration of non-competitive N M D A antagonists also causes significant apoptosis in areas such as the hippocampus, thalamus, cortex, and striatum 24 to 48 hours after treatment (Wang et al., 2001 ;Lai et al., 2002;Beninger et al., 2002;Harris et al., 2003-/Fredriksson et al., 2004). Given that cell loss or disarray in these areas is reported in schizophrenia (Pakkenberg, 1990;Jones, 1997;Harrison, 1999;Gothelf et al., 2000), these findings strengthen the 130 validity of neonatal non-competitive N M D A antagonist administration as a model of schizophrenia, and may explain their behavioral effects. It is not known whether Ro25-6981 administration causes similar neuropathological effects, although the potential exists given that various competitive and non-competitive N M D A antagonists cause apoptosis in the developing brain (Dconomidou et al., 1999). Conclusion The convulsions caused by Ro25-6981 on PND 6 and 7 failed to affect PPI or locomotor responding in the present experiments. In contrast, febrile seizures early in life are associated with increased risk for schizophrenia (Vestergaard et al., 2005) and psychosis (Kanemoto et al., 2001). Additionally, kainic acid-induced seizures on PND 7 in rats result in the delayed emergence of subtle PPI disruptions in early adulthood (Howland et al., 2004) and have short and long-term effects on temporal lobe electrophysiology, especially within the hippocampus (Tremblay et al., 1984;Stafstrom et al., 1992;Khalilov et al., 1999b;Lynch et al., 2000;Silveira et al., 2002). As the electrographic characteristics of the convulsions induced by Ro25-6981 are presently unknown, experiments designed to address this issue may enable more specific hypotheses to be generated regarding the brain areas important for long-term behavioral changes following neonatal seizures and their relationship to schizophrenia. 131 References Akbarian S, Sucher NJ, Bradley D, Tafazzoli A , Trinh D, Hetrick WP, Potkin SG, Sandman C A , Bunney WE, Jr., Jones E G (1996) Selective alterations in gene expression for N M D A receptor subunits in prefrontal cortex of schizophrenics. J Neurosci 16: 19-30. Auberson YP, Allgeier H , Bischoff S, Lingenhoehl K, Moretti R, Schmutz M (2002) 5-Phosphonomethylquinoxalinediones as competitive N M D A receptor antagonists with a preference for the human 1A/2A, rather than 1A/2B receptor composition. Bioorg Med Chem Lett 12: 1099-1102. Bast T, Feldon J (2003) Hippocampal modulation of sensorimotor processes. Prog Neurobiol 70: 319-345. Ben Ar i Y , Khazipov R, Leinekugel X , Caillard O, Gaiarsa JL (1997) G A B A A , N M D A and A M P A receptors: a developmentally regulated 'menage a trois'. Trends Neurosci 20: 523-529. Beninger RJ, Jhamandas A, Aujla H , Xue L, Dagnone R V , Boegman RJ, Jhamandas K (2002) Neonatal exposure to the glutamate receptor antagonist MK-801: effects on locomotor activity and pre-pulse inhibition before and after sexual maturity in rats. Neurotox Res 4: 477-488. Boyce-Rustay J M , Holmes A (2005) Functional roles of N M D A receptor NR2A and NR2B subunits in the acute intoxicating effects of ethanol in mice. Synapse 56: 222-225. Braff DL, Geyer M A , Swerdlow NR (2001) Human studies of prepulse inhibition of startle: normal subjects, patient groups, and pharmacological studies. Psychopharmacology (Berl) 156: 234-258. Brake WG, Zhang T Y , Diorio J, Meaney MJ , Gratton A (2004) Influence of early postnatal rearing conditions on mesocorticolimbic dopamine and behavioural responses to psychostimulants and stressors in adult rats. Eur J Neurosci 19: 1863-1874. Chaperon F, Muller W, Auberson YP, Tricklebank M D , Neijt HC (2003) Substitution for PCP, disruption of prepulse inhibition and hyperactivity induced by N-methyl-D-aspartate receptor antagonists: preferential involvement of the NR2B rather than NR2A subunit. Behav Pharmacol 14: 477-487. Clinton S M , Meador-Woodruff JH (2004) Abnormalities of the N M D A Receptor and Associated Intracellular Molecules in the Thalamus in Schizophrenia and Bipolar Disorder. Neuropsychopharmacology 29: 1353-1362. Coyle JT, Tsai G (2004) N M D A receptor function, neuroplasticity, and the pathophysiology of schizophrenia. Int Rev Neurobiol 59: 491-515. Cull-Candy S, Brickley S, Farrant M (2001) N M D A receptor subunits: diversity, development and disease. Curr Opin Neurobiol 11: 327-335. 132 De Vry J, Jentzsch K R (2003) Role of the N M D A receptor NR2B subunit in the discriminative stimulus effects of ketamine. Behav Pharmacol 14: 229-235. Duncan GE, Moy SS, Perez A, Eddy D M , Zinzow W M , Lieberman JA, Snouwaert JN, Roller B H (2004) Deficits in sensorimotor gating and tests of social behavior in a genetic model of reduced N M D A receptor function. Behav Brain Res 153: 507-519. Facchinetti F, Ciani E , Dall'Olio R, Virgili M , Contestable A , Fonnum F (1993) Structural, neurochemical and behavioural consequences of neonatal blockade of N M D A receptor through chronic treatment with CGP 39551 or MK-801. Brain Res Dev Brain Res 74: 219-224. Fischer G, Mutel V , Trube G, Malherbe P, Kew JN, Mohacsi E, Heitz MP, Kemp JA (1997) Ro 25-6981, a highly potent and selective blocker of N-methyl-D-aspartate receptors containing the NR2B subunit. Characterization in vitro. J Pharmacol Exp Ther 283: 1285-1292. Fox K, Schlaggar B L , Glazewski S, O'Leary DD (1996) Glutamate receptor blockade at cortical synapses disrupts development of thalamocortical and columnar organization in somatosensory cortex. Proc Natl Acad Sci U S A 93: 5584-5589. Fredriksson A , Archer T, Aim H , Gordh T, Eriksson P (2004) Neurofunctional deficits and potentiated apoptosis by neonatal N M D A antagonist administration. Behav Brain Res 153: 367-376. Gao X M , Sakai K, Roberts RC, Conley RR, Dean B , Tamminga C A (2000) Ionotropic glutamate receptors and expression of N-methyl-D-aspartate receptor subunits in subregions of human hippocampus: effects of schizophrenia. A m J Psychiatry 157: 1141-1149. Goff DC, Coyle JT (2001) The emerging role of glutamate in the pathophysiology and treatment of schizophrenia. Am J Psychiatry 158: 1367-1377. Gorter JA, de Bruin JP (1992) Chronic neonatal MK-801 treatment results in an impairment of spatial learning in the adult rat. Brain Res 580: 12-17. Gothelf D, Soreni N , Nachman RP, Tyano S, Hiss Y , Reiner O, Weizman A (2000) Evidence for the involvement of the hippocampus in the pathophysiology of schizophrenia. Eur Neuropsychopharmacol 10: 389-395. Gould E, Cameron HA, McEwen BS (1994) Blockade of N M D A receptors increases cell death and birth in the developing rat dentate gyrus. J Comp Neurol 340: 551-565. Grimwood S, Slater P, Deakin JF, Hutson PH (1999) NR2B-containing N M D A receptors are up-regulated in temporal cortex in schizophrenia. Neuroreport 10: 461-465. Guilarte TR, McGlothan JL (1998) Hippocampal N M D A receptor mRNA undergoes subunit specific changes during developmental lead exposure. Brain Res 790: 98-107. 133 Harris L W , Sharp T, Gartlon J, Jones D N , Harrison PJ (2003) Long-term behavioural, molecular and morphological effects of neonatal N M D A receptor antagonism. Eur J Neurosci 18: 1706-1710. Harrison PJ (1999) The neuropathology of schizophrenia. A critical review of the data and their interpretation. Brain 122 (Pt 4): 593-624. Higgins G A , Ballard T M , Enderlin M , Haman M , Kemp JA (2005) Evidence for improved performance in cognitive tasks following selective NR2B N M D A receptor antagonist pre-treatment in the rat. Psychopharmacology (Berl) 179: 85-98. Holmes GL, Khazipov R, Ben Ari Y (2002) New concepts in neonatal seizures. Neuroreport 13: A3-A8. Howland JG, Hannesson DK, Phillips A G (2004) Delayed onset of prepulse inhibition deficits following kainic acid treatment on postnatal day 7 in rats. Eur J Neurosci 20: 2639-2648. ' . Ikonomidou C, Bosch F, Miksa M , Bittigau P, Vockler J, Dikranian K, Tenkova TI, Stefovska V , Turski L, Olney JW (1999) Blockade of N M D A receptors and apoptotic neurodegeneration in the developing brain. Science 283: 70-74. Itokawa M , Yamada K, Yoshitsugu K, Toyota T, Suga T, Ohba H , Watanabe A , Hattori E, Shimizu H , Kumakura T, Ebihara M , Meerabux J M , Toru M , Yoshikawa T (2003) A microsatellite repeat in the promoter of the N-methyl-D-aspartate receptor 2A subunit (GRIN2A) gene suppresses transcriptional activity and correlates with chronic outcome in schizophrenia. Pharmacogenetics 13: 271-278. Jentsch JD, Roth R H (1999) The neuropsychopharmacology of phencyclidine: from N M D A receptor hypofunction to the dopamine hypothesis of schizophrenia. Neuropsychopharmacology 20: 201-225. Jones E G (1997) Cortical development and thalamic pathology in schizophrenia. Schizophr Bull 23: 483-501. Kanemoto K, Tsuji T, Kawasaki J (2001) Reexamination of interictal psychoses based on D S M IV psychosis classification and international epilepsy classification. Epilepsia 42: 98-103. Kapur S, Seeman P (2002) N M D A receptor antagonists ketamine and PCP have direct effects on the dopamine D(2) and serotonin 5-HT(2)receptors-implications for models of schizophrenia. Mol Psychiatry 7: 837-844. Khalilov I, Dzhala V , Ben Ari Y , Khazipov R (1999a) Dual role of G A B A in the neonatal rat hippocampus. Dev Neurosci 21: 310-319. 134 Khalilov I, Dzhala V , Medina I, Leinekugel X , Melyan Z, Lamsa K, Khazipov R, Ben Ar i Y (1999b) Maturation of kainate-induced epileptiform activities in interconnected intact neonatal limbic structures in vitro. Eur J Neurosci 11: 3468-3480. Khazipov R, Khalilov I, Tyzio R, Morozova E, Ben Ar i Y , Holmes G L (2004) Developmental changes in GABAergic actions and seizure susceptibility in the rat hippocampus. Eur J Neurosci 19: 590-600. Kofman O (2002) The role of prenatal stress in the etiology of developmental behavioural disorders. Neurosci Biobehav Rev 26: 457-470. Lai M C , Liou CW, Yang SN, Wang C L , Hung PL, Wu CL, Tung Y R , Huang LT (2002) Recurrent Bicuculline-Induced Seizures in Rat Pups Cause Long-Term Motor Deficits and Increase Vulnerability to a Subsequent Insult. Epilepsy Behav 3: 60-66. Lee J, Rajakumar N (2003) Role of NR2B-containing N-methyl-D-aspartate receptors in haloperidol-induced c-Fos expression in the striatum and nucleus accumbens. Neuroscience 122: 739-745. Leinekugel X , Khalilov I, McLean H , Caillard O, Gaiarsa JL, Ben Ari Y , Khazipov R (1999) G A B A is the principal fast-acting excitatory transmitter in the neonatal brain. Adv Neurol 79: 189-201. Lewis D A , Levitt P (2002) Schizophrenia as a disorder of neurodevelopment. Annu Rev Neurosci 25: 409-432. Lipska B K , Weinberger DR (2000) To model a psychiatric disorder in animals: schizophrenia as a reality test. Neuropsychopharmacology 23: 223-239. Luthi A , Schwyzer L, Mateos J M , Gahwiler B H , McKinney R A (2001) N M D A receptor activation limits the number of synaptic connections during hippocampal development. Nat Neurosci 4: 1102-1107. Lynch M , Sayin U , Bownds J, Janumpalli S, Sutula T (2000) Long-term consequences of early postnatal seizures on hippocampal learning and plasticity. Eur J Neurosci 12: 2252-2264. Mallon AP, Auberson Y P , Stone TW (2005) Selective subunit antagonists suggest an inhibitory relationship between NR2B and NR2A-subunit containing N-methyl-D: -aspartate receptors in hippocampal slices. Exp Brain Res 162: 374-383. Miyamoto Y , Yamada K, Noda Y , Mori H, Mishina M , Nabeshima T (2001) Hyperfunction of dopaminergic and serotonergic neuronal systems in mice lacking the N M D A receptor epsilonl subunit. J Neurosci 21: 750-757. Miyatake R, Furukawa A , Suwaki H (2002) Identification of a novel variant of the human NR2B gene promoter region and its possible association with schizophrenia. Mol Psychiatry 7: 1101-1106. 135 Mohn AR, Gainetdinov RR, Caron M G , Roller B H (1999) Mice with reduced N M D A receptor expression display behaviors related to schizophrenia. Cell 98: 427-436. Monyer H , Burnashev N , Laurie DJ, Sakmann B, Seeburg PH (1994) Developmental and regional expression in the rat brain and functional properties of four N M D A receptors. Neuron 12: 529-540. Murray F, Kennedy J, Hutson PH, Elliot J, Huscroft I, Mohnen K, Russell M G , Grimwood S (2000) Modulation of [3H]MK-801 binding to N M D A receptors in vivo and in vitro. Eur J Pharmacol 397: 263-270. Mutel V , Buchy D, Klingelschmidt A , Messer J, Bleuel Z, Kemp JA, Richards JG (1998) In vitro binding properties in rat brain of [3H]Ro 25-6981, a potent and selective antagonist of N M D A receptors containing NR2B subunits. J Neurochem 70: 2147-2155. Olney JW, Newcomer JW, Farber NB (1999) N M D A receptor hypofunction model of schizophrenia. J Psychiatr Res 33: 523-533. Pakkenberg B (1990) Pronounced reduction of total neuron number in mediodorsal thalamic nucleus and nucleus accumbens in schizophrenics. Arch Gen Psychiatry 47: 1023-1028. Qin S, Zhao X , Pan Y , Liu J, Feng G, Fu J, Bao J, Zhang Z, He L (2005) An association study of the N-methyl-D-aspartate receptor NR1 subunit gene (GRIN1) and NR2B subunit gene (GRIN2B) in schizophrenia with universal D N A microarray. Eur J Hum Genet., in press. Rabacchi S, Bailly Y , Delhaye-Bouchaud N , Mariani J (1992) Involvement of the N -methyl D-aspartate (NMDA) receptor in synapse elimination during cerebellar development. Science 256: 1823-1825. Ritter L M , Vazquez D M , Meador-Woodruff JH (2002) Ontogeny of ionotropic glutamate receptor subunit expression in the rat hippocampus. Brain Res Dev Brain Res 139: 227-236. Semba J, Tanaka N , Wakuta M , Suhara T (2001) Neonatal phencyclidine treatment selectively attenuates mesolimbic dopamine function in adult rats as revealed by methamphetamine-induced behavior and c-fos mRNA expression in the brain. Synapse 40: 11-18. Silveira DC, Sogawa Y , Holmes G L (2002) The expression of Fos following kainic acid-induced seizures is age-dependent. Eur J Neurosci 15: 329-344. Stafstrom CE, Chronopoulos A, Thurber S, Thompson JL, Holmes G L (1993) Age-dependent cognitive and behavioral deficits after kainic acid seizures. Epilepsia 34: 420-432. 136 StafStrom CE, Tandon P, Hori A , Liu Z, Mikati M A , Holmes G L (1997) Acute effects of MK801 on kainic acid-induced seizures in neonatal rats. Epilepsy Res 26: 335-344. Stafstrom CE, Thompson JL, Holmes G L (1992) Kainic acid seizures in the developing brain: status epilepticus and spontaneous recurrent seizures. Brain Res Dev Brain Res 65: 227-236. Stefani M R , Moghaddam B (2005) Transient N-methyl-D-aspartate receptor blockade in early development causes lasting cognitive deficits relevant to schizophrenia. Biol Psychiatry 57: 433-436. Swerdlow NR, Platten A, Kim Y K , Gaudet I, Shoemaker J, Pitcher L , Auerbach P (2001) Sensitivity to the dopaminergic regulation of prepulse inhibition in rats: evidence for genetic, but not environmental determinants. Pharmacol Biochem Behav 70: 219-226. Tremblay E, Nitecka L, Berger M L , Ben Ari Y (1984) Maturation of kainic acid seizure-brain damage syndrome in the rat. I. Clinical, electrographic and metabolic observations. Neuroscience 13: 1051-1072. Vallano M L (1998) Developmental aspects of N M D A receptor function. Crit Rev Neurobiol 12: 177-204. van den B M , Garner B , Koch M (2003) Neurodevelopmental animal models of schizophrenia: effects on prepulse inhibition. Curr Mol Med 3: 459-471. Vestergaard M , Pedersen CB, Christensen J, Madsen K M , Olsen J, Mortensen PB (2005) Febrile seizures and risk of schizophrenia. Schizophr Res 73: 343-349. Wang C, Mclnnis J, Ross-Sanchez M , Shinnick-Gallagher P, Wiley JL, Johnson K M (2001) Long-term behavioral and neurodegenerative effects of perinatal phencyclidine administration: implications for schizophrenia. Neuroscience 107: 535-550. Weinberger DR (1987) Implications of normal brain development for the pathogenesis of schizophrenia. Arch Gen Psychiatry 44: 660-669. Zhang X Y , Liu AP, Ruan D Y , Liu J (2002) Effect of developmental lead exposure o n the expression of specific N M D A receptor subunit mRNAs in the hippocampus of neonatal rats by digoxigenin-labeled in situ hybridization histochemistry. Neurotoxicol Teratol 24: 149-160. 137 C H A P T E R F I V E : G E N E R A L D I S C U S S I O N The experiments contained within the present dissertation were designed to address two main objectives. First, Chapter Two detailed a number of experiments conducted to test the effects of electrical stimulation of discrete sub-regions of the hippocampus on PPI. Results of these experiments suggest brief periods of higher frequency activity that result in increased locomotor activity and ventral striatal dopamine efflux reversibly disrupt PPI when applied to the ventral, but not dorsal, hippocampus. The experiments described in Chapters Three and Four were designed to further the hypothesis that a period at the end of the first postnatal week is particularly sensitive for the development of the circuits mediating PPI and locomotor activity (Lipska and Weinberger, 2000). The experiments summarized in Chapter Three support this hypothesis by demonstrating that a single exposure to the kainate receptor agonist kainic acid on postnatal day seven disrupted PPI in early adulthood, but not before puberty. In Chapter Four, the effects of administration of the NR2B-subunit selective N M D A receptor antagonist Ro25-6981 on postnatal day six and seven were also assessed. Serendipitously, Ro25-6981 was observed to induce convulsions at this age; however, no consistent effects of this treatment on PPI or locomotor activity were noted either before puberty or in early adolescence. Although the implications of these results have been discussed at some length in the proceeding chapters, the present chapter will serve to further integrate these data with contemporary strategies of modeling schizophrenia in rodents. 138 The Role of the Hippocampus in the Regulation of Prepulse Inhibition and Locomotor Activity As detailed in the discussion section of Chapter Two, hippocampal regulation of PPI is complex and incompletely understood. However, a great deal of evidence using both N M D A (Wan et al., 1996;Klarner et al., 1998;Zhang et a l , 1999;Swerdlow et al., 2001b;Zhang et al., 2002) and higher frequency electrical (Howland et al., 2004b) stimulation protocols, indicates that over-activity of the ventral, but not dorsal, hippocampus disrupts PPI. Numerous studies have also assessed the role of hippocampal activity in locomotor behavior patterns of adult rats. Similar to the effects described for PPI, stimulation of the ventral hippocampus with either N M D A (Mogenson and Nielsen, 1984;Bardgett and Henry, 1999;Bast et al., 2001;Zhang et al., 2002) or higher frequency electrical current (Taepavarapruk et al., 2000) increases locomotion, whereas comparable stimulation of the dorsal hippocampus has no effect (Zhang et al., 2002). Here it is important to note that the dorsal hippocampus can influence locomotor activity under some conditions as dorsal hippocampal infusions of carbachol (Flicker and Geyer, 1982a;Mogenson and Nielsen, 1984), picrotoxin (Flicker and Geyer, 1982b) or other agents (Bast and Feldon, 2003), can alter the behavior. Thus, although the regulation of PPI and locomotor activity by the dorsal and ventral hippocampus is complex, both behaviors are clearly more sensitive to stimulation of the ventral than the dorsal region (Zhang et al., 2002;Bast and Feldon, 2003;Howland et al., 2004b). Previous research supports functional differentiation within the hippocampus, especially in regards to spatial learning and memory (supported by the dorsal hippocampus) and fear-related behavior (ventral hippocampus) (Moser and Moser, 1998; Kjelstrup et al., 2002). 139 When the roles of dopamine and glutamate in mediating the effects of either N M D A or electrical stimulation of the ventral hippocampus on PPI or locomotor activity are considered, an interesting dissociation has emerged (Bast and Feldon, 2003). As was previously described for PPI, neither dopamine antagonists (systemically administered) nor glutamate antagonists (infused into the nucleus accumbens) effectively block the disruption of PPI induced by infusion of N M D A into the ventral hippocampus (Wan et al., 1996;Bast et al., 2001). In contrast, both dopamine and glutamate antagonists block the increases in locomotion observed following stimulation of the ventral hippocampus (Bardgett and Henry, 1999;Taepavarapruk et al., 2000;Bast et al., 2001). Additionally, locomotor activity in a novel environment increases dopamine efflux in the nucleus accumbens, an effect that can be reversed by inactivation of the ventral hippocampus or administration of a glutamate receptor antagonist into the ventral tegmental area (Legault and Wise, 2001). Thus, these findings indicate that dopamine and glutamate transmission is critical for the ventral hippocampal modulation of locomotor activity, but not for PPI. Although the circuitry underlying the role of dopamine and glutamate in the ventral hippocampal stimulation-elicited increases in locomotor behavior is still being investigated, evidence suggests that dopamine and glutamate receptors in the nucleus accumbens (Taepavarapruk et al., 2000) and ventral tegmental area (Legault and Wise, 2001) play an important role, although other brain regions may also be involved. Stimulation of the ventral hippocampus with N M D A increases dopamine efflux in the medial prefrontal cortex (Peleg-Raibstein et al., 2005), and co-administration of dopamine antagonists with N M D A into the ventral hippocampus blocks the expected increase in locomotor behavior observed when N M D A is administered alone (Gimenez-140 Llort et al., 2002). Thus, strong parallels exist between the neural circuitry and pharmacology underlying the regulation of locomotor activity by the ventral hippocampus and that commonly described for schizophrenia. On the other hand, the mechanisms underlying the disruption of PPI following stimulation of the ventral hippocampus are less clear. As neither dopamine nor glutamate antagonists reverse the PPI disruptions following ventral hippocampal stimulation (Wan et al., 1996;Bast et al., 2001), direct activation of glutamatergic afferents from the ventral hippocampus to the nucleus accumbens is unlikely to underlie these effects. A recent study provides further support for this notion by demonstrating that lesions of the fornix, a tract containing the ventral hippocampal efferents to the nucleus accumbens, fail to block the disruption of PPI following stimulation of the ventral hippocampus with N M D A (Swerdlow et al., 2004). Therefore, the modulation of PPI by the ventral hippocampus likely involves either (1) interactions with brain areas other than those directly implicated in locomotor control (i.e., areas other than the nucleus accumbens) or (2) poly-synaptic pathways to the nucleus accumbens that do not include projections via the fornix. One potential circuit that may underlie the effects of stimulation of the ventral hippocampus includes the ventral hippocampal projections to the perirhinal and entorhinal cortex (Swerdlow et al., 2004). These areas have been implicated in the modulation of PPI (Swerdlow et al., 2001b;Goto et a l , 2002) and are connected, via non-fornical routes, to structures such as the nucleus accumbens (Totterdell and Meredith, 1997), basolateral amygdala (Pitkanen et al., 2000), and medial prefrontal cortex (Insausti et al., 1997) that are also involved in the modulation of PPI (Swerdlow et al., 2001a). 141 Importantly, given that the mechanisms through which stimulation of the ventral hippocampus disrupts PPI are currendy unknown, it has been suggested that further understanding of this phenomenon may aid in the development of novel therapeutic strategies for schizophrenia (Bast and Feldon, 2003). The Utility of Adult Animal Models of Schizophrenia As was discussed in Chapter One, animal models of schizophrenia created by manipulations performed during adulthood generally have poor construct validity given numerous lines of evidence that support a developmental component to the etiology of the disorder. However, adult models of schizophrenia can still contribute to a better understanding of the disorder in a number of different ways. For example, novel insights into the neural substrates underlying the behavioral symptoms of schizophrenia can be gained by performing experiments using adult animals. Support for this assertion is provided by data presented in Chapter Two that suggest increased ventral hippocampal activity in adulthood may underlie some symptoms of schizophrenia. Interestingly, results from brain imaging studies confirm the importance of these preclinical findings by demonstrating that basal levels of hippocampal activity are increased in schizophrenia (Heckers et al., 1998;Benes, 2000;Heckers, 2001). Additionally, the present results (Howland et al., 2004b), and those of other researchers (Zhang et al., 2002), suggest that the ventral hippocampus may be more involved in the regulation of sensorimotor processes such as PPI and locomotor activity than the dorsal hippocampus. As a result, examining alterations in specific sub-regions of the hippocampus in patients with schizophrenia may reveal previously unappreciated characteristics of the neurobiology of the disorder (Goldman and Mitchell, 2004). In 142 humans, the anterior portion of the hippocampus corresponds to the rodent ventral hippocampus, and some studies indicate that this region may be preferentially altered in schizophrenia (Csernansky et al., 1998;Szeszko et al., 2003;Narr et al., 2004; but see also Narr et al., 2001;Velakoulis et al., 2001). Furthermore, experiments with adult rats enable basic knowledge of the neural mechanisms underlying performance of behavioral tasks to be generated quickly. This knowledge can then be used to assess the potential mechanisms underlying behavioral deficits following developmental manipulations. This type of strategy was used to demonstrate that the PPI disruptions observed in socially isolated rats are dependent on dopamine levels in the nucleus accumbens (Powell et al., 2003) and extensively by Lipska and colleagues regarding various aspects of the altered neural circuitry and pharmacology underlying changes in the neonatal ventral hippocampal lesion model (Lipska et al., 1998;Lipska and Weinberger, 2000). Thus, combining information gained from both adult and developmental approaches will likely enable a more rapid and thorough understanding of the disorder (Floresco et al., 2005). Developmental Models of Schizophrenia - Effects of Early Postnatal Glutamate Manipulations Clearly, the results gained from both the neonatal kainic acid and Ro25-6981 manipulations are not as strong as was desired at the outset of the experiments. However, a number of interesting observations have been gained from these data, many of which should be verified in future experiments. The most important result was the subtle and reliable decrease in PPI observed following kainic acid administration in Chapter Three. Consistent with the ventral hippocampal lesion model, the PPI deficits were observed 143 only after the animals had reached early adulthood. However, these deficits were not augmented by apomorphine administration or accompanied consistently by changes in locomotor activity, as is the case with the PPI deficits following neonatal ventral hippocampal lesions (Lipska et al., 1993;Lipska et al., 1995). As was discussed in the introduction, some first degree relatives of patients with schizophrenia exhibit lower PPI responses than matched controls (Cadenhead et al., 2000). Thus, disrupted PPI may be considered a candidate marker for a 'trait' (or underlying predisposition) to develop schizophrenia. Accordingly, the kainic acid model, while not reproducing the full-blown expression of schizophrenia, may be useful as a method of producing an underlying predisposition for the disorder in rodents. Further support for this assertion is gained from studies suggesting that the hippocampus is also altered in first degree relatives of schizophrenic patients (Seidman et al., 2002) and in adult rats by neonatal kainic acid administration (Lynch et al., 2000). Assessing the interaction of neonatal kainic acid administration with additional manipulations may support the notion that schizophrenia results from multiple 'hits' over the course of development (McCarley et al., 1999;Bayer et al., 1999;Lewis and Levitt, 2002;Wong and Van Tol, 2003;Ellenbroek, 2003). To date, such strategies have been rarely used in an attempt to model the disorder although in one report, rats with neonatal ventral hippocampal lesions treated repeatedly with PCP in early adulthood displayed enhanced locomotor responses when compared to lesioned rats that had not been treated with PCP (Hori et al., 2000). Support for this experimental design is provided by observations indicating that neonatal kainic acid administration renders rats more 144 sensitive to morphological damage and behavioral impairments following kainic acid administration in adulthood (Koh et al., 1999). It is disappointing that the long-term behavioral experiments performed with Ro25-6981 failed to yield positive results. However, investigating novel mechanisms of brain development in the context of schizophrenia will improve understanding of the disorder. The rational for the use of Ro25-6981 was conceived from detailed knowledge surrounding the dynamic changes of specific N M D A subunits during early development, and similar experiments developed to test mechanistically-driven hypotheses will likely be an important component of preclinical schizophrenia research in the future. Further consideration of the null results of the experiments assessing the behavioral effects of neonatal administration of the NR2B antagonist Ro25-6981 can be focused in two main directions: (1) why the experiments performed fail to yield significant results and (2) what conclusions may be drawn from these experiments with respect to the existing literature regarding early developmental animal models of schizophrenia. Although the experiments using Ro25-6981 were carefully designed, the effects of this drug have not been extensively studied, especially in regard to the developing mammalian brain. In contrast, the acute effects of kainic acid administration are well known during development, and fit logically with some conceptions of the etiology of schizophrenia (Howland et al., 2004a). For example, kainic acid administration early in the neonatal period alters the activity of the hippocampus (Stafstrom et al., 1992;Khalilov et al., 1999;Lynch et al., 2000;Silveira et al., 2002) and additional limbic and cortical areas including the entorhinal cortex (Khalilov et al., 1999) implicated in schizophrenia. 145 Additionally, rats treated neonatally with kainic acid exhibit subtle physiological alterations in the hippocampus during adulthood, which may underlie some of the behavioral alterations observed in these animals (Lynch et al., 2000). To my knowledge, the present experiments are the first to assess the effects of Ro25-6981 administration in neonatal rats. In retrospect, it may have been profitable to more carefully assess the acute effects of Ro25-6981 administration on the neonatal brain before the long-term behavioral experiments described herein were performed. Two main avenues will likely be especially profitable for understanding both the acute and long-term effects of neonatal Ro25-6981 administration on brain and behavior. Initially, determining which brain areas are activated with either convulsive or sub-convulsive doses of the drug will enable the development of a more detailed hypothesis-driven approach regarding the potential long-term behavioral effects of the convulsions induced by Ro25-6981. I expect that the activity of structures such as the hippocampus, cortex, and striatum will be altered by administration of Ro25-6981 due to high levels of NR2B expression in those areas neonatally (Monyer et al., 1994;Loftis and Janowsky, 2003), and the well documented role of these areas in seizures (Ben Ar i and Cossart, 2000). However, high levels of NR2B-containing N M D A receptors are also expressed in other areas including the thalamus and cerebellum at this developmental age (Loftis and Janowsky, 2003). Interestingly, connections between these areas and the frontal cortex have been implicated in schizophrenia (Andreasen et al., 1999;Konarski et al., 2005). Thus, altered activity patterns in distributed neural circuits may underlie the convulsions produced by Ro25-6981. 146 Secondly, an assessment of the effects of Ro25-6981 administration on cell survival in the developing brain is essential. As detailed in Chapter Four, a number of competitive and non-competitive N M D A receptor antagonists induce massive cell death in the developing brain, a factor which may be important in the long-term behavioral effects of their administration (Ikonomidou et al., 1999;Wang et a l , 2001;Harris et al., 2003;Fredriksson et al., 2004). The influence of Ro25-6981 on cell survival in the developing brain is currently unknown, however the high proportion of NR2J3-containing N M D A receptors early in development supports the hypothesis that treatment with Ro25-6981 may cause apoptosis during this developmental age. Although a detailed understanding of the effects of Ro25-6981 administration is lacking, the results of the behavioral experiments conducted prior to and after puberty are still surprising as the induction of three strong convulsions during the end of the postnatal week failed to alter PPI or locomotor responding. One factor that could be especially important in the different behavioral effects observed between the convulsions elicited by kainic acid and Ro25-6981 is that convulsions elicited by kainic acid are significantly longer in duration than those elicited with Ro25-6981. Longer convulsions may disturb developing neural networks to a significantly greater extent than shorter ones (Jensen and Baram, 2000;Holmes, 2004). Previous studies using other convulsive agents such as flourothyl, which have a short duration of action (10 to 15 minutes), support this assertion. These experiments indicate that a series of 25 to 50 convulsions during the first week of life must be elicited for long-term alterations in behavior to be observed (Holmes et al., 1998;Huang et al., 1999). Further support for this hypothesis is gained from data suggesting that a history of prolonged febrile seizures is a significant risk factor 147 for psychosis in adulthood epileptics (Kanemoto et al., 2001) and that prolonged febrile seizures result in long-term changes in hippocampal excitability in rodents (Chen et al., 1999;Jensen and Baram, 2000). Therefore, it is tempting to speculate that repeated convulsions induced by Ro25-6981 during the first postnatal week would be related to alterations in PPI, locomotor activity, and cognitive deficits in adult rats. Developmental Models of Schizophrenia - The Time Course of Symptom Emergence Although developmental animal models of schizophrenia are likely more ecologically valid than adult models, they are much more difficult to establish. One of the cardinal features of the majority of patients with schizophrenia is the delayed onset of many symptoms of the disorder (Marcotte et al., 2001;Wong and Van Tol, 2003), however, this feature of the disorder remains elusive in animal models (Marcotte et al., 2001). Research examining recovery of function after brain injury generally supports the assertion that behavioral impairment is less severe if the injury is suffered early in life - a phenomenon commonly termed the Kennard principle (Kolb et al., 2000;Marcotte et al., 2001). However, it is important to note that the pattern of recovery from injuries sustained during early brain development show some exceptions to the Kennard principle. For example, lesions of the frontal cortex induced at specific developmental stages (i.e. late during the embryologic period or after the first postnatal week) result in significant recovery over time, while other periods exist (i.e. the first postnatal week) where recovery from similar injuries is significantly poorer than expected (Kolb and Whishaw, 1989;Kolb et al., 2000). In the case of schizophrenia, the relation between adverse events early in development and subsequent manifestation of symptoms suggest a delayed response in 148 which behavioral deficits emerge after a period of relative normality. The neonatal ventral hippocampal lesion model provides important proof that alterations in the neural substrates implicated in schizophrenia during certain developmental stages may result in the delayed onset of various behavioral and electrophysiological alterations related to the disorder (Lipska et al., 1993;Lipska et al., 1995;Wood et al., 1997;Lipska and Weinberger, 2000;Lipska et a l , 2002;Goto and O'Donnell, 2002). Additional research suggests that manipulations other than ventral hippocampal lesions such as immune activation (Zuckerman et al., 2003), administration of the anti-mitotic agent arabinoside (Elmer et al., 2004), or kainic acid (Howland et al., 2004a) result in the delayed onset of behavioral abnormalities associated with schizophrenia (unfortunately, unlike the design employed in this thesis, the behavioral effects of many early neonatal treatments developed have not been tested before adulthood). Taken together, these data suggest that certain critical periods exist in the neonatal brain for the normal development of the neural circuits mediating behaviors commonly ascribed to schizophrenia in animals (Lipska and Weinberger, 2000;Marcotte et al., 2001;Howland et al., 2004a). Future work related to the mechanisms underlying these changes will likely provide novel insights into the etiology of the disorder. Criteria for Establishing Validity in Behavioral Models of Schizophrenia The present experiments relied heavily on PPI and locomotor behavior in assessing the experimental manipulations performed in modeling schizophrenia. Although these behaviors have been used extensively by researchers in the field, they have a number of shortcomings. Importantly, PPI is disrupted in a number of neurological and psychiatric disorders in addition to schizophrenia including 149 Huntington's disease, Tourette's syndrome, obsessive-compulsive disorder, and pathological gambling (Braff et al., 2001). Given the complexity of the neural circuitry mediating PPI this is not particularly surprising; however, it is problematic in that disrupted PPI may not be characteristic of schizophrenia per se, but rather of alterations in the distributed circuitry mediating it (Swerdlow et al., 2001a). As previously discussed, increased locomotor activity is often used as an indirect measure of ventral striatal dopamine activity, especially when combined with the administration of dopamine agonists (Kelly et al., 1975;Castall et al., 1977;Porrino et al., 1984;Lipska and Weinberger, 2000;Marcotte et al., 2001). Unfortunately, altered locomotor behavior is a very general phenomenon and can be influenced by a number of factors including anxiety levels, alterations in sensory processing, and memory disruptions (Bast and Feldon, 2003). Although some parallels between increased locomotor activity and certain symptoms of schizophrenia have been suggested (Bast and Feldon, 2003), these assertions are clearly speculative (Marcotte et al., 2001). As a result of these shortcomings, testing PPI and locomotor activity levels should be viewed as an important 'first-pass' in the assessment of potential animal models of schizophrenia. Future experiments with an array of behaviors relevant to schizophrenia will serve to strengthen the validity of putative models of schizophrenia (Lipska and Weinberger, 2000). Recent interest has been generated around preclinical correlates of the negative and cognitive symptoms of schizophrenia as they have traditionally been ignored in much of the literature (Ellenbroek and Cools, 2000;Floresco et al., 2005). Tests assessing social interaction, reward sensitivity, and executive functions such as cognitive set shifting and working memory are likely to be especially useful in further 150 understanding the more complicated and treatment-resistant symptoms of the disorder (Ellenbroek and Cools, 2000;Robbins, 2004;Floresco et al., 2005). It is worth pointing out, however, that many individuals with schizophrenia do not exhibit all symptoms of the disorder (Wong and Van Tol, 2003). Therefore, reliance on strategies that successfully model many aspects of the disorder in animals may be counterproductive in some instances. Interestingly, some manipulations, such as neonatal ventral hippocampal lesions, produce changes in many behaviors resembling schizophrenia (Lipska and Weinberger, 2000), while others, such as certain neonatal N M D A antagonist treatment regimes, produce selective deficits on certain behavioral tasks relevant to one class of symptoms (e.g. cognitive symptoms) (Stefani and Moghaddam, 2005). The present data do not indicate how animals treated with either kainic acid or Ro25-6981 may respond if further testing was conducted. However, in some studies, rats treated neonatally with kainic acid display deficits in learning and memory (Lynch et al., 2000), although null findings have also been reported (Stafstrom et al., 1993). Therefore, in the future, it may be profitable to greatly extend the test battery beyond the tests presently employed. Finally, testing the efficacy of antipsychotic drugs at reversing the behavioral disturbances observed following neonatal kainic acid administration could greatly aid in assessing its validity as a model of schizophrenia. As has been noted throughout the present dissertation, both typical and atypical antipsychotics are effective at ameliorating the behavioral disturbances, including PPI deficits, in numerous animal models of schizophrenia (Lipska and Weinberger, 2000;Marcotte et al., 2001;Bast et al., 2001 ;Le Pen and Moreau, 2002;Bast and Feldon, 2003;Van den et al., 2003;Le Pen et al., 2003). 151 Conclusion In the introduction, three main goals for developing animal models of schizophrenia were introduced: (1) advancing the understanding of the symptoms of the disorder, (2) furthering the understanding of schizophrenia's etiology, and (3) aiding in the development of pharmaceutical therapies (Lipska and Weinberger, 2000). The present dissertation explored two general strategies for developing behaviorally-oriented animal models of schizophrenia. Although the results were somewhat mixed, the experiments were successful at providing novel insights into the symptoms and etiology of the disorder. In general, they support the assertion that short periods of altered activity in the limbic system, and hippocampus in particular, at different points during development may underlie the expression of some of the most basic symptoms of schizophrenia. These data also suggest that the nature and anatomical location of these alterations critically determines their long-term functional effects. 152 References Andreasen NC, Nopoulos P, O'Leary DS, Miller DD, Wassink T, Flaum M (1999) Defining the phenotype of schizophrenia: cognitive dysmetria and its neural mechanisms. Biol Psychiatry 46: 908-920. Bardgett M E , Henry JD (1999) Locomotor activity and accumbens Fos expression driven by ventral hippocampal stimulation require D l and D2 receptors. Neuroscience 94: 59-70. Bast T, Feldon J (2003) Hippocampal modulation of sensorimotor processes. Prog Neurobiol 70: 319-345. Bast T, Zhang WN, Heidbreder C, Feldon J (2001) Hyperactivity and disruption of prepulse inhibition induced by N-methyl-D-aspartate stimulation of the ventral hippocampus and the effects of pretreatment with haloperidol and clozapine. Neuroscience 103: 325-335. Bayer TA, Falkai P, Maier W (1999) Genetic and non-genetic vulnerability factors in schizophrenia: the basis of the "Two hit hypothesis". Journal of Psychiatric Research 33: 543-548. Ben Ar i Y , Cossart R (2000) Kainate, a double agent that generates seizures: two decades of progress. Trends in Neurosciences 23: 580-587. Benes F M (2000) Emerging principles of altered neural circuitry in schizophrenia. Brain Res Brain Res Rev 31: 251-269. Braff DL, Geyer M A , Swerdlow NR (2001) Human studies of prepulse inhibition of startle: normal subjects, patient groups, and pharmacological studies. Psychopharmacology (Berl) 156: 234-258. Cadenhead KS, Swerdlow NR, Shafer K M , Diaz M , Braff D L (2000) Modulation of the startle response and startle laterality in relatives of schizophrenic patients and in subjects with schizotypal personality disorder: evidence of inhibitory deficits. Am J Psychiatry 157: 1660-1668. Castall B , Marsden CD, Naylor RJ, Pycock CJ (1977) Stereotyped behaviour patterns and hyperactivity induced by amphetamine and apomorphine after discrete 6-hydroxydopamine lesions of extrapyramidal and mesolimbic nuclei. Brain Res 123: 89-111. Chen K, Baram TZ, Soltesz I (1999) Febrile seizures in the developing brain result in persistent modification of neuronal excitability in limbic circuits. Nat Med 5: 888-894. Csernansky JG, Joshi S, Wang L, Haller JW, Gado M , Miller JP, Grenander U , Miller MI (1998) Hippocampal morphometry in schizophrenia by high dimensional brain mapping. Proc Natl Acad Sci U S A 95: 11406-11411. 153 Ellenbroek B A (2003) Animal models in the genomic era: possibilities and limitations with special emphasis on schizophrenia. Behav Pharmacol 14: 409-417. Ellenbroek B A , Cools A R (2000) Animal models for the negative symptoms of schizophrenia. Behav Pharmacol 11: 223-233. Elmer G l , Sydnor J, Guard H, Hercher E, Vogel M W (2004) Altered prepulse inhibition in rats treated prenatally with the antimitotic Ara-C: an animal model for sensorimotor gating deficits in schizophrenia. Psychopharmacology (Berl) 174: 177-189. Flicker C, Geyer M A (1982a) Behavior during hippocampal microinfusions. II. Muscarinic locomotor activation. Brain Res 257: 105-127. Flicker C, Geyer M A (1982b) Behavior during hippocampal microinfusions. III. Lidocaine versus picrotoxin. Brain Res 257: 129-136. Floresco SB, Geyer M A , Gold L H , Grace A A (2005) Developing Predictive Animal Models and Establishing a Preclinical Trials Network for Assessing Treatment Effects on Cognition in Schizophrenia. Schizophr Bull. Fredriksson A, Archer T, Aim H , Gordh T, Eriksson P (2004) Neurofunctional deficits and potentiated apoptosis by neonatal N M D A antagonist administration. Behavioural Brain Research 153: 367-376. Gimenez-Llort L , Wang FH, Ogren SO, Ferre S (2002) Local dopaminergic modulation of the motor activity induced by N-methyl-D-aspartate receptor stimulation in the ventral hippocampus. Neuropsychopharmacology 26: 737-743. Goldman M B , Mitchell CP (2004) What is the functional significance of hippocampal pathology in schizophrenia? Schizophr Bull 30: 367-392. Goto K, Ueki A , Iso H , Morita Y (2002) Reduced prepulse inhibition in rats with entorhinal cortex lesions. Behav Brain Res 134: 201-207. Goto Y , O'Donnell P (2002) Delayed mesolimbic system alteration in a developmental animal model of schizophrenia. J Neurosci 22: 9070-9077. Harris LW, Sharp T, Gartlon J, Jones DNC, Harrison PJ (2003) Long-term behavioural, molecular and morphological effects of neonatal N M D A receptor antagonism. European Journal of Neuroscience 18: 1706-1710. Heckers S (2001) Neuroimaging studies of the hippocampus in schizophrenia. Hippocampus 11: 520-528. Heckers S, Rauch SL, Goff D, Savage CR, Schacter DL, Fischman AJ , Alpert N M (1998) Impaired recruitment of the hippocampus during conscious recollection in schizophrenia. Nat Neurosci 1: 318-323. 154 Holmes G L (2004) Effects of early seizures on later behavior and epileptogenicity. Ment Retard Dev Disabil Res Rev 10: 101-105. Holmes GL, Gairsa JL, Chevassus-Au-Louis N , Ben Ari Y (1998) Consequences of neonatal seizures in the rat: morphological and behavioral effects. Ann Neurol 44: 845-857. Hori T, Subramaniam S, Srivastava L K , Quirion R (2000) Behavioral and neurochemical alterations following repeated phencyclidine administration in rats with neonatal ventral hippocampal lesions. Neuropharmacology 39: 2478-2491. Howland JG, Hannesson DK, Phillips A G (2004a) Delayed onset of prepulse inhibition deficits following kainic acid treatment on postnatal day 7 in rats. Eur J Neurosci 20: 2639-2648. Howland JG, MacKenzie E M , Yim TT, Taepavarapruk P, Phillips A G (2004b) Electrical stimulation of the hippocampus disrupts prepulse inhibition in rats: frequency- and site-dependent effects. Behav Brain Res 152: 187-197. Huang L, Cilio MR, Silveira DC, McCabe B K , Sogawa Y , Stafstrom CE, Holmes G L (1999) Long-term effects of neonatal seizures: a behavioral, electrophysiological, and histological study. Brain Res Dev Brain Res 118: 99-107. Ikonomidou C, Bosch F, Miksa M , Bittigau P, Vockler J, Dikranian K, Tenkova TI, Stefovska V, Turski L, Olney JW (1999) Blockade of N M D A receptors and apoptotic neurodegeneration in the developing brain. Science 283: 70-74. Insausti R, Herrero MT, Witter M P (1997) Entorhinal cortex of the rat: cytoarchitectonic subdivisions and the origin and distribution of cortical efferents. Hippocampus 7: 146-183. Jensen FE, Baram TZ (2000) Developmental seizures induced by common early-life insults: short- and long-term effects on seizure susceptibility. Ment Retard Dev Disabil Res Rev 6: 253-257. Kanemoto K, Tsuji T, Kawasaki J (2001) Reexamination of interictal psychoses based on D S M IV psychosis classification and international epilepsy classification. Epilepsia 42: 98-103. Kelly PH, Seviour PW, Iversen SD (1975) Amphetamine and apomorphine responses in the rat following 6-OHDA lesions of the nucleus accumbens septi and corpus striatum. Brain Res 94: 507-522. Khalilov I, Dzhala V , Medina I, Leinekugel X , Melyan Z, Lamsa K, Khazipov R, Ben Ar i Y (1999) Maturation of kainate-induced epileptiform activities in interconnected intact neonatal limbic structures in vitro. Eiir J Neurosci 11: 3468-3480. 155 Kjelstrup K G , Tuvnes FA, Steffenach HA, Murison R, Moser EI, Moser M B (2002) Reduced fear expression after lesions of the ventral hippocampus. PNAS 99:10825-10830. Klarner A , Koch M , Schnitzler H U (1998) Induction of Fos-protein in the forebrain and disruption of sensorimotor gating following N-methyl-D-aspartate infusion into the ventral hippocampus of the rat. Neuroscience 84: 443-452. Koh S, Storey TW, Santos TC, Mian A Y , Cole A J (1999) Early-life seizures in rats increase susceptibility to seizure-induced brain injury in adulthood. Neurology 53: 915-921. Kolb B , Gibb R, Gorny G (2000) Cortical plasticity and the development of behavior after early frontal cortical injury. Dev Neuropsychol 18: 423-444. Kolb B , Whishaw IQ (1989) Plasticity in the neocortex: mechanisms underlying recovery from early brain damage. Prog Neurobiol 32: 235-276. Konarski JZ, Mclntyre RS, Grupp L A , Kennedy SH (2005) Is the cerebellum relevant in the circuitry of neuropsychiatric disorders? J Psychiatry Neurosci 30: 178-186. Le Pen G, Kew J, Alberati D, Borroni E, Heitz MP, Moreau JL (2003) Prepulse inhibition deficits of the starde reflex in neonatal ventral hippocampal-lesioned rats: reversal by glycine and a glycine transporter inhibitor. Biol Psychiatry 54: 1162-1170. Le Pen G, Moreau JL (2002) Disruption of prepulse inhibition of startle reflex in a neurodevelopmental model of schizophrenia: reversal by clozapine, olanzapine and risperidone but not by haloperidol. Neuropsychopharmacology 27: 1-11. Legault M , Wise R A (2001) Novelty-evoked elevations of nucleus accumbens dopamine: dependence on impulse flow from the ventral subiculum and glutamatergic neurotransmission in the ventral tegmental area. Eur J Neurosci 13: 819-828. Lewis DA, Levitt P (2002) Schizophrenia as a disorder of neurodevelopment. Annu Rev Neurosci 25:409-432. Lipska B K , A l Amin HA, Weinberger DR (1998) Excitotoxic lesions of the rat medial prefrontal cortex. Effects on abnormal behaviors associated with neonatal hippocampal damage. Neuropsychopharmacology 19: 451-464. Lipska B K , Halim ND, Segal PN, Weinberger DR (2002) Effects of reversible inactivation of the neonatal ventral hippocampus on behavior in the adult rat. J Neurosci 22: 2835-2842. Lipska B K , Jaskiw GE, Weinberger DR (1993) Postpubertal emergence of hyperresponsiveness to stress and to amphetamine after neonatal excitotoxic hippocampal damage: a potential animal model of schizophrenia. Neuropsychopharmacology 9: 67-75. 156 Lipska B K , Swerdlow NR, Geyer M A , Jaskiw GE, Braff DL, Weinberger DR (1995) Neonatal excitotoxic hippocampal damage in rats causes post-pubertal changes in prepulse inhibition of startle and its disruption by apomorphine. Psychopharmacology (Berl) 122: 35-43. Lipska B K , Weinberger DR (2000) To model a psychiatric disorder in animals: schizophrenia as a reality test. Neuropsychopharmacology 23: 223-239. Loftis J M , Janowsky A (2003) The N-methyl-D-aspartate receptor subunit NR2B: localization, functional properties, regulation, and clinical implications. Pharmacol Ther 97:55-85. Lynch M , Sayin U , Bownds J, Janumpalli S, Sutula T (2000) Long-term consequences of early postnatal seizures on hippocampal learning and plasticity. Eur J Neurosci 12: 2252-2264. Marcotte ER, Pearson D M , Srivastava L K (2001) Animal models of schizophrenia: a critical review. J Psychiatry Neurosci 26: 395-410. McCarley RW, Wible CG, Frumin M , Hirayasu Y , Levitt JJ, Fischer IA, Shenton M E (1999) MRI anatomy of schizophrenia. Biol Psychiatry 45: 1099-1119. Mogenson GJ, Nielsen M (1984) A study of the contribution of hippocampal-accumbens-subpallidal projections to locomotor activity. Behav Neural Biol 42: 38-51. Monyer H , Burnashev N , Laurie DJ, Sakmann B , Seeburg PH (1994) Developmental and regional expression in the rat brain and functional properties of four N M D A receptors. Neuron 12: 529-540. Moser M B , Moser EI (1998) Functional differentiation in the hippocampus. Hippocampus 8: 593-600. Narr K L , Thompson P M , Sharma T, Moussai J, Blanton R, Anvar B , Edris A , Krupp R, Rayman J, Khaledy M , Toga A W (2001) Three-dimensional mapping of temporo-limbic regions and the lateral ventricles in schizophrenia: gender effects. Biol Psychiatry 50: 84-97. Narr K L , Thompson P M , Szeszko P, Robinson D, Jang S, Woods RP, Kim S, Hayashi K M , Asunction D, Toga A W , Bilder R M (2004) Regional specificity of hippocampal volume reductions in first-episode schizophrenia. Neuroimage 21: 1563-1575. Peleg-Raibstein D, Pezze M A , Ferger B, Zhang W N , Murphy C A , Feldon J, Bast T (2005) Activation of dopaminergic neurotransmission in the medial prefrontal cortex by N-methyl-d-aspartate stimulation of the ventral hippocampus in rats. Neuroscience 132: 219-232. 157 Pitkanen A , Pikkarainen M , Nurminen N , Ylinen A (2000) Reciprocal connections between the amygdala and the hippocampal formation, perirhinal cortex, and postrhinal cortex in rat. A review. Ann N Y Acad Sci 911: 369-391. Porrino LJ , Lucignani G, Dow-Edwards D, Sokoloff L (1984) Correlation of dose-dependent effects of acute amphetamine administration on behavior and local cerebral metabolism in rats. Brain Res 307: 311-320. Powell SB, Geyer M A , Preece M A , Pitcher L K , Reynolds GP, Swerdlow NR (2003) Dopamine depletion of the nucleus accumbens reverses isolation-induced deficits in prepulse inhibition in rats. Neuroscience 119: 233-240. Robbins TW (2004) Animal models of the psychoses. In: Neurobiology of Mental Illness, 2nd. Ed. (Charney DS, Nestler EJ, eds), pp 263-286. New York: Oxford University Press. Seidman LJ , Faraone SV, Goldstein J M , Kremen WS, Horton NJ, Makris N , Toomey R, Kennedy D, Caviness VS, Tsuang M T (2002) Left hippocampal volume as a vulnerability indicator for schizophrenia: a magnetic resonance imaging morphometric study of nonpsychotic first-degree relatives. Arch Gen Psychiatry 59: 839-849. Silveira DC, Sogawa Y, Holmes G L (2002) The expression of Fos following kainic acid-induced seizures is age-dependent. Eur J Neurosci 15: 329-344. Stafstrom CE, Chronopoulos A , Thurber S, Thompson JL, Holmes G L (1993) Age-dependent cognitive and behavioral deficits after kainic acid seizures. Epilepsia 34: 420-432. Stafstrom CE, Thompson JL, Holmes G L (1992) Kainic acid seizures in the developing brain: status epilepticus and spontaneous recurrent seizures. Brain Res Dev Brain Res 65: 227-236. Stefani M R , Moghaddam B (2005) Transient N-methyl-D-aspartate receptor blockade in early development causes lasting cognitive deficits relevant to schizophrenia. Biol Psychiatry 57: 433-436. Swerdlow NR, Geyer M A , Braff D L (2001a) Neural circuit regulation of prepulse inhibition of startle in the rat: current knowledge and future challenges. Psychopharmacology (Berl) 156: 194-215. Swerdlow NR, Hanlon F M , Henning L, Kim Y K , Gaudet I, Halim N D (2001b) Regulation of sensorimotor gating in rats by hippocampal N M D A : anatomical localization. Brain Res 898: 195-203. Swerdlow NR, Shoemaker JM, Noh HR, Ma L, Gaudet I, Munson M , Crain S, Auerbach PP (2004) The ventral hippocampal regulation of prepulse inhibition and its disruption by apomorphine in rats are not mediated via the fornix. Neuroscience 123: 675-685. 158 Szeszko PR, Goldberg E, Gunduz-Bruce H , Ashtari M , Robinson D, Malhotra A K , Lencz T, Bates J, Crandall DT, Kane J M , Bilder R M (2003) Smaller anterior hippocampal formation volume in antipsychotic-naive patients with first-episode schizophrenia. Am J Psychiatry 160: 2190-2197. Taepavarapruk P, Floresco SB, Phillips A G (2000) Hyperlocomotion and increased dopamine efflux in the rat nucleus accumbens evoked by electrical stimulation of the ventral subiculum: role of ionotropic glutamate and dopamine D l receptors. Psychopharmacology (Berl) 151: 242-251. Totterdell S, Meredith GE (1997) Topographical organization of projections from the entorhinal cortex to the striatum of the rat. Neuroscience 78: 715-729. Van den B M , Garner B , Koch M (2003) Neurodevelopmental animal models of schizophrenia: effects on prepulse inhibition. Curr Mol Med 3: 459-471. Velakoulis D, Stuart GW, Wood SJ, Smith DJ, Brewer WJ, Desmond P, Singh B , Copolov D, Pantelis C (2001) Selective bilateral hippocampal volume loss in chronic schizophrenia. Biol Psychiatry 50: 531-539. Wan FJ, Caine SB, Swerdlow NR (1996) The ventral subiculum modulation of prepulse inhibition is not mediated via dopamine D2 or nucleus accumbens non-NMDA glutamate receptor activity. Eur J Pharmacol 314: 9-18. Wang C, Mclnnis J, Ross-Sanchez M , Shinnick-Gallagher P, Wiley JL, Johnson K M (2001) Long-term behavioral and neurodegenerative effects of perinatal phencyclidine administration: Implications for schizophrenia. Neuroscience 107: 535-550. Wong A H , Van Tol H H (2003) Schizophrenia: from phenomenology to neurobiology. Neurosci Biobehav Rev 27: 269-306. Wood GK, Lipska B K , Weinberger DR (1997) Behavioral changes in rats with early ventral hippocampal damage vary with age at damage. Brain Res Dev Brain Res 101: 17-25. Zhang W, Pouzet B , Jongen-Relo A L , Weiner I, Feldon J (1999) Disruption of prepulse inhibition following N-methyl-D-aspartate infusion into the ventral hippocampus is antagonized by clozapine but not by haloperidol: a possible model for the screening of atypical antipsychotics. Neuroreport 10: 2533-2538. Zhang W N , Bast T, Feldon J (2002) Effects of hippocampal N-methyl-D-aspartate infusion on locomotor activity and prepulse inhibition: differences between the dorsal and ventral hippocampus. Behav Neurosci 116: 72-84. Zuckerman L , Rehavi M , Nachman R, Weiner I (2003) Immune activation during pregnancy in rats leads to a postpubertal emergence of disrupted latent inhibition, dopaminergic hyperfunction, and altered limbic morphology in the offspring: a novel neurodevelopmental model of schizophrenia. Neuropsychopharmacology 28: 1778-1789. 159 

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