THE DEVELOPMENT OF NEURODEGENERATION AND BEHAVIOURAL ALTERATIONS FOLLOWING LITHIUM/PILOCARPINE-INDUCED STATUS EPILEPTICUS IN RATS

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1 THE DEVELOPMENT OF NEURODEGENERATION AND BEHAVIOURAL ALTERATIONS FOLLOWING LITHIUM/PILOCARPINE-INDUCED STATUS EPILEPTICUS IN RATS by Crystal Maureen Dykstra A thesis submitted in conformity with the requirements for the degree of doctor of philosophy Institute of Medical Science University of Toronto Copyright by Crystal Maureen Dykstra (2011)

2 THE DEVELOPMENT OF NEURODEGENERATION AND BEHAVIOURAL ALTERATIONS AFTER LITHIUM/PILOCARPINE-INDUCED STATUS EPILEPTICUS IN RATS Crystal Maureen Dykstra Doctor of Philosophy Institute of Medical Science University of Toronto 2011 The lithium/pilocarpine model of epilepsy mimics mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) in humans. Systemic injection of pilocarpine in lithium chloride (LiCL) pretreated adult rats results in an acute episode of severe continuous seizure activity (status epilepticus, SE). SE causes a latent period, whereby the animal appears neurologically normal, with subsequent development of spontaneous recurrent seizures (SRSs). Neuropathological changes that occur during the latent period are believed to contribute to the epileptic condition. The present thesis characterized the development of neuronal death and behavioural alterations in rats after SE induced by the repeated low-dose pilocarpine procedure (RLDP), and investigated the causal relationship between these two processes. Our data demonstrated that the RLDP procedure for the induction of SE results in widespread neurodegeneration and behavioural alterations comparable to the pilocarpine and low-dose pilocarpine (LDP) procedures. However, the advantage to using this protocol was straindependent as it reduced mortality in Wistar, but not in Long Evans Hooded (LEH), rats. Stereological analysis of neurons (stained for the neuronal specific marker [NeuN]) at various times (1 hr to 3 months) following SE showed that different brain regions within the ii

3 hippocampus, amygdala, thalamus and piriform cortex exhibited differential rates of neuronal loss, with the majority of SE-induced neuronal death present by 24 hours. SE resulted in decreased exploratory behavior as assessed in the open field test, increased aggression to handling, increased hyperreactivity as assessed in the touch-response test, and anxiolytic effects as measured in the elevated-plus maze. Furthermore, deficits in search strategies used, as well as impaired spatial learning and memory, contributed to poor Morris water maze (MWM) performance. Partial neuroprotection within the hippocampus (by tat-nr2b9c) had no effect on the number of rats developing SRSs or on behavioural alterations; this argues against a causal relationship between neurodegeneration within this region, genesis of SRSs, and behavioural morbidity. iii

4 Acknowledgments This thesis would not have been possible without the support of many people. First and foremost, I offer my sincerest gratitude to Dr. James Gurd, whose encouragement, guidance and support from the initial to the final level enabled me to complete this thesis. I greatly appreciate his patience and knowledge whilst allowing me the room to work in my own way. I would like to extend my gratitude to Dr. Bill Milgram, who was abundantly helpful and offered invaluable assistance, support and guidance. I would also like to thank my program advisory committee members, Drs. Mac Burnham and Mike Tymianski, for their time, guidance and constructive criticism. There are many individuals who have offered their time and expertise in training me in specific laboratory techniques. I would like to thank Dr. Gwen Ivy for teaching me histological techniques that were invaluable for this work and for sharing her lab space. I would like to express my gratitude to Raymond Or for training on the microscopes, and to Candace Ikeda- Douglas for introducing me to the lithium/pilocarpine seizure model. I am grateful to Dr. Stephen Reid for sharing his time and expertise in animal surgeries. I am extremely grateful to Nankie Bissoon for not only sharing her vast knowledge of experimental techniques, but also for her guidance and friendship in the lab. I would like to extend my gratitude to Dr. Janelle Leboutillier for her knowledge, guidance and support. Finally, I owe my deepest gratitude to my parents for their love and support and to the rest of my family for their encouragement. iv

5 List of Publications Some of the material presented in this thesis has been published. This is to certify that I, Crystal Dykstra, carried out the research documented in the following publications: Dykstra CM, Ratnam M, Gurd JW (2009) Neuroprotection after status epilepticus by targeting protein interactions with postsynaptic density protein 95. J Neuropathol Exp Neurol 68: v

6 Table of Contents Acknowledgments... iv List of Publications... v Table of Contents... vi List of Tables... xv List of Figures... xvi List of Appendices... xix List of Abbreviations... xx Chapter 1 General Introduction Main features of mesial temporal lobe epilepsy with hippocampal sclerosis Animal models of seizure development and epilepsy Kindling Post-status epilepticus models Background information on the pilocarpine and lithium/pilocarpine models The pilocarpine model Convulsive effects of pilocarpine are mediated by activation of M1 receptors The cholinergic system is involved in the initiation but not the maintenance of SE The lithium/pilocarpine model The proconvulsive mechanisms of lithium Use of diazepam to control SE duration and reduce mortality Behavioural and clinical features of seizure development in the lithium/pilocarpine model The acute phase Behavioural seizures during the acute phase Electroencephalographic patterns during motor limbic seizures and SE Scoring of pilocarpine-induced seizures vi

7 Effect of SE duration on mortality and neuropathology Epileptogenesis Duration of the latent phase The chronic phase Behaviour during the chronic phase Electroencephalographic patterns observed during SRSs Neuropathology Neurodegeneration SE-induced neurodegeneration Progression and severity of neuronal loss following SE Mechanisms underlying SE-induced neuronal death Types of cell death mechanisms initiated by SE Factors determining the extent and phenotype of SE-induced neuronal death Synaptic reorganization Reactive gliosis Neurogenesis Co-morbid interictal disorders in mesial temporal lobe epilepsy The relationship between epilepsy and cognitive and interictal behavioural alterations Shared neurodevelopmental, genetic or environmental causes predispose subjects to develop both epilepsy and co-morbid behavioural and cognitive disturbances Neuropathological changes underlying the genesis of interictal behavioural disturbances are closely related to those mediating epileptogenesis itself Spontaneous recurrent seizures contribute to interictal behavioural and cognitive impairment in post-se models Other factors affecting severity of interictal behavioural and cognitive impairment Interictal behavioural disturbances following SE Anxiety vii

8 Exploration Aggression The effect of SE on spatial learning and memory The Morris water maze as a test of visual-spatial learning and memory The effect of SE on performance in the MWM task The goals of the thesis Chapter 2 Hypotheses and Specific Objectives Comparison of procedures for the induction of SE The effect of recovery time on SE-induced neurodegeneration The effect of tat-nr2b9c on SE-induced neuropathology and cognitive impairment The effect of SE on behavioural performance in tasks assessing anxiety, exploration and aggression The effect of SE on search strategy use in the Morris water maze Chapter A comparison between Long-Evans hooded and Wistar rats related to the induction and severity of status epilepticus in the low-dose and repeated low-dose lithium/pilocarpine procedures Introduction Methods Animals Induction of status epilepticus Monitoring of seizure activity Post-seizure animal care: Histology and Stereological analysis: Drugs: Statistical Analysis: Results viii

9 3.3.1 Induction of SE in Wistars and Long Evans Hooded rats The effect of SE on mortality Severity of seizures in LEH and Wistar rats SE-induced neuropathology in LEH and Wistar rats following SE induced with the RLDP procedure Comparison of SE-induced neuropathology resulting from the LDP and RLDP protocols in Wistar rats Discussion Differential effects of induction procedure in LEH and Wistar rats Comparison of SE-induced neuropathology in LEH and Wistar rats following SE induction with the RLDP procedure Comparison of the effect of the LDP and RLDP protocols on SE-induced neurodegeneration in Wistar rats Conclusion Chapter Temporal profile of neuronal death following lithium/pilocarpine-induced status epilepticus Introduction Methods Animals Induction of status epilepticus Post-seizure care Detection of SRSs Histology and Stereological analysis Fluoro-Jade B staining Statistical Analysis: Results SE induction and survival rates: ix

10 4.3.2 Spontaneous seizures after lithium/pilocarpine induced SE Neuropathology following status epilepticus: Overview SE-induced neurodegeneration in the hippocampus: SE-induced neurodegeneration in amygdaloid nuclei: SE-induced neurodegeneration in the piriform cortex: Detection of Fluoro-jade B stained neurons Discussion The neuropathological effect of increasing survival time following status epilepticus The relationship between SE, SRSs and delayed neuronal death Differences in the severity and spatial pattern of neuronal death following SE The type of cell death produced by SE Conclusion Chapter Neuroprotection following status epilepticus by targeting protein interactions with PSD Introduction Methods Induction of status epilepticus Administration of peptides NeuN Immunohistochemistry Statistical Analysis Results Induction of status epilepticus SE induced by repeated low doses of pilocarpine results in neurodegeneration in the hippocampus and piriform cortex Tat-NR2B9c reduces SE-induced neurodegeneration in the hippocampus x

11 5.3.4 Preferential neuroprotection of Tat-NR2B9c is found within specific regions of the CA1 and CA Tat-NR2B9c did not provide neuroprotection in CA1 when administered during SE Discussion Tat-NR2B9c provided significant neuroprotection in the hippocampus Regional specificity of neuroprotection by Tat-NR2B9c within CA1 and CA Neuroprotective effect of tat-nr2b9c is dependent on time of administration Conclusion Chapter Long-lasting behavioural and anxiolytic changes in rats following status epilepticus Introduction Methods Animals Induction of status epilepticus and administration of peptides Behavioural tests Results SE induction Open field test Hyperexcitability tests Elevated-plus maze: The effect of Tat-NR2B9c on behaviour following SE Discussion SE causes anxiolytic changes in behaviour and increased hyperexcitability Behavioural changes in rats following SE are long-lasting Treatment with tat-nr2b9c did not have neuroprotective effects as assessed behaviourally xi

12 6.4.4 Conclusion Chapter The effect of SE on performance in the Morris water maze and use of exploratory strategies Introduction Methods Animals Induction of status epilepticus and administration of peptides Morris water maze testing Results SE induction Visible platform testing The effect of SE on spatial acquisition The effect of SE on spatial reversal Acquisition and reversal probe tests Effect of SE on search strategy use during spatial acquisition Effect of SE on search strategy use during spatial reversal Quantitative assessment of the contribution of search strategy to overall performance SE results in differential impairment in Morris water maze performance and search strategy use The effect of Tat-NR2B9c on visual-spatial learning and use of search strategies following SE Discussion SE rats exhibit impaired performance in the MWM and improve during prolonged training SE rats use less efficient strategies in the MWM Improvement in search strategy selection contributed to improved performance epileptic rats xii

13 7.4.4 The pathological effects of SE may interfere with the selection of more efficient search strategies Rats following SE exhibited variability in behaviour during MWM testing Neuroprotection of the dorsal hippocampus by tat-nr2b9c did not modify performance in the MWM Conclusion Chapter General Discussion The lithium/pilocarpine model of mesial temporal lobe epilepsy Comparison of the low-dose (LDP) and repeated low dose lithium/pilocarpine (RLDP) procedures The severity and pattern of neuronal death in the lithium/pilocarpine model Pattern of neuronal death in the hippocampus Pattern of neuronal death in extrahippocampal structures Differences in the pattern of neuronal loss between human MTLE and rats after SE The effect of increasing survival time on SE-induced neuronal death Majority of neuronal death occurs early in rats following SE Majority of neuronal death is the consequence of SE and not of spontaneous recurrent seizures (SRSs) Cognitive and behavioural alterations following lithium/pilocarpine-induced SE The effect of SE on spatial learning and memory The effect of SE on use of behavioural search strategies Impaired use of behavioural strategies in human MTLE-HS The temporal relationship between neuronal death, behavioural alterations and cognitive impairment following status epilepticus The effect of neuroprotection on epileptogenesis, behavioural alterations, and cognitive impairment Neuroprotection within the hippocampus xiii

14 8.7.2 Effect of neuroprotection in extrahippocampal regions Conclusion Chapter Future directions Cell death mechanisms contributing to differential rates of neuronal loss following SE Previous literature Summary of our findings Proposed studies Specific cognitive alterations in rats following SE Previous literature Summary of our findings Proposed studies The causal relationship between neurodegeneration, genesis of SRSs and behavioural alterations Previous literature Summary of our findings Proposed studies References Appendices Appendix I: Literature comparison Appendix II: Temporal reduction in neuron densities within regions of the hippocampus, thalamus, amygdala and piriform cortex Appendix III: Convolution analyses Assessment of performance based on shift in strategy use Assessment of performance based on improved efficacy within each strategy xiv

15 List of Tables Table 1.1 Scoring system for pilocarpine-induced seizures Table 3.1 Comparison of rat strain and SE-inducing protocols between SE induction and mortality rates at 3 days following SE Table 4.1 The effect of SE on the area (mm 2 ) of the hippocampus, thalamus and amygdala Table 4.2 Temporal progression of brain regions exhibiting initial neuronal loss significantly different from corresponding shams Table 4.3 Temporal progression of brain regions exhibiting maximal neuronal death following Table 5.1 Comparison of the effect of treatment on mortality, seizure severity and weight gain following SE Table 5.2 Comparison of SE-induced pyramidal cell loss in individual counting frames Table 6.1 Comparison of experimental groups in seizure susceptibility and mortality at 3 months following SE Table 7.1 Performance as a function of strategy use in hidden platform Morris water maze testing Table 7.2 Comparison of RLDP SE rats that exhibit differences in Morris water maze performance Table 8.1 Consequences of neuroprotective drug treatment xv

16 List of Figures Figure 1.1 Epileptogenesis is caused by an initial precipitating injury... 4 Figure 3.1 Placement of counting frames within the hippocampus, hilus and piriform cortex Figure 3.2 Comparison of behavioural seizure activity between rat strain and SE-inducing protocol Figure 3.3 Comparison of neuron cell densities in the hippocampus and piriform cortex of LEH and Wistar rats following SE Figure 3.4 Comparison of neuronal cell densities in the hippocampus and piriform cortex of Wistar rats following SE Figure 4.1 Placement of counting frames Figure 4.2 The area size of different brain regions assessed Figure 4.3 Anti-NeuN immunohistochemical staining decreases following SE in the dorsal and ventral hippocampus Figure 4.4 Confocal micrographs (400X) of NeuN stained cells in hippocampal subfields Figure 4.5 Total length of dentate gyrus remains constant following lithium/pilocarpine induced SE Figure 4.6 Temporal profiles of neuronal loss in hippocampal subfields following lithium/pilocarpine induced SE Figure 4.7 Anti-NeuN immunohistochemical staining decreases following SE in several thalamic nuclei Figure 4.8 Confocal micrographs (400X) of NeuN stained cells in several thalamic nuclei Figure 4.9 Temporal profiles of neuronal loss in several thalamic nuclei following lithium/pilocarpine induced status epilepticus (SE) Figure 4.10 Anti-NeuN immunohistochemistry staining decreases following SE in several amygdaloid nuclei and in the posterior piriform cortex Figure 4.11 Confocal micrographs (400X) of NeuN stained cells in several amygdaloid nuclei and in the piriform cortex Figure 4.12 Temporal profiles of neuronal loss in several amygdaloid nuclei and in the posterior piriform cortex Figure 4.13 Confocal micrographs (400X) of Fluoro-jade B (FJB) stained cells present at 24 hours but not at 3 months after SE in the hippocampus, thalamus and amygdala xvi

17 Figure 4.14 The number of damaged brain regions as a function of increasing recovery time after 60-min of SE Figure 5.1 Neurodegeneration depicted in NeuN-stained coronal sections of the rat dorsal hippocampus and posterior piriform cortex (PPC) 14 days following SE Figure 5.2 Tat-NR2B9c reduces pyramidal cell loss in the dorsal hippocampus when administered 3 hours after SE Figure 5.3 Tat-NR2B9c exhibits differential neuroprotection within different regions of the CA1 and CA3 subfields of the hippocampus Figure 5.4 Tat-NR2B9c is not neuroprotective when administered 10 minutes following the onset of SE Figure 6.1 Schematic of SE-induction protocols and treatments in SE and non-se groups Figure 6.2 The effect of SE on behaviour in the open field Figure 6.3 The effect of SE on behaviour in the four hyperexcitability tests Figure 6.4 The effect of SE on behaviour in the elevated-plus maze Figure 6.5 Tat-NR2B9c had no effect on behaviour in the open field Figure 6.6 Tat-NR2B9c had no effect on behaviour in the four hyperexcitability Figure 6.7 Tat-NR2B9c had no effect on behaviour in the elevated-plus maze Figure 7.1 Behavioural categories Figure 7.2 Number of trials performed to reach criterion during visible platform testing Figure 7.3 The effect of SE on hidden platform testing in the Morris water maze Figure 7.4 Number of platform crossings in spatial acquisition and spatial reversal probe trials and swim speed Figure 7.5 The effect of SE on the distribution of search strategies used during spatial acquisition and spatial reversal testing Figure 7.6 Summary of search strategy use between groups during Morris water maze testing 196 Figure 7.7 SE results in differential impairment in Morris water maze performance Figure 7.8 SE results in differential use of search strategies during Morris water maze testing 206 Figure 7.9 Tat-NR2B9c did not improve performance in SE rats during hidden platform learning xvii

18 Figure 7.10 Tat-NR2B9c has no effect on the distribution of search strategies used during spatial acquisition in rats following SE xviii

19 List of Appendices Appendix I: Literature comparison Table A1-1 Summary of studies assessing severity and progression of neuronal loss after pilocarpine-induced SE in the hippocampus, thalamus, amygdala and piriform cortex Table A1-2 Summary of studies investigating the effect of status epilepticus on behaviours of anxiety, aggression and exploration in rodents Table A1-3 Summary of studies investigating the effect of status epilepticus on visual-spatial learning and memory Appendix II: Temporal reduction in neuron densities within regions of the hippocampus, thalamus, amygdala and piriform cortex Table A2-1 Reduction in neuronal densities within hippocampal regions following 60 min of SE Table A2-2 Reduction in neuronal densities within several thalamic nuclei following 60 min of SE Table A2-3 Reduction in neuronal densities within several amygdaloid nuclei and posterior piriform cortex following 60 min of SE Appendix III: Convolution analyses Assessment of performance based on shift in strategy use Assessment of performance based on improved efficacy within each strategy xix

20 List of Abbreviations AD afterdischarges Ave average ºC degree Celsius ACh acetylcholine AMPA α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid ANOVA one-way analysis of variance BDZ benzodiazepine CNS central nervous system Cy5 cyanine dye 5 DAG diacylglycerol DGCs dentate granule cells EEG electroencephalogram EPM elevated plus maze FJ fluoro-jade FJB fluoro-jade B g gram GABA gamma-aminobutyric acid GAERS genetic absence epilepsy rats from Strasbourg GCL granule cell layer GFAP glial fibrillary acidic protein hr hour HS hippocampal sclerosis ILAE The international league against epilepsy IMPase inositol monophosphatase i.p. intraperitoneal injection IPI initial precipitating injury KA kainic acid kg kilogram LTP long-term potentiation LiCl lithium chloride LDP low-dose lithium/pilocarpine LEH Long Evans hooded M molar (mole/liter) meq milliequivalent MPEP 2-methyl-6(pehnylethynyl)pyridine MTLE mesial temporal lobe epilepsy MTLE-HS mesial temporal lobe epilepsy with hippocampal sclerosis mg milligram mglur5 metabotropic glutamate receptor 5 antagonist M molarity min minutes ml milliliter mm millimeter mm millimolar MWM Morris water maze NeuN neuronal nuclear protein xx

21 nnos NSC NMDA NMDAR PBS PTLE IP3 PI PIP2 PLC β PSD-95 RLDP s.c. SE SRS TLE SD SEM SE μg μm w/v (v/v) neuronal nitric oxide synthase neuronal stem cells N-methyl-D-aspartate N-methyl-D-aspartate receptor phosphate buffered saline paradoxical temporal lobe epilepsy inositial triphosphate phosphatidylinositol cycle phosphatidylinositol 4,5-bisphosphate phospholipase C-beta postsynaptic density protein-95 Repeated low-dose lithium/pilocarpine subcutaneous status epilepticus spontaneous recurrent seizure temporal lobe epilepsy standard deviation standard error of means status epilepticus microgram micrometer weight/volume volume/volume For brain regions: BLP basolateral amygdaloid nucleus, posterior part BMP basomedial amygdaloid nucleus, posterior part CM central medial thalamic nucleus DG dentate gyrus DMD dorsomedial hypothalamic nucleus, dorsal part DMV dorsomedial hypothalamic nucleus, ventral part d3v dorsal 3 rd ventricle 3V 3 rd ventricle LaDL lateral amygdaloid nucleus, dorsolateral part LaVM lateral amygdaloid nucleus, ventrolateral part LDDM laterodorsal thalamic nucleus, dorsomedial part LDVL laterodorsal thalamic nucleus, ventrolateral part LV lateral ventricle MD mediodorsal thalamic nucleus MePV medial amygdaloid nucleus, posteroventral part MePD medial amygdaloid nucleus, posterodorsal part MTu medial tuberal nucleus Rt reticular thalamus nucleus Pir Pirform cortex PMCo posteromedial cortical amygdaloid nucleus xxi

22 Po PoDG` PPC pyr STh STIA VPL VPM posterior thalamic nuclei polymorph layer of the dentate gyrus posterior piriform cortex pyramidal cell layer subthalamic nucleus the bed nucleus of the stria terminalis, intraamygdaloid division ventral posterolateral thalamic nucleus ventral posteromedial thalamic nucleus xxii

23 1 Chapter 1 General Introduction 1.1 Main features of mesial temporal lobe epilepsy with hippocampal sclerosis Epilepsy is the most frequent neurodegenerative disease after stroke (Acharya et al., 2008), and accounts for a significant portion of the disease burden worldwide (de Boer et al., 2008). It afflicts more than 170,000 Canadians ( per 1000 population) (Tellez-Zenteno et al., 2004) and at least 50 million people globally (de Boer et al., 2008). The International League against Epilepsy (ILAE) proposed the definitions of epileptic seizures and epilepsy, stating that an epileptic seizure is a transient occurrence of signs and/or symptoms due to abnormal excessive or synchronous neuronal activity in the brain. Epilepsy is a disorder of the brain characterized by an enduring predisposition to generate epileptic seizures and by the neurobiological, cognitive, psychological, and social consequences of this condition. The definition of epilepsy requires the occurrence of at least one epileptic seizure (Fisher et al., 2005). The current ILAE classification of epileptic seizures was approved in 1981, and later extended for the classification of epilepsies and epilepsy syndromes in 1989 (Commission on classification and terminology of the international league against epilepsy. 1989). Revisions have since been proposed to update the nomenclature to reflect modern neuroimaging, genomic technologies and basic sciences (reviewed in: Engel, 2001; Seino, 2006; Berg et al., 2010). The 1989 classification divides epilepsies into generalized and focal epileptic seizures, depending on whether the characteristic seizures begin simultaneously on both sides of the brain or are confined to one hemisphere (Commission on classification and terminology of the international league against epilepsy, 1989). Focal epileptic seizures may spread to both hemispheres and become secondarily generalized. Epilepsy syndromes are further classified into idiopathic, symptomatic and cryptogenic categories based on their underlying etiologies; these terms were recently changed to genetic, structural/metabolic and unknown cause, respectively (Berg et al., 2010). More than half of the epilepsies are classified as structural/metabolic disturbances (Engel and Schwartzkroin, 2006); the most common of these, and also the most common form of human epilepsy is mesial temporal lobe epilepsy with hippocampal sclerosis

24 2 (MTLE-HS) (Wieser, 2004). MTLE-HS is characterized by focal epileptic seizures with or without secondary generalization originating from the mesial temporal lobe (Margerison and Corsellis, 1966; King and Spencer, 1995; Bartolomei et al., 2005; Bertram, 2009). While this review focuses on the characteristics of MTLE-HS, an extensive list of other epilepsies, epileptic syndromes and related seizure disorders are extensively reviewed elsewhere (Engel, 2001; Chabolla, 2002; Engel and Schwartzkroin, 2006; Seino, 2006; Berg et al., 2010). The main features of MTLE-HS are: (i) the localization of seizure foci in the limbic system, particularly in the hippocampus, entorhinal cortex, and amygdala (Margerison and Corsellis, 1966; King and Spencer, 1995; Bartolomei et al., 2005; Bertram, 2009); (ii) the frequent finding of an initial precipitating injury (IPI) that precedes the appearance of mesial temporal lobe epilepsy (MTLE) (Mathern et al., 2002; Mathern et al., 1996); (iii) a seizure-free time interval following the IPI known as epileptogenesis or latent phase ( Engel, 1993; Wieser, 2004); (iv) a high incidence of hippocampal sclerosis (HS) (also referred to as mesial temporal sclerosis or Ammon s horn sclerosis) (Babb, 1986; Babb and Brown, 1986; Babb et al., 1991; Sharma et al., 2007) and; (v) a high prevalence of interictal behavioural disturbances and cognitive impairment (Boro and Haut, 2003; Devinsky, 2004a; Gaitatzis et al., 2004; Swinkels et al., 2005; Cornaggia et al., 2006; Marcangelo and Ovsiew, 2007; Garcia-Morales et al., 2008). As illustrated in Figure 1.1, neuronal loss and synaptic reorganization are proposed to be the cause of chronic epileptic seizures and interictal behavioural and cognitive morbidity (Wieser, 2004; Sharma et al., 2007; Acharya et al., 2008). Even though these conditions exist prior to development of MTLE-HS as a consequence of the IPI (Mathern et al., 1996; Wieser, 2004), several studies have suggested that repeated ictal events may contribute to further deterioration (Mathern et al., 1996; Wieser, 2004; Bernasconi et al., 2005). The main features of MTLE-HS are recapitulated in chronic animal models of temporal lobe epilepsy (TLE), particularly in the kindling and status epilepticus (SE) models. The subject of this thesis, the lithium/pilocarpine model, belongs to SE models. The lithium/pilocarpine model has been used in many laboratories to investigate the pathogenesis of MTLE-HS and to evaluate the efficacy of anti-epileptogenic drugs (reviewed in: Leite et al., 2002; Curia et al., 2008; Scorza et al., 2009). This introduction begins with a brief overview of the commonly used animal models of TLE. The subsequent sections provide an overview on background information and important features of the lithium/pilocarpine model, including: (i) the

25 3 occurrence of SE as the IPI, (ii) the presence of a latent period followed by the appearance of spontaneous recurrent seizures (SRSs), (iii) the occurrence of widespread neurodegeneration (including hippocampal sclerosis) and synaptic reorganization, and (iv) the development of interictal behavioural disturbances and cognitive impairment.

26 4 Figure 1.1: Epileptogenesis is caused by an initial precipitating injury. During this period, neurodegeneration and synaptic reorganization occur and contribute to the development of recurrent seizures and behavioural and cognitive dysfunction. Recurrent seizures may also contribute to additional morbidity and pathophysiological changes. Different factors can affect seizure development and cognitive and behavioural outcomes.

27 5

28 6 1.2 Animal models of seizure development and epilepsy The two most commonly used animal models to investigate epileptogenesis and MTLE-HS are kindling and SE. Although major procedural differences exist between the two models, and each has its own characteristics, both are capable of inducing a persistent, epileptic-like condition (reviewed in: Leite et al., 2002; Loscher, 2002; McIntyre et al., 2002; Morimoto et al., 2004; Martin and Pozo, 2006; Sharma et al., 2007). The major emphasis of this thesis is SE. The ILAE defined SE in 1964 as a seizure that persists for a sufficient length of time or is repeated frequently enough to produce a fixed and enduring epileptic condition (Arnautova and Nesmeianova, 1964). Although a specific length of time is not specified in the definition, many investigators have traditionally identified SE as prolonged seizures lasting 30 minutes or longer, the time necessary to produce neuronal death in animal models of SE (DeLorenzo et al., 1999; Fujikawa, 2005; Knake et al., 2009); others have argued, however, that SE should be defined as seizures lasting 5 minutes or longer for several reasons: (1) the relationship between seizure activity and neuronal loss in humans is not well understood, (2) self-terminating seizures rarely last longer than 2 minutes, and (3) earlier medical treatment of SE is likely to reduce the mortality and morbidity associated with this condition (Lowenstein et al., 1999; Meldrum, 1999). Despite this ambiguity, SE rodent models have significantly contributed to our understanding of the pathophysiological mechanisms underlying SE, and this is discussed in detail in the subsequent sections. Several extensive reviews related to the kindling model are available (McIntyre et al., 2002; Morimoto et al., 2004; Sutula and Ockuly, 2006), and this model is only briefly described here Kindling Induction of epilepsy by kindling involves periodic application of a brief stimulus that evokes repetitive epileptic spikes (an afterdischarge, or AD) (reviewed in: McIntyre et al., 2002; Morimoto et al., 2004; Sutula and Ockuly, 2006). Over repeated stimulations, the duration of the evoked ADs and the intensity of behavioural seizures increase, while the stimulus threshold to evoke epileptiform activity decreases. This process results in an overall increase and longlasting susceptibility of the animal to additional seizures. Most studies terminate kindling once an animal exhibits a few secondary generalized (stage v) seizures (kindling scale described by Racine, 1972). The gradual development and progression of epileptogenesis by kindling allows

29 7 investigators to reliably quantify both electrographic measures (AD number and duration) and behavioural responses (seizure stages and number of class v seizures). Because this process occurs in the absence of overt brain damage, it may be an ideal model for studying paradoxical TLE (defined in section 1.5). A limitation of the kindling model may be that the standard stage v criterion results in an incomplete epileptogenesis in which SRSs do not occur. A minimum of 90 to 100 kindled seizures beyond the stage v criterion is required in rats for development of SRSs (Sutula and Ockuly, 2006). This process, referred to as over-kindling, is rarely used because of the intensive work and time involved. Of particular interest, over-kindled rats exhibit similar neuropathological changes to those found in SE models (Sutula and Ockuly, 2006) Post-status epilepticus models In contrast to kindling, SE is easier to produce and reliably generates epileptic animals. SE can be induced by electrical stimulation of limbic brain structures (e.g., amygdala and hippocampus), or by systemic administration of chemoconvulsants (e.g., kainate or pilocarpine) (reviewed in: Leite et al., 2002; Loscher, 2002; Morimoto et al., 2004; Martin and Pozo, 2006; Sharma et al., 2007). Loscher (2002) referred to models that involve SE as post status epilpeticus models, since the latent phase that follows SE and precedes SRSs is of most interest to many researchers. Pathological changes that occur during this period are proposed to contribute to the development of epilepsy; therefore, therapeutic treatments that affect these changes may also be antiepileptogenic, and prevent at risk individuals from developing epilepsy (Loscher, 2002). The morphological damage that occurs in animal models of SE is very similar to that seen in human MTLE-HS, although the damage in SE models can be more severe and widespread (Sharma et al., 2007). The use of electrical stimulation to induce SE is described as being more labourintensive and thus less desirable as a model as compared to the use of chemoconvulsants (Sharma et al., 2007). However, high mortality rates are a disadvantage in some kainate and pilocarpine models, but can be minimized by administering lower doses of the chemoconvulsant (Hellier et al., 1998; Glien et al., 2001). In the present thesis we used pilocarpine in conjunction with lithium chloride (LiCl) to induce SE.

30 8 1.3 Background information on the pilocarpine and lithium/pilocarpine models The pilocarpine model, especially in combination with LiCl, reproduces many clinical and morphological aspects of MTLE-HS in rodents. These features are outlined in section 1.1 and are further described in the subsequent sections. A major drawback to the pilocarpine and lithium/pilocarpine models is high mortality. However, mortality can be limited by modification of the SE-inducing procedure and by controlling SE duration. In this section, background information and cellular mechanisms underlying SE induction in the pilocarpine and lithium/pilocarpine models are reviewed The pilocarpine model The pilocarpine model was first used in rats (Turski et al., 1983a; Turski et al., 1983b) and then in mice (Turski et al., 1984) to produce limbic seizures. acetylcholine receptor agonist. Pilocarpine is a muscarinic Several extensive reviews describe a variety of protocols available for administering pilocarpine to induce SE, and these are briefly described here (Cavalheiro et al., 2006; Curia et al., 2008; Scorza et al., 2009). With systemic administration, the pilocarpine dose necessary to induce SE ranges from 300 to 400 mg/kg in adult rats (Clifford et al., 1987; Liu et al., 1994; Melloa and Mendez-Oterobs, 1996). Lower pilocarpine doses ( mg/kg) occasionally produce brief limbic seizures, but do not result in SE (Turski et al., 1983b). The dose of pilocarpine administered to induce SE significantly affects seizure development, survival rates, and neuropathology. When compared to lower doses of pilocarpine (350 mg/kg, i.p.), higher doses ( mg/kg, i.p.) have resulted in a reduced latency to SE onset and a greater percentage of rats developing SRSs (Clifford et al., 1987; Liu et al., 1994). However, higher doses of pilocarpine have also resulted in greater mortality rates, and this may be caused by the greater convulsive seizure severity and brain damage observed in these animals (Clifford et al., 1987; Liu et al., 1994). Pilocarpine can also induce SE in rats after intracerebroventricular (Croiset and De Wied, 1992) and intrahippocampal (Furtado et al., 2002) administration.

31 Convulsive effects of pilocarpine are mediated by activation of M1 receptors The cholinergic system is involved in the initiation of SE following pilocarpine treatment, as pretreatment of the animals with scopolamine (a muscarinic antagonist) prevents the development of convulsive seizures (Turski et al., 1983a). Other cholinomimetics, such as carbachol and oxotremorine, are also able to induce seizures and seizure-induced brain damage (Turski et al., 1983a; Olney et al., 1986). Subsequent studies have showed that the ability of pilocarpine to induce SE is dependent on activation of the M1 muscarinic receptor subtype, since M1 receptor knockout mice do not develop seizures in response to pilocarpine (Hamilton et al., 1997), and epileptic activity is blocked by pirenzepine, an M1-specific antagonist (Maslansky et al., 1994). Various mechanisms downstream of M1 receptor activation has been proposed to account for the effect of pilocarpine on neuronal excitability, including activation of phospholipase C-beta (PLC β ) (Scarr, 2009) and/or Src kinase (Rosenblum et al., 2000; Murthy, 2008), both of which activate subsequent downstream events resulting in enhanced excitability. Each of these signaling mechanisms is briefly described. 1. M1 receptors are coupled to Gq/G11-type G proteins, which are in turn coupled to the activation of PLC β (reviewed in: Wess et al., 2007; Scarr, 2009). PLC β cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) generates inositial triphosphate (IP3) and diacylglycerol (DAG) (Berridge, 2009), resulting in an alteration in a Ca 2+ and K + current and increasing the excitability of the brain (Segal, 1988). A well-known mechanism of cholinergic excitation involves the M1-mediated depletion of PIP2, which results in closure of voltage-gated K + channels (termed M-channels), since the presence of PIP2 is necessary for the open state of these channels to remain stabilized (reviewed in: Brown, 2010). 2. An alternative mechanism by which M1 receptors may generate excitatory responses independent of PLC β signaling involves Src kinase activation (Rosenblum et al., 2000). Activation of M1 receptors can induce elevation of intracellular Ca 2+, which stimulates Src kinase activation (Felder, 1995). Src kinase is able to subsequently phosphorylate other signaling molecules, including soluble guanylyl cyclase (Murthy, 2008) and extracellular signal-regulated kinase (ERK) (Rosenblum et al., 2000), both of which have

32 10 been implicated in cholinergic excitation (Rosenblum et al., 2000; Kuzmiski and MacVicar, 2001) The cholinergic system is involved in the initiation but not the maintenance of SE The development of SE requires a pool of neurons capable of initiating and sustaining abnormal firing (Noe and Manno, 2005). The generation of synchronized neuronal activity is facilitated by the loss of inhibitory synaptic transmission mediated by GABA and sustained by the excitatory transmission mediated by glutamate (Smolders et al., 1997; Priel and Albuquerque, 2002; Noe and Manno, 2005; Meurs et al., 2008). Experiments in cultured hippocampal neurons have demonstrated that pilocarpine acting through muscarinic receptors causes an imbalance between excitatory and inhibitory transmission resulting in the generation of SE (Priel and Albuquerque, 2002). Furthermore, in-vivo microdialysis has showed that pilocarpine induces an elevation in glutamate levels in the hippocampus following the appearance of seizures (Smolders et al., 1997; Meurs et al., 2008). The increase in neuronal activity leads to a loss of inhibition by accelerated internalization of the GABA A receptors (reviewed in: Goodkin et al., 2005). Once seizures are initiated, their maintenance depends on mechanisms distinct from muscarinic receptors, since atropine becomes ineffective in controlling established seizures (Clifford et al., 1987; Curia et al., 2008). Substantial evidence now supports the idea that following the activation of muscarinic receptors, SE is maintained by activation of the N-methyl-D-aspartate (NMDA) receptors (Nagao et al., 1996; Smolders et al., 1997; Deshpande et al., 2008) The lithium/pilocarpine model Honchar et al., (1983) reported that pretreatment of rats with LiCl (3 meq/kg) permits the dose of pilocarpine to be decreased approximately 10-fold (30 mg/kg, i.p.; referred to as the low-dose lithium/pilocarpine (LDP) protocol in this thesis). This procedure resulted in lower mortality rates and produced SE more reliably when compared to systematic administration of pilocarpine alone (Clifford et al., 1987; Sharma et al., 2007). The effects of lithium are specific to the cholinergic system since SE induced by the nerve agent soman, a cholinesterase inhibitor, is also potentiated by lithium; however, SE induced by glutamate agonists NMDA and kainic acid, or the gamma-aminobutyric acid (GABA) antagonists bicuculline and pentylenetetrazole are not (McDonough et al., 1987; Ormandy et al., 1991). Neither lithium (3 meq/kg) nor pilocarpine

33 11 (30 mg/kg) causes abnormal electrographic responses when administered alone (Clifford et al., 1987; Jope and Gu, 1991). Lithium pretreatment is only effective if pilocarpine is administered within 24 hours; the proconvulsive effects of lithium becomes less effective if pilocarpine is administered after 24 hours, and no seizures develop after 48 hours (Clifford et al., 1987). The lithium/pilocarpine and pilocarpine models produce similar behavioural, electrographical and neuropathological alterations in rats following SE (Clifford et al., 1987). Glien et al., (2001) further modified the lithium/pilocarpine model. If pilocarpine was administered as a single dose of 30 mg/kg in lithium-pretreated rats, and SE duration was limited to 90 min, mortality was 45% and 80% of survivors developed SRSs. However, if pilocarpine was administered in divided doses of 10 mg/kg at 30-min interval until SE ensued (referred to as the repeated low dose lithium/pilocarpine (RLDP) protocol in the present thesis), the mortality rate was reduced to 7% and 85% of animals that survived SE developed SRSs. The reduced mortality is attributed to the titrated administration of pilocarpine that accommodates for intrastrain differences in sensitivities of individual rats to the chemoconvulsive properties of pilocarpine (Glien et al., 2001). There was no significant difference in the induction rates of SE between rats treated with the LDP (total of 73%) and the RLDP (total of 61%) procedures The proconvulsive mechanisms of lithium Recent interest in determining lithium s physiological role in potentiating the convulsive effects of pilocarpine stems from its role in the treatment of manic-depressive illness (Belmaker and Bersudskya, 2007; Agam et al., 2009). Here, the main hypothesis regarding lithium s therapeutic and prophylactic effect in affective disorder is that inhibition of inositol monophosphatase (IMPase) by lithium impairs the operation of the phosphatidylinositol cycle (PI cycle) (see reviews: Osborne et al., 1988; Berridge and Irvine, 1989; Jope and Williams, 1994; Haim and Belmaker, 2001; Belmaker and Bersudskya, 2007). Lithium at therapeutic doses (3 meq/kg) inhibits rat brain IMPase, thereby resulting in a reduction in inositol and accumulation of inositol monophosphate (Belmaker and Bersudskya, 2007). In the absence of IMPase, lithium no longer potentiates the effect of pilocarpine. For instance, Impa1 (encoding IMPase) knock-out mice without lithium pre-treatment behaved similarly to lithium pre-treated rats receiving a single i.p. subthreshold dose of pilocarpine (30 mg/kg, i.p.) (Agam et al., 2009). Because lithium inhibits IMPase, inositol becomes less available for re-synthesis of PIP2 and the consequently reduces

34 12 PIP2 levels (Belmaker and Bersudskya, 2007; Brown, 2010); this increases membrane excitability of the neuron in response to pilocarpine as described in section Use of diazepam to control SE duration and reduce mortality In the pilocarpine model, SE spontaneously remits within 5 to 6 hours after initiation (Turski et al., 1989; Lemos and Cavalheiro, 1995); however, this is often associated with exceedingly high mortality rates (Curia et al., 2008). Limiting SE duration can improve long-term survival in rats following SE; this is often accomplished by administrating diazepam, a GABA A agonist (Glien et al., 2001). Curia et al., (2008) reported that, following induction of pilocarpine-induced SE, diazepam effectively decreased mortality when administered 30, 60, 120 or 180 min after SE induction. In addition, diazepam terminated behavioural and electrographic seizures when administered up to 4 hrs following the initiation of SE in several seizure models (Brandt et al., 2003a; Brandt et al., 2006; Goffin et al., 2007). The anticonvulsant action of diazepam against pilocarpine-induced seizures was associated with prompt attenuation of extracellular glutamate concentrations in the hippocampus (Khan et al., 1999). 1.4 Behavioural and clinical features of seizure development in the lithium/pilocarpine model Retrospective studies show that a large proportion of patients with mesial temporal lobe epilepsy (MTLE) undergo an IPI, including febrile convulsions, SE, encephalitis, stroke or traumatic brain injury (Mathern et al., 1996; Mathern et al., 2002). Up to 80% of adults with MTLE are reported to have presented childhood SE or prolonged febrile seizures (French et al., 1993; Cendes and Andermann, 2002). In the majority of cases, SRSs appeared after a 5- to 10-year latent period (Engel, 1993). Similarly, an epidemiologic study reported that up to 42% of individuals with SE as their first seizure (mean age 39.7) developed epilepsy over the next 10 years (Hesdorffer et al., 1998). The pilocarpine model closely mimics the clinical manifestations of MTLE in humans, in which an acute triggering process is frequently followed by a latent phase and subsequent development of recurrent seizures (see Figure 1.1). An acute episode of SE serves as the IPI (Cavalheiro et al., 2006; Curia et al., 2008; Scorza et al., 2009). Three stages of seizure development following SE have been described below. These are the acute phase, epileptogenesis, and the chronic phase.

35 The acute phase The acute phase includes the initial occurrence of repetitive seizures, including SE, and is typically considered to involve the initial 24-hours following seizure induction (Scorza et al., 2009) Behavioural seizures during the acute phase The progression of behavioural changes following pilocarpine injection is very similar in the pilocarpine (Turski et al., 1983b) and lithium/pilocarpine models (Clifford et al., 1987; Glien et al., 2001). Behavioural manifestations increase with time following pilocarpine and may be divided into 4 phases (Turski et al., 1983b; Clifford et al., 1987): 1. The first phase of behaviour changes occurs from peripheral cholinergic stimulation within the first several minutes after pilocarpine injection. These include piloerection, salivation, tremor, chromadacryorrhea and diarrhea. 2. In the second phase, a series of stereotyped behaviours including oro-facial movements, salivation, eye-blinking, twitching of vibrissae and yawning subsequently develops and persists for up to 45 min following pilocarpine injection. Animals also show a predilection for remaining in one corner of the cage with upward extension of the nose and neck. During the first and second phases of behavioural changes, animals are able to be distracted by tactile or sound stimulation (Clifford et al., 1990). 3. Behavioural alterations in phase 1 and 2 subside with development of limbic motor seizures in phase 3. These seizures are characterized by intense salivation, rearing, upper extremity clonus, and falling, and reoccur every 3 to 15 min. 4. During phase 4 of behavioural changes, SE typically develops soon following the initial limbic seizures (Turski et al., 1983b; Clifford et al., 1987). During SE, rats are unresponsive to external touch and sound stimulation (Clifford et al., 1987). In the absence of specific measures to terminate seizures (see section 1.3.6) SE spontaneously remits within 5 to 6 hours (Turski et al., 1983b; Clifford et al., 1987). Animals are typically found to be in a comatose state for the 24 hour period following cessation of SE.

36 Electroencephalographic patterns during motor limbic seizures and SE Pilocarpine produces both ictal and interictal epileptiform activity in the EEG recordings, and changes in electrographic patterns correlate well with the sequence of behavioural alterations described in section (Clifford et al., 1987; Turski et al., 1989; Leite et al., 1990). Immediately following pilocarpine injection, low-voltage, fast activity with spikes appears in the neocortex and amygdala, while a clear theta rhythm appears in the hippocampus. This EEG pattern is correlated with phase 1 of behavioural changes (see ). When behavioural manifestations become more severe (phase 2), high voltage, fast EEG activity replaces the hippocampal theta rhythm. Electrographic seizures characterized by high voltage, fast activity and prominent spiking precede limbic motor seizures (phase 3), and are proposed to result from muscarinic system activation (van Der Linden et al., 1999). This activity originates in the hippocampus and propagates to the neocortex and amygdala (Turski et al., 1983b; Turski et al., 1989; Leite et al., 1990). Sustained electrographic discharges occur during SE (phase 4), and after several hours, evolve to a pattern of periodic discharges on a relatively flat background. Studies using 14 C-2-deoxyglucose functional mapping show that specific brain structures are associated with the different behavioural changes (Lothman and Collins, 1981; Handforth and Ackermann, 1988; Lothman et al., 1991; Handforth and Ackermann, 1995; Handforth and Treiman, 1995). Table 1.1 summarizes the relationship between seizure severity and the activation of specific brain regions (see review: Veliskova, 2006). Of particular interest, brain regions that show the highest metabolic activation during SE also exhibit the most severe neuronal loss in the latent phase (Ingvar, 1986; Ingvar et al., 1987; Handforth and Ackermann, 1992; Handforth and Ackermann, 1995; Fernandes et al., 1999; Bouilleret et al., 2000) Scoring of pilocarpine-induced seizures In this thesis, the severity of seizures was assessed using a modified version of the Racine s scale (1973), which at first was developed to score kindled seizures in adult rats. As summarized in Table 1.1, stages I to V are the classical stages described by Racine (1972). Levels VI and VII have been additionally added to the modified scale. Level VI is achieved with the occurrence of two or more level V seizures (rearing and multiple falls) (Cammisuli et al., 1997). Level VII consists of tonic-clonic seizures (Veliskova, 2006). The scale is based on specific characteristics

37 15 of epileptic seizures in the pilocarpine model, and the progression of seizure activity resulting from gradual involvement of distinct neural networks (Veliskova, 2006). Stages I and II involve the spread of paroxysmal activity from the original structure (e.g., hippocampus) to other limbic regions (Lothman and Collins, 1981; Handforth and Ackermann, 1988; Lothman et al., 1991; Handforth and Ackermann, 1995; Handforth and Treiman, 1995). In stage III, the occurrence of clonic seizures indicates activation of structures beyond the limbic system, namely the neocortex, thalamus and basal ganglia (Engel et al., 1978; Lothman and Collins, 1981; Browning and Nelson, 1986; Handforth and Ackermann, 1995; Handforth and Treiman, 1995; Veliskova et al., 2005). Clonic seizures consist of rhythmic movement of forelimbs that are often accompanied by facial clonus. The forelimb clonus can be either unilateral (stage III) or coordinated bilateral clonus (stage IV) with or without rearing or tail erection (Straub tail). In stage IV, the hind limbs are usually spaced apart and the animal appears as if in a kangaroo position (Ono et al., 1990). During seizure stages V and VI, the hind limbs can also be involved and the rat may lose balance temporarily; however, the animal will immediately make an effort to get back to the upright position. The tonic-clonic seizures (stage VII) represent a spread of paroxysmal activity from the forebrain to the brainstem (Browning and Nelson, 1986). During this type of seizures, the righting reflex is lost. The tonic phase typically involves tonic flexion and then extension of the forelimb, hindlimb or both with variable duration. The tonic extension of hindlimbs represents the most severe seizure phase (Swinyard, 1973). After the tonic phase, long-lasting clonus of all limbs may develop. Wild running and jumping in some rats may also be observed. Scoring systems specific to the type of seizures generated by other models exist and are reviewed by others (Veliskova, 2006).

38 16 Table 1.1: Scoring system for pilocarpine-induced seizures Behavioural expression Righting Seizure Structures involved 2 stage 1 reflex I Staring with mouth clonus Preserved Limbic structures II Head nodding, automatisms (i.e., scratching, sniffing orientation) III Unilateral forelimb clonus Preserved Other forebrain regions, specifically neocortex, thalamus and basal ganglia IV Bilateral forelimb clonus (rearing) V Forelimb clonus with rearing and one fall VI Forelimb clonus with rearing and multiple falls VII Tonic/clonic seizures. Infrequent observation of wild running and Brief loss of postural control Lost Brainstem jumping with vocalization 1. Seizure stages as described by Cammisuli et al., 1997 and by Veliskova, Structures mapped by 2-deoxyglucose studies and EEG patterns of specific brain regions (see section ) Effect of SE duration on mortality and neuropathology SE duration is considered a critical factor in predicting outcome of SE in humans ( DeLorenzo et al., 1992; Scholtes et al., 1994; Towne et al., 1994; DeLorenzo et al., 1999; Drislane et al., 2009;). Towne et al., (1994) highlighted the prognostic significance of SE duration in reporting a 2.7% mortality for episodes lasting less than an hour, but 32% for those lasting more than an hour. DeLorenzo et al., (1999) reported that seizures lasting 10 to 29 min often stop on their own, but very few seizures lasting over 30 min do so; mortality is substantially higher in the latter group (DeLorenzo et al., 1999). Thirty to 60 min appears to be a critical time frame after which the brain loses its ability to autonomously terminate seizure activity; beyond this time, neurodegeneration and SE-related morbidity ensue (Scholtes et al., 1994; DeGiorgio et al., 1995; DeGiorgio et al., 1999; DeLorenzo et al., 1999; Fujikawa, 2005; Nandhagopal, 2006; Knake et al., 2009). In accord with human data, a higher mortality rate is correlated with longer SE duration in the pilocarpine and lithium/pilocarpine models (reviewed in section 1.3.6). Prolonged seizures are

39 17 also associated with increased refractoriness to benzodiazepine (BDZ) treatment, and may account for the higher mortality since SE activity becomes more difficult to control with passing time. Several extensive reviews related to pharmacoresistance to BDZ in several SE animal models and in patients with MTLE or SE are available (Macdonald and Kapur, 1999; Avoli et al., 2005; Loscher, 2007; Goodkin and Kapur, 2009). A minimum of 40 to 60 minutes of continuous electrographic seizure discharges is required for neuronal death to occur in rats (Nevander et al., 1985; Fujikawa, 1996). Fujikawa et al., (1996) showed that no neuronal injury was present in rats after 20 min of SE, but increased substantially between 40 and 60 min, and only slightly between 1 and 3 hrs. The proposed mechanisms of neuronal death include excitotoxicity, energy impairment, free radical generation and apoptosis (Fujikawa, 2005; Niquet et al., 2005); these processes are discussed in section 1.5. The duration of SE is also critical to the development of SRSs and is discussed in section Epileptogenesis Epileptogenesis, also referred to as the latent phase, is the period between the acute phase and the occurrence of SRSs. This stage is characterized by a progressive normalization of EEG and an absence of seizure activity. The animals are often in poor physical shape following cessation of SE, and post-seizure care is critical to increase survival rates (Glien et al., 2001). Body weight typically decreases after SE (10-20%), but recovers to pre-treatment values during the early latent phase (Turski et al., 1989) Duration of the latent phase Several studies investigating the latency to development of SRSs have used a single high dose of pilocarpine ( mg/kg) to induce SE, and allowed SE to spontaneously remit (Cavalheiro et al., 1991; Liu et al., 1994; Arida et al., 1999; Goffin et al., 2007). In these studies, continuous video-eeg recordings were used to detect for the onset and progression of SRSs. The end of the latent period was defined as the first occurrence of a stage IV or greater epileptic seizure. Latent periods ranging between 3 days and 44 days were reported (Cavalheiro et al., 1991; Liu et al., 1994; Arida et al., 1999). Arida et al., (1999) additionally showed a relationship between the frequency of SRSs and the duration of the latent period. Shorter latent periods (3-5 days) were

40 18 associated with a greater number of SRSs (range seizures in 135 days) than longer (28 30 days) latent periods (range seizures in 135 days) (Arida et al., 1999). Two lines of evidence indicate that SE duration affects the epileptogenic process. Unless otherwise indicated, the following studies investigating this relationship have used the high-dose pilocarpine model. 1. First, a minimum duration of SE is required for the development of chronic epilepsy. Using diazepam to terminate SE, Klitgaard et al., (2002) reported that SRSs developed in rats with a SE duration of 30 min, but not in rats with a SE duration of 7.5 and 15 min (Klitgaard et al., 2002). On the other hand, Lemos and Cavalheiro (1995) reported that SRSs developed in rats with a SE duration of 60 min, but not in rats with only 30 min of SE; in this study, SE was terminated with combined treatment of diazepam (10 mg/kg, i.p.) and pentobarbital (30 mg/kg, i.p.). A possible rationale for the difference in results between studies is the difference in procedures used to terminate SE. 2. Second, studies have shown that SE duration can affect the duration of the latency period and frequency of SRSs. Longer durations of SE were correlated with increased latencies to the development of SRSs, and this was suggested to be related to the increased production of neurosteroids from activated glial cells (Biagini et al., 2006; Biagini et al., 2009). Because neurosteroids, such as allopregnanolone, act as positive GABA A receptor modulators, an increase in their induction was proposed to retard epileptogenesis (Biagini et al., 2006; Biagini et al., 2009). Using the lithium/pilocarpine model, Glien et al., (2001) also showed that a longer SE duration was associated with increased latency to the occurrence of SRSs. In both of these studies, SE duration was controlled with diazepam (10 20 mg/kg, i.p.). In contrast, Lemos and Cavalheiro (1995) obtained different results by terminating SE in rats with combined treatment of diazepam (10 mg/kg, i.p.) and pentobarbital (30 mg/kg, i.p.). This study reported a progressive increase in the latent period and a decrease in seizure frequency in animals with shorter SE. In addition, animals with shorter SE (1 and 2 hrs) exhibited less neuronal loss and axonal sprouting in the hippocampal formation. Consequently, the severity of neuropathology was suggested to affect the length of the latent period. The combined treatment of diazepam (10 mg/kg) and pentobarbital (30 mg/kg) has been reported to reduce mossy fiber sprouting and neuronal loss after SE (Luongo et al., 2009). It is possible that this interference with

41 19 histopathological changes contributes to the difference in results between this study and others that used only diazepam to control SE duration (Glien et al., 2001; Klitgaard et al., 2002; Biagini et al., 2006; Goffin et al., 2007; Biagini et al., 2009) The chronic phase The chronic phase is characterized by the appearance of SRSs, which persist for the life of the animal Behaviour during the chronic phase As proposed by Goffin et al., (2007), the classification of pilocarpine-induced seizures (see Table 1.1) can be simplified by referring to stages I - III as partial seizures, and stages IV VII as secondarily generalized seizures. According to this classification, recurrent seizures start appearing approximately 7 days after SE as partial seizures, and develop into generalized seizures in the following days (Goffin et al., 2007). The evolution of SRSs in the pilocarpine and lithium/pilocarpine models mimic the behavioural and electrographic stages of kindling (see section ) (Leite et al., 1990; Cavalheiro et al., 2006; Goffin et al., 2007). Once the SRSs resemble a stage V seizure of amygdala kindling (Racine, 1972), the majority of subsequent seizures are also generalized (Cavalheiro et al., 2006). At this point, SRSs appear to recur in clusters with cyclicity, peaking every 5 to 8 days (Goffin et al., 2007) or more (Arida et al., 1999). The frequency of SRSs increases and then remains constant 2 months after SE, and persists throughout the lifetime of the animal (Priel et al., 1996; Arida et al., 1999; Goffin et al., 2007) Electroencephalographic patterns observed during SRSs The first spontaneous seizures are partial seizures characterized by paroxysmal activity in the hippocampus without changes in cortical recordings (Leite and Cavalheiro, 1995). Subsequent seizures show a gradual spreading of paroxysmal activity from the hippocampus to cortical recordings and longer duration of ictal events. The fully developed generalized seizures are characterized by bursts of spiking activity in the hippocampus that spread to the neocortex (Leite and Cavalheiro, 1995; Goffin et al., 2007). Electrographic seizures rarely last more than 60 sec and are followed by depressed background activity with frequent EEG interictal spikes. The interictal spikes are more intense when animals are seizure-free and in the period of slow-wave

42 20 sleep and are nearly non-existent during motor activity and paradoxical sleep (Arida et al., 1999). Consequently, a higher seizure frequency (i.e., lower seizure threshold) tends to occur during daylight hours (Arida et al., 1999; Goffin et al., 2007). 1.5 Neuropathology Several pathophysiological phenomena that occur during the latency phase are suggested to contribute to epileptogenesis. These include neurodegeneration, synaptic reorganization, glial cell activation, and ectopic cell proliferation (see reviews: Fujikawa, 2005; Dalby and Mody, 2001; McNamara et al., 2006; Pitkänen and Lukasiuk, 2009). In the present thesis, we have specifically focused on neurodegeneration following SE, and discussed this process in the subsequent sections. The other histopathological changes have been briefly described here but extensive reviews on each of these are available Neurodegeneration SE-induced neurodegeneration In most patients, MTLE is believed to be initiated by brain damage and synaptic reorganization secondary to an IPI (Babb, 1986; Babb and Brown, 1986; Babb et al., 1991; Sharma et al., 2007). MTLE can be subclassified histopathologically as paradoxical temporal lobe epilepsy (PTLE) or hippocampal sclerosis (HS) (Sharma et al., 2007). No lesions are observed in PTLE, whereas in 70% of MTLE patients with HS, lesions include neuronal degeneration, gliosis and aberrant mossy fiber sprouting in the inner molecular layer of the dentate gyrus. Neuronal loss is also frequently observed in the amygdala and the surrounding entorhinal, perirhinal and parahippocampal cortices, and in extra-temporal areas, including the thalamus and cerebellum (Jutila et al., 2002; Bernasconi et al., 2003; Bonilha et al., 2010). In the absence of systematic complications or pre-existing epilepsy, SE in humans produces similar widespread neuronal loss and reactive gliosis in the hippocampus, amygdala, thalamus, piriform and entorhinal cortices and Purkinje cell layer of the cerebellum (Fujikawa et al., 2000). This pattern of neuronal loss is replicated in the pilocarpine and lithium/pilocarpine models of SE (Honchar et al., 1983; Turski et al., 1983a; Turski et al., 1983b; Fujikawa, 1996; Covolan and Mello, 2000), and is generalized in other chemoconvulsant models of epilepsy including kainic acid, bicuculline, picrotoxin and

43 21 pentetrazole (Lothman and Collins, 1981; Ben-Ari et al., 1981; Ben-Ari, 1985; Turski et al., 1985) Progression and severity of neuronal loss following SE Previous studies assessing the progression of neuronal death in rats after SE have been limited by a semi-quantitative assessment of tissue damage and/or by the incomplete nature of the time frame examined (Fujikawa, 1996; Motte et al., 1998; Covolan and Mello, 2000; Peredery et al., 2000; Poirier et al., 2000). For example, timeframes are restricted to less than 3 to 5 days (Fujikawa, 1996; Covolan and Mello, 2000) or timeframes examined are spaced far apart (Motte et al., 1998; Peredery et al., 2000; Poirier et al., 2000b). A summary of these studies is provided in appendix I, Table A1-1. SE has been shown to primarily contribute to neuronal death (Peredery et al., 2000; Pitkänen et al., 2002; Gorter et al., 2004; Deshpande et al., 2007), with the majority of neuronal injury/death occurring in the several days following SE (Fujikawa, 1996; Motte et al., 1998; Covolan and Mello, 2000; Peredery et al., 2000; Poirier et al., 2000). The effect of SRSs on neurodegeneration, however, remains unclear. While some studies report no correlation of neuronal death and the frequency of SRSs (Pitkänen et al., 2002; Gorter et al., 2004), others have demonstrated progressive neuronal loss during the chronic stage of seizure development (Roch et al., 2002). In the kindling model, brief recurrent seizures can cause neuronal loss and hippocampal sclerosis (Cavazos et al., 1991; Cavazos et al., 1994), supporting a potential role of SRSs in neuronal damage. The difficulty in verifying whether the chronic epileptic seizures that follow SE lead to brain damage is delineating what extent of neuronal loss is attributed to SRSs, and what occurs because of delayed damage from the IPI (reviewed in: Dudek et al., 2002). In the present thesis, a detailed quantitative time course of neurodegeneration between 1 hr and 3 months following SE was completed: (1) to improve our understanding and to compare the evolution of neuronal death in different brain structures, (2) to determine which of these structures exhibit delayed neuronal loss extending past epileptogenesis, and (3) to establish a critical timeframe for the feasibility of neuroprotective strategies Mechanisms underlying SE-induced neuronal death Early studies demonstrated that electrographic seizures can cause neuronal death in artificially ventilated animals, even in the absence of underlying systematic physiological complications (Meldrum and Brierley, 1973; Meldrum et al., 1974; Nevander et al., 1985). Prolonged seizures

44 22 initiate neuronal death by excitotoxicity (reviewed in: Fujikawa, 2005). This occurs when glutamate receptors are excessively stimulated. The specific role of NMDA receptors in SEinduced neurodegeneration is strongly supported by two main findings. (1) NMDA receptor antagonists administered in rats before or after SE provide significant neuroprotection (Fariello et al., 1989; Clifford et al., 1990; Fujikawa et al., 1994; Fujikawa, 1995). (2) In an in-vitro model of SE, specific entry of Ca 2+ through NMDA receptors results in more cell death as opposed to Ca 2+ entering through non-nmda glutamate receptors or voltage-gated calcium channels (Deshpande et al., 2008). Similar findings have been demonstrated in other models of glutamate neurotoxicity (Tymianski et al., 1993; Sattler et al., 1998). During SE, excessive synaptic release of glutamate can cause excitotoxicity by binding to NMDA receptors and allowing high levels of Ca 2+ to enter the cells. Ca 2+ influx into the cells activates a number of enzymes, including neuronal nitric synthase, phospholipases, endonucleases, and cysteine proteases (i.e., calpains and caspases). These enzymes subsequently damage cell structures such as components of the cytoskeleton, membrane and DNA (reviewed in: Fujikawa, 2005; Forder and Tymianski, 2009; Hardingham, 2009; Lau and Tymianski, 2010; Wang and Qun, 2010), leading to different forms of cell death (see section ) Types of cell death mechanisms initiated by SE Even though it is generally accepted that neuronal death following SE is excitotoxic in nature (Fountain, 2000; Fujikawa, 2005; Deshpande et al., 2008), it is not clear if the cell death phenotype is primarily necrotic or apoptotic (reviewed in: Fujikawa, 2005; Lau and Tymianski, 2010; Wang and Qun, 2010). A detailed discussion of necrosis and apoptotsis is beyond the scope of this thesis, but numerous reviews of this topic are available (Saraste and Pulkki, 2000; Manning and Zuzel, 2003; Goldstein and Kroemer, 2007; Doonan and Cotter, 2008; Vanlangenakker et al., 2008). Briefly, necrosis results from loss of cellular homeostasis and membrane integrity and acute mitochondrial dysfunction, all leading to massive energy failure (reviewed in: Goldstein and Kroemer, 2007; Vanlangenakker et al., 2008). Apoptosis is characterized by cell shrinkage, membrane blebbing, DNA internucleosomal degradation, chromosome condensation and formation of membrane-bound apoptotic bodies, and relies on

45 23 preserved mitochondrial functioning and integrity and ATP synthesis (reviewed in: Saraste and Pulkki, 2000; Doonan and Cotter, 2008). A myriad of neurotoxic signaling cascades are capable of initiating apoptosis and/or necrosis, and several extensive reviews of these pathways are available (Yakovlev and Faden, 2004; Harwood et al., 2005; Henshall, 2007; Henshall and Murphy, 2008; Vosler et al., 2008). This section briefly describes the roles of caspases and calpains in apoptotic and necrotic cell death, respectively. Specific caspases are suggested to mediate apoptosis and can be activated by either intrinsic (death receptor-mediated) or extrinsic (mitochondria-mediated) pathways (Harwood et al., 2005; Henshall, 2007). In response to a stimulus, the initiator caspases (e.g., caspase-8 and caspase-9) activate executioner (or effector) caspases (e.g., caspase-3), which subsequently mediate the biochemical and morphological features of apoptosis (Harwood et al., 2005; Henshall, 2007). On the other hand, calpain is activated by an increase in intracellular Ca 2+ and cleaves multiple substrates including cytoskeletal and associated proteins, kinases, phosphatases, membrane receptors and transporters; excessive calpain activity leads to cytoskeletal protein breakdown and subsequent loss of structural integrity and disturbances of axonal transport, accelerating cell death and contributing to necrotic morphology (Pang et al., 2003 and reviewed in: Fujikawa, 2005; Vosler et al., 2008). Still, generalizing the involvement of caspases and calpains to specific cell death morphologies is not straightforward. Extensive cross-talk between the cysteine proteases occurs, and this causes the boundary between necrotic and apoptotic cell death phenotypes to become blurred (Harwood et al., 2005). Instead, a widely accepted view is that excitotoxic cell death occurs along a continuum of necrotic and apoptotic morphologies (Fujikawa et al., 2000b; Doonan and Cotter, 2008; Kotariya et al., 2010; Wang and Qun, 2010). Necrosis is generally considered the principal morphological phenotype of dying cells after SE (Fujikawa, 2005; Kotariya et al., 2010). Despite the predominance of necrotic morphology in degenerated neurons, others suggest that apoptosis may play a critical role in seizure-induced brain damage (reviewed in: Bengzon et al., 2002; Henshall, 2007; Henshalla and Murphy, 2008). Often, a mixed form of neuronal death with apoptotic and necrotic features following SE is reported (Sloviter et al., 1996; Becker et al., 1999; Fujikawa et al., 1999; Covolan et al., 2000; Fujikawa et al., 2000b; Weise et al., 2005). Autophagy is also up-regulated and contributes to neuronal death following prolonged seizures (Cao et al., 2009a; Cao et al., 2009b); this process can induce cell death through excessive self-digestion and degradation of essential cellular

46 24 constituents (reviewed in: Levine and Klionsky, 2004; Codogno and Meijer, 2005), and is initiated with increased oxidative stress (Chu, 2006). A heterogeneous population of cell death morphologies is similarly reported in animal models of stroke (Snider et al., 1999) and traumatic brain injury (Singleton and Povlishock, 2004) Factors determining the extent and phenotype of SE-induced neuronal death Several factors have been shown to affect the extent and phenotype of neuronal death following prolonged seizures. These include severity of SE, intracellular ATP levels, and regional and neuronal specific cell-death signaling pathways. Each of these factors is briefly described. (1) The intensity of continuous convulsive seizures in Wistar rats was shown to affect cell death morphology of hippocampal pyramidal cells; more severe SE (induced by 12 mg/kg of KA, i.p.) led to an increase of necrotic cell death, whereas milder SE (induced by 9 mg/kg of KA, i.p.) increased the presence of dying neurons exhibiting apoptotic features (Tokuhara et al., 2007 and reviewed in: Meldrum, 2002). In contrast, Fujikawa et al., (2010) only detected necrotic cell death in Wistar rats subjected to different durations of pilocarpine-induced SE (1 hr or 3 hrs), failing to establish a relationship between SE duration and cell death morphology. (2) A critical determinant of the cell death mechanism is intracellular ATP concentration, the production of which depends on the structural and functional integrity of the mitochondria. Whereas ATP depletion is associated with necrosis, ATP is required for the development of apoptosis. Cheung et al., (2009) demonstrated apoptotic cell death in the CA1 and CA3 pyramidal cell layer of Sprague Dawley rats following 40 min of SE (induced by unilateral microinjection of KA into CA3). Of particular interest, these regions had maintained ATP levels, and the majority of degenerating pyramidal cells exhibited intact structural integrity of the mitochondria. In the pilocarpine and lithium/pilocarpine models, variability in the severity of mitochondrial damage following SE is reported; the ultrastructural damage of the mitochondria in hippocampal pyramidal cells ranges from no visible changes, to mitochondrial swelling, which is most frequently detected, and/or severe damage characterized by rupture of the inner and outer mitochondrial membranes ( Fujikawa et al., 1999; Jing et al., 2007).

47 25 (3) Repetitive or prolonged seizures can lead to region-specific cell death pathways. For example, Sloviter et al., (1996) demonstrated that repetitive seizures caused apoptotic cell death in dentate granule cells (DGCs) and necrotic cell death in the CA1 and CA3 pyramidal cells. Of particular interest, Lopez-Meraz et al., (2010) showed that in immature rats following KA-induced SE, caspase-9 was up-regulated in DGCs exhibiting apoptotic cell death, while caspase-8 was up-regulated in pyramidal CA1 cells exhibiting necrotic cell death (caspases described in section ). Differences in post-synaptic cell-death pathways can occur within the same neuronal population. KA or pilocarpineinduced SE in adult rats resulted in two types of pyramidal cell death: early necrosis (1 day after SE) and delayed cell death with apoptotic features (3 to 7 days after SE). One possibility is that the morphological differences in neuronal death may be attributed to differential activation of cystein proteases (see section ). For example, the expression and activation of calpain was detected in early necrotic cell death following SE (Araújo et al., 2008; Wang et al., 2008), whereas caspase-3 was detected in delayed neuronal death exhibiting apoptotic features (Narkilahti et al., 2003; Weise et al., 2005; Wang et al., 2008) The role of neurodegeneration in epileptogenesis Although SE results in significant neurodegeneration and in the development of epilepsy, the contribution of neuronal death in the genesis of SRSs is unclear. Two lines of evidence suggest neuronal loss is not required for the genesis of chronic epilepsy. First, several studies have showed that nearly complete protection of limbic structures, including the hippocampus and amygdala, did not prevent the development of epilepsy (Ebert et al., 2002; Loscher, 2002; Brandt et al., 2006). Second, several animal models of epilepsy can lead to development of SRSs in the absence of overt brain damage. For example, seizures produced by electroconvulsive shock repeated for several days initiate mossy fiber sprouting and development of SRSs without neuronal loss. Similarly, in immature rats, febrile seizures can lead to SRSs without neuronal death (Baram et al., 2002; Kapur, 2006). Still, others have reported that SE-induced neurodegeneration can exacerbate the epileptogenic process and functional outcome. For instance, Zhang et al., (2002) compared two groups of rats treated differently with kainic acid. Rats in one group experienced two short sessions of priming

48 26 seizures and one sustained episode of SE; this priming effect resulted in no obvious neuronal loss or mossy fiber sprouting. The second group experienced only a single episode of SE. Though both groups developed epilepsy, the massive neuronal damage and mossy fiber sprouting in the latter group resulted in more frequent and intensified SRSs (Zhang et al., 2002). Similarly, Persinger and Dupont (2004) showed that the extent of neuronal loss in the temporal cortex, dentate gyrus, hilus and CA3 region correlated with the frequency of SRSs (Persinger and Dupont, 2004). Others showed that neuroprotective agents improved behavioural and cognitive functioning in rats following SE (Rice et al., 1998; dos Santos et al., 2005; Brandt et al., 2006; Cunha et al., 2009; Jun et al., 2009). Thus, although it seems that neurodegeneration is not a prerequisite for the development of epilepsy, neuroprotective strategies may have a role in modifying the disease outcome (Loscher, 2002; Pitkanen and Kubova, 2004; Naegele, 2007; Acharya et al., 2008), and in mitigating interictal behavioural and cognitive morbidity (see section 1.6). In the present thesis, we investigated the effectiveness of a neuroprotective strategy in rats following lithium/pilocarpine-induced SE. As previously described in section , excitotoxicity mediated via the NMDAR is recognized as a major mechanism in neurodegeneration resulting from SE. Consistent with a critical role of NMDARs in SE-induced neurodegeneration, previous studies showed that systematic administration of NMDAR antagonists are neuroprotective in rodent models of epilepsy, even when given after the onset of SE (Fariello et al., 1989; Clifford et al., 1990; Fujikawa et al., 1994; Fujikawa, 1995). The clinical efficacy of NMDAR antagonists is limited, however, due to psychomimetic side-effects in humans (Krystal et al., 1994; Lahti et al., 1995; Malhotra et al., 1996; Rowland et al., 2005). An alternative approach to preventing NMDAR-mediated excitotoxicity would be to disrupt the interaction of the receptor with downstream signaling molecules (see section 5.1). PSD-95 is a critical scaffolding protein that links the NMDAR to signaling enzymes within the postsynaptic density, and suppression of the expression of PSD-95 selectively attenuated excitotoxicity triggered via NMDARs (Sattler et al., 1999). Tat-NR2B9c is a synthetic peptide, consisting of the C-terminal 9 amino acids of the NR2B subunit of NMDARs fused to the membrane transduction domain of the HIV-1-Tat protein, that was designed to disrupt excitotoxic signaling from the NMDAR by interfering with protein interactions involving the PDZ1 and PDZ2 domains of PSD-95(see section 5.2.2). Tat-NR2B9c was previously shown to provide

49 27 significant neuroprotection and preserve cognitive function after transient stroke in rats (Aarts et al., 2002; Sun et al., 2008). Here, we assessed the ability of tat-nr2b9c to provide neuroprotection (see chapter 5) and mitigate behavioural morbidity (see chapters 6 and 7) following lithium/pilocarpine-induced SE in rats. The effectiveness of other neuroprotective strategies assessed in post-status epilpeticus rodent models is further discussed in section 8.7 and compared in Table Synaptic reorganization Mossy fiber sprouting is observed in the human epileptic temporal lobe (Sutula et al., 1989; Houser et al., 1990; Franck et al., 1995), and has been extensively studied in animal models of TLE (reviewed in: Dalby and Mody, 2001; Sutula, 2002; Nadler, 2003; Cavazos and Cross, 2006; Pitkänen and Lukasiuk, 2009). Axons of granule cells (mossy fibers) are most often described as sprouting aberrantly into the dentate supragranular layer; new synapses on granule cell dendrites increase the overall excitatory connection between granule cells (Okazaki et al., 1995; Buckmaster et al., 2002). Additional mossy fiber reorganization includes axonal growth in the hilus, development of infrapyramidal and suprapyramidal (interblade) connectivity, and expansion of the terminal field of the mossy fiber pathway in CA3 and along the septotemporal axis of the hippocampus over distances as long as 700 to 800 microns (Sutula et al., 1998; Buckmaster and Dudek, 1999; Holmes et al., 1999). Seizure-induced reorganization along the septotemporal axis provides a mechanism for translamellar synchronization of cellular hyperexcitability within the hippocampus to occur (reviewed in: Cavazos and Cross, 2006). Although mossy fiber sprouting can occur in the absence of neurodegeneration caused by repeated seizures (e.g., in the kindling model; reviewed in Morimoto et al., 2004), neuronal death (e.g., in the hilus and CA3 pyramidal cell layer) can also initiate mossy fiber reorganization following an IPI (reviewed in: Sutula, 2002; Nadler, 2003). Mossy fiber sprouting appears prior to the occurrence of SRSs and persists for the lifetime of the animal (Cavazos et al., 1991; Nissinen et al., 2001; Wuarin and Dudek, 2001). Several studies have demonstrated a direct correlation between the degree of neuronal loss, mossy fiber sprouting into the dentate supragranular layer and granule cell hyperexcitability (Sutula, 2002; Cavazos and Cross, 2006; Sutula and Dudek, 2007). Recent studies indicate that the functional effects of the recurrent excitatory circuits formed by mossy fiber reorganization may only be apparent under conditions

50 28 of reduced inhibition, or alterations in the extracellular ionic environment (e.g., elevation of [K + ] o ) (reviewed in: Sutula, 2002; Sutula and Dudek, 2007). Similar to SE-induced neurodegeneration, the development of mossy fiber sprouting following prolonged seizures can exacerbate the epileptogenic process and functional outcome (see figure 1.1). Jing et al., (2009) showed that suppression of mossy fiber sprouting, by combined treatment consisting of intrahippocampal transplantation of adult neural stem cells and intraventricular erythropoietin- infusion, reduced hippocampal excitability and prevented development of SRSs in kainate-treated rats (Jing et al., 2009). In other studies, pre-treatment with cycloheximide in rats has been shown to reduce mossy fiber reorganization (Longo and Mello, 1997; dos Santos et al., 2005). It also concomitantly reduced behavioural alterations (Lee et al., 2003; dos Santos et al., 2005), indicating a connection between mossy fiber sprouting and SE-induced behavioural morbidity. Synaptic reorganization has been detected elsewhere, including the CA1, entorhinal cortex and neocortex (Esclapez et al., 1999; Wuarin and Dudek, 2001; Smith and Dudek, 2002; Cavazos et al., 2004) Reactive gliosis Reactive gliosis is a prominent characteristic in the epileptic brains of humans (Spencer et al., 1999; D'Ambrosio, 2004) and is recapitulated in rodent models of TLE. It is characterized by the hypertrophy (intensive outgrowth of cellular processes) of astroctyes as well as by the proliferation of microglial cells and astrocytes (Ridet et al., 1997). Glial cells perform many functions, including structural support, water and ionic homeostasis, regulation of neurotransmission, inflammatory responses and neurogenic potential (Ridet et al., 1997); reactive gliosis results in acute and long-term alterations of these functions, which in turn contribute to the epileptogenic process. Several extensive reviews on the role of glial cells in epilepsy are available (D'Ambrosio, 2004; Eid et al., 2008; Jabs et al., 2008; Binder and Steinhäuser, 2006; Seifert et al., 2010). Of particular interest, reactive astrocytes were shown to contribute to delayed neuronal death of cortical and hippocampal pyramidal cells following pilocarpine-induced SE (Ding et al., 2007). In this study, prolonged elevation of astrocytic Ca 2+ signaling correlated with the temporal profile of neuronal death, which was maximal at 3 days following SE. Astrocytic Ca 2+ signaling leads to gliotransmission of glutamate and D-serine; administration of MPEP (2-methyl-6(pehnylethynyl)pyridine), a metabotropic glutamate receptor

51 29 5 antagonist (mglur5), blocked astrocytic Ca 2+ signaling and provided significant neuroprotection, linking astrocytic gliotransmission to delayed neuronal death. The neuroprotective effect of ifenprodil, an antagonist specific for NR2B-containing NMDA receptors, indicates that gliotransmission induces neuronal excitotoxicity by activation of these receptors (Ding et al., 2007) Neurogenesis Adult neurogenesis is the generation of new neurons in the central nervous system through division of neural stem cells (NSCs). These stem cells divide to form neuroblasts, which eventually differentiate into neuronal or glial phenotypes and are integrated into functional networks. The majority of NSCs are located in two neurogenic regions: (1) Neuroblasts from the subventricular zone lining the lateral ventricles migrate along the rostral migratory stream into the olfactory bulb, where the majority differentiate into interneurons (Whitman and Greer, 2009). (2) Neuroblasts in the subgranular zone of the DG, a lamina between the granule cell layer (GCL) and the hilus, migrate into the GCL and mainly mature into functional granule cells (Parent, 2007). In the pilocarpine model, SE increases the rate of neurogenesis in the DG for a 2-week period (Parent et al., 1997). While the majority of cells migrate appropriately into the DG following SE, approximately 20% migrate aberrantly to ectopic locations, most notably the dentate hilus (Walter et al., 2007). Hilar ectopic granule cells are invariably bipolar, in contrast to normally situated granule cells, which have only an apical dendrite. Even though their membrane properties closely resemble normally positioned granule cells, they are notable for bursting in synchrony with CA3 pyramidal cells (Scharfman et al., 2000). The increased excitability of these cells is attributed to their abnormal innervations; hilar ectopic granule cells receive mossy fiber input from other granule cells and innervate other granule cells, in addition to the typical targets of the mossy fiber pathway (CA3 pyramidal cells and inhibitory interneurons, hilar mossy cells and inhibitory interneurons) (Parent et al., 1997; Scharfman et al., 2000; Dashtipour et al., 2001). These cells also receive few inhibitory synapses (Dashtipour et al., 2001). Of particular interest, Gong et al., (2007) demonstrated that loss of a subset of interneurons expressing reelin, a migration guidance cue that influences neuronal migration, contributes to the ectopic location of granule cells following SE.

52 30 The altered innervations of hilar ectopic granule cells and the recurrent synapses between CA3 pyramidal neurons combine to form a reverberatory loop, which is proposed to participate in synchronization and propagation of seizure activity (Nadler, 2003; Parent and Murphy, 2008). During the chronic phase of seizure development a decrease in neurogenesis occurs (Hattiangady et al., 2004). The increase in neurogenesis following SE and the decrease in neurogenesis during chronic epilepsy both contribute to the epileptogenic process and to the development cognitive and behavioural impairments (reviewed in: Hattiangady and Shetty, 2008; Kuruba et al., 2009). Several extensive reviews on the role of neurogenesis in epilepsy are available (Parent, 2007; Danzer, 2008; Hattiangady and Shetty, 2008; Parent and Murphy, 2008; Kuruba et al., 2009). 1.6 Co-morbid interictal disorders in mesial temporal lobe epilepsy MTLE is often associated with interictal behavioural disturbances, including anxiety, depression, and psychosis, as well as learning and memory impairments ( Boro and Haut, 2003; Devinsky, 2004; Gaitatzis et al., 2004; Swinkels et al., 2005; Cornaggia et al., 2006; Marcangelo and Ovsiew, 2007; Garcia-Morales et al., 2008). Because of several confounding variables in the clinical setting, however, the causal relationships between epilepsy, affective disturbances and cognitive impairments are poorly understood. These confounders preclude direct demonstration that seizures themselves are responsible for behavioural and cognitive deficits, and include the underlying etiology and brain pathology, present and past anticonvulsant treatments, degree of seizure control, and developmental status of the patient (Heinrichs and Seyfried, 2006). Many of these problems are circumvented by the use of animal models of epilepsy, which allow for a more direct correlation of behavioural outcomes with physiological and histological data (reviewed in: Majak and Pitkanen, 2004; Post, 2004; Heinrichs and Seyfried, 2006). The present section reviews current hypotheses regarding the relationship between epilepsy and interictal behavioural and cognitive disturbances, and specifically focuses on data derived from post-status epilepticus animal models The relationship between epilepsy and cognitive and interictal behavioural alterations Based on animal models of epilepsy, three hypotheses regarding the causal relationship between seizures and behavioural morbidity have been proposed:

53 31 1. Shared neurodevelopmental, genetic or environmental causes predispose subjects to develop both epilepsy and co-morbid behavioural and cognitive disturbances 2. Neuropathological changes underlying the genesis of interictal behavioural disturbances are closely related to those mediating epileptogenesis itself 3. SRSs contribute to cognitive and interictal behavioural impairment Shared neurodevelopmental, genetic or environmental causes predispose subjects to develop both epilepsy and comorbid behavioural and cognitive disturbances Evidence for this hypothesis is supported by studies showing cognitive and interictal behavioural disturbances prior to seizure development in genetically-predisposed epileptic rats (Kelly et al., 2003; Sarkisova et al., 2003; Midzyanovskaya et al., 2005; Jones et al., 2008; Runke and McIntyre, 2008). For instance, Jones et al., (2008) illustrated that genetic absence epilepsy rats from Strasbourg (GAERS, a genetic model of absence epilepsy in rats; epilepsy model reviewed in: Marescaux et al., 1992; Danober et al., 1998) exhibit an increase in anxiety- and depressivelike behaviours compared to their non-epileptic controls. This behavioural phenotype of GAERS is present prior to the occurrence of spontaneous spike-and-wave discharges (non-convulsive absence seizures), which develop between postnatal days 30 to 60, and persists throughout the life of the animal. Similarly, FAST kindling rats that have been bred to exhibit enhanced rates of amygdala kindling, a model of temporal lobe epileptogenesis (model reviewed in: McIntyre et al., 1999; Racine et al., 1999; McIntyre et al., 2002), display different behaviours of stress responsivity and anxiety prior to kindling compared to SLOW rats (McIntyre et al., 2002; Kelly et al., 2003; McIntyre and Gilby, 2007; Runke and McIntyre, 2008). For example, FASTkindling rats exhibit less anxiety in the elevated-plus maze, and increased exploratory behaviour without habituation over repeated trials in the open field (McIntyre et al., 2002). Additionally, FAST-kindling rats display inferior performance in the Morris water maze prior to kindling, suggesting both working and reference memory impairments (Anisman and McIntyre, 2002).

54 Neuropathological changes underlying the genesis of interictal behavioural disturbances are closely related to those mediating epileptogenesis itself The second hypothesis states that neuropathological changes underlying the genesis of interictal behavioural and cognitive disturbances are closely related to those mediating epileptogenesis itself. This is strongly supported by studies of post-status epilepticus animal models. As described previously, epilepsy can be seen as a process whereby an IPI (like SE) results in a latent phase (epileptogenesis) and, eventually, the occurrence of SRSs (see section 1.4). As described in section 1.5, neuronal death and synaptic reorganization caused by an initial brain injury contribute to the genesis of recurrent seizures (Fujikawa, 2005; Acharya et al., 2008; Sharma et al., 2008), and have also been proposed to underlie interictal behavioural and cognitive morbidity (Majak and Pitkanen, 2004; Sayin et al., 2004; de Oliveira et al., 2008). For example, experimental febrile seizures cause long-lasting behavioural and cognitive disturbances and epileptogenesis, and are accompanied by neuropathological changes including neuronal loss, mossy fiber sprouting, and gliosis (Kubova et al., 2004; Sayin et al., 2004; de Oliveira et al., 2008; and reviewed in Stafstrom, 2002). Similar results are obtained in adult rats following SE induced by epileptogenic agents (Milgram et al., 1988; Detour et al., 2005; dos Santos et al., 2006; Cardoso et al., 2008; de Oliveira et al., 2008; Cardoso et al., 2009; Cardoso et al., 2009). The severity of behavioural and cognitive deficits is correlated with the extent of neuronal death (Milgram et al., 1988; Niessen et al., 2005; Cilio et al., 2003) and mossy fiber sprouting (de Oliveira et al., 2008; de Rogalski et al., 2001; Sogawa et al., 2001). Neuroprotection within selective brain regions or a reduction in mossy fiber sprouting has been demonstrated to mitigate SE-induced morbidity(bolanos et al., 1998; Rice et al., 1998; Cilio et al., 2001; dos Santos et al., 2005; Brandt et al., 2006; Frisch et al., 2007; Cunha et al., 2009; Jun et al., 2009), providing direct support for the hypothesis that neuropathological changes during epileptogenesis cause interictal behavioural and cognitive impairments in rats after SE.

55 Spontaneous recurrent seizures contribute to interictal behavioural and cognitive impairment in post-se models The temporal onset of behavioural and cognitive impairments has been shown to occur as early as 24 hours following SE induced by kainic acid or pilocarpine, and precedes development of SRSs (Milgram et al., 1988; Rice et al., 1998; Mikati et al., 2001; Sogawa et al., 2001; Chauviere et al., 2009). These data are consistent with neuropathological changes contributing to epileptogenesis and early cognitive and behavioural disturbances. As illustrated in Figure 1.1, however, an alternative hypothesis suggests that SRSs contribute to neuronal death, synaptic reorganization, and interictal behavioural and cognitive decline. SE-induced epileptogenesis results in the appearance of SRSs within a few weeks (discussed in sections and 1.4.3). Hort et al., (1999) and Sun et al., (2009) demonstrated that although MWM performance is impaired after SE, learning and memory are further deteriorated with development of SRSs (see appendix I, Table A1-2 for details). In another study, the impaired performance in the MWM was less severe in rats with sporadic seizures (<1/day) than in frequently seizing animals ( 1/day) (Nissinen et al., 2000). In spontaneous seizing animals, however, it is difficult to differentiate the contribution of SE-induced damage to cognitive and behavioural impairment from the damage caused by SRSs, particularly because the rats with frequent seizures begin with greater SE-induced damage (Roch et al., 2002; Majak and Pitkanen, 2004). Additionally, brain damage in specific brain regions can progress weeks to months after SE, complicating the assessment of neuronal loss caused by SRSs in post-status epilepticus models (Fujikawa, 1996; Pitkänen et al., 2002) Other factors affecting severity of interictal behavioural and cognitive impairment In various animal models of epilepsy, additional factors are shown to affect the severity of interictal behavioural and/or cognitive impairment. These include genetic background (Hort et al., 2000), age at the time of the epileptogenic insult (Stafstrom et al., 1993; Cilio et al., 2003; Kubova et al., 2004), extent of brain damage (Milgram et al., 1988; Mohajeri et al., 2003), location of seizure focus (Becker et al., 1997), seizure frequency (Nissinen et al., 2000) and environmental conditions (Faverjon et al., 2002; Wang et al., 2007) (also reviewed in: Post, 2004; Majak and Pitkanen, 2004). Many of these factors also influence the severity of behavioural disturbances in human epilepsies (Cornaggia et al., 2006; Titlic et al., 2009).

56 Interictal behavioural disturbances following SE The effect of SE and epilepsy on a variety of different behaviours has been assessed. Because behavioural tasks assessing anxiety, exploration and aggression have been shown to be particularly affected in rodents following prolonged seizures, we focused our attention on the analyses of these behaviours Anxiety Anxiety disorders are commonly reported in patients with epilepsy (Cornaggia et al., 2006; Titlic et al., 2009). Previous researchers have proposed that the behavioural symptoms of human anxiety disorders, which include hypervigilance, exaggerated startle, avoidance and escape reactions (Vazquez and Devinsky, 2003), reflect the inappropriate activation or exaggeration of normally adaptive defence responses (Rosen and Schulkin, 1998). Because the neural mechanisms underlying anxiety are conserved across mammalians, this behaviour may be investigated using behavioural tasks that are ethologically designed to evaluate defence responses in rodents; the repertoire of defence reactions in rodents can differ between tasks, and include flight/escape, freezing, startle, and defensive threat/attack (Rodgers, 1997). Defence responses are believed to have originated as anti-predator strategies, but mammals additionally exhibit defensive reactions to other threatening stimuli, including those associated with aggressive conspecifics and dangerous/potentially dangerous objects or environments (Griebel, 1995; Rodgers, 1997; Rodgers et al., 1997). Over 30 different types of behavioural tasks designed to assess anxiety have been developed (Rodgers, 1997; Rodgers and Dalvi, 1997). Wall and Messier (2001) caution that in using behavioural tests of anxiety, fundamental differences between anxiety in humans and animals may exist. In humans, anxiety can also be described as an affective, emotional state that may or may not have an externally observable correlate. In testing anxiety in rodents, we must rely on observable behaviours and assume that these reflect the internal emotional state (Griebel, 1995). Because the elevated plus maze (EPM) and open field are the most commonly used tests of anxiety and exploration (see section ) in animal models of epilepsy (Wall and Messier, 2001; Heinrichs and Seyfried, 2006; Ramos, 2008), we have primarily focused our discussion on the effect of SE on behaviour in these tasks. The EPM consists of two elevated, open (brightly lit) arms perpendicular to two enclosed (dark) arms (Rodgers and Dalvi, 1997; Stafstrom, 2006). Rodents prefer dark, enclosed spaces to

57 35 brightly lit, open areas, but they are also spontaneously exploratory by nature. Because rodents have an innate fear of heights, elevating the EPM off the floor also contributes to the anxiety level. The EPM is regarded as an unconditioned spontaneous behavioural conflict test that uses an ethologically relevant situation to measure the conflict between the drive to explore a new environment, and the tendency to avoid a potentially dangerous area (File, 1993; Treit et al., 1993; Carobrez and Bertoglio, 2005). It has also been suggested that a rodent s aversion to open spaces can be attributed to thigmotaxis, a reaction in which the animal remains close to the vertical surfaces, and which is part of their natural defensive repertoire (Carobrez and Bertoglio, 2005). Similar to the EPM, the open field is another unconditioned spontaneous behavioural conflict test (Falter et al., 1992; Treit et al., 1993). The open field test evaluates the conflict between exploration of a novel environment and aversion to open spaces from which escape is prevented by a surrounding wall. Typically, rodents will explore a new environment but become less active on subsequent exposure to the environment, a process referred to as habituation (Walsh and Cummins, 1976; Prut and Belzung, 2003). SE induced by pilocarpine or kainic acid results in a long-lasting anxiolytic response as assessed by the EPM test, with adult (Choleris et al., 2001; Prut and Belzung, 2003; Chauviere et al., 2009) and immature rats (Santos et al., 2000; Sayin et al., 2004; Kubova et al., 2004; Detour et al., 2005; dos Santos et al., 2005; Cardoso et al., 2009; Sun et al., 2009) spending a greater amount of time in the unprotected open arm. In contrast, Groticke et al., (2008) failed to find behavioural changes with this task in mice. Less consistent results are reported in the open field test. Brandt et al., (2006) reported an anxiolytic response in rats following self-sustaining status epilepticus (SSSE) induced by electrical stimulation of the basolateral amygdala, with an increased amount of time spent in the central zone of the open field. In contrast, others found that SE caused by pilocarpine or kainic acid resulted in an anxiogenic response, whereby rats (Santos et al., 2000; dos Santos et al., 2000; Sayin et al., 2004) or mice (Cardoso et al., 2009) exhibited increased thigmotaxis (e.g., increased locomotor activity and/or time spent in the periphery of the open field). Finally, several studies have reported no differences in locomotor activity or time spent in the central or peripheral zones of the open field (Groticke et al., 2008; Cardoso et al., 2009; Müller et al., 2009). Because behavioural responses of rodents in these tasks are extremely sensitive to differences in the species/strain used, the environmental conditions (e.g., lighting) and the procedures used, these factors are likely to have contributed to

58 36 differences in results between the studies. Impaired anxiety in rodents following SE has been reported in other behavioural tasks, which include the hole-board test (Kubova et al., 2004; Sayin et al., 2004; Szyndler et al., 2005; de Oliveira et al., 2008; dos Santos et al., 2006), the light-dark box test (Groticke et al., 2008; Müller et al., 2009), the novel-object recognition test (de Oliveira et al., 2008; Groticke et al., 2008; Müller et al., 2009), and the elevated T-maze test (Groticke et al., 2008; Müller et al., 2009). Results are summarized in appendix I, Table A Exploration Although exploratory activity is less routinely assessed in the open field and EPM tests, it can provide critical information on different aspects of behavior (Rodgers et al., 1997; Heinrichs and Seyfried, 2006 Milgram et al., 1988). This includes the well-being of the animal, the presence or increase of inadaptive behaviour, and the reactivity of the animal to a novel environment. In these tasks, exploratory activity can be analyzed by the frequency and/or duration of rearing activity (also referred to as vertical activity), by the frequency of risk assessments, and by locomotor activity (Rodgers and Dalvi, 1997; Carobrez and Bertoglio, 2005). Risk assessments include stretched-attend postures, and involve the reluctance of the animals to leave the confines of the enclosed protected arm of the EPM (Rodgers and Dalvi, 1997; Carobrez and Bertoglio, 2005). In the EPM test, studies have demonstrated that SE causes a long-lasting increase in exploratory behaviour (e.g., frequency of rearing and risk assessments) (Detour et al., 2005; Sun et al., 2009). While some authors have similarly reported an increase in rearing activity in the open field test (Kubova et al., 2004; Erdogan et al., 2005; Sun et al., 2009), others have found diminished rearing activity (dos Santos et al., 2005; Groticke et al., 2008). An increase in locomotor activity has also been reported in the EPM test (e.g., number of closed or total arm entries) and/or the open field test (e.g., total distanced travelled) ( Milgram et al., 1988; Santos et al., 2000; Kubova et al., 2004; Sayin et al., 2004; Detour et al., 2005; dos Santos et al., 2005; Szyndler et al., 2005; Brandt et al., 2006; Cardoso et al., 2009; Müller et al., 2009; Sun et al., 2009). Impaired exploratory activity in rodents following SE has been detected in other behavioural tests, including the hole-board test and the novel object recognition test (Groticke et al., 2008; Müller et al., 2009). Results are summarized in appendix I, Table A1-2.

59 37 A change in exploratory and/or locomotor activity in rats following SE can indicate a decrease in the well-being of the animal (Heinrichs and Seyfried, 2006). In a series of comprehensive articles on animal well-being, Clark and colleagues defined specific indicators of wellness in multiple species (Clark et al., 1997a; Clark et al., 1997b; Clark et al., 1997c; Clark et al., 1997d). For instance, wellness is determined by the presence of certain species-typical behaviour, such as grooming and exploratory activity (e.g., rearing and risk assessments) in rats. An absence of abnormal behaviours is also required, such as non-goal directed activities; in rodents, this can include motor stereotypes, increased non-directed locomotion, flight and immobility, social withdrawal and aggression (Heinrichs and Seyfried., 2006). A human analog of the well-being assessment for epileptic animals is the Quality of Life in Epilepsy inventory (QOLIE) (Heinrichs and Seyfried., 2006), which includes assessment of physical, affective, and social behaviours (Devinsky et al., 1995). Not surprisingly, patients with epilepsy show impairment in these aspects of their daily lives (Devinsky et al., 1995; Devinsky., 2000). As outlined previously, a change in exploratory behavior can indicate the presence of an inadaptive response. For instance, a decrease in exploratory behaviour can result from changes of other behaviours, such as increased non-directed locomotor activity (Heinrichs and Seyfried, 2006). In the EPM, Groticke et al., (2008) and Müller et al., (2009) showed that decreased exploratory behaviour (i.e., stretch-attend postures) was accompanied by increased locomotion in mice following pilocarpine-induced SE. A decrease in exploratory behaviour and riskassessments can also indicate an animal exhibiting hyporeactivity to a stressful environment (dos Santos et al., 2005). In support of this view, several studies showed a decrease in rearing activity in rats and mice following SE (dos Santos et al., 2005; Groticke et al., 2008). In several studies, animals were repeatedly assessed in the open field test (Krsek et al., 2001; Kubova et al., 2004; Sun et al., 2009). In a control (or non-epileptic) animal, the behavioural responses to the open field normally habituates over subsequent exposures (e.g., attenuation of rearing activity and/or locomotion). However, pilocarpine-treated rats exhibited no signs of habituation, suggesting impairment in a simple nonassociative form of learning and memory (Krsek et al., 2001; Kubova et al., 2004; Sun et al., 2009).

60 Aggression Increased aggressiveness is reported in patients with epilepsy (Geschwind, 1983; Cornaggia et al., 2006). Psychological stress, the effects of anticonvulsant therapy and the actual occurrence of seizures are ruled out as possible causes (Geschwind., 1983). Geschwind (1983) speculated that interictal emotional disturbances in patients with TLE are the result of an intermittent spike focus (i.e., increased hyperexcitability) in the temporal lobe that leads to a permanent alteration in the responsiveness of the limbic system. In particular, the temporolimbic structures of the brain that subserve emotional representation are highly epileptogenic, and are involved in the hypothalamic-pituitary-adrenal axis, which modulates hormonal secretion and mediates hormonal feedback (Herzog, 1999). These alterations in neural signaling may produce a heightened emotional response to many stimuli as well as a decrease in sexual responsiveness (Herzog, 1999). Of particular interest, brain damage to the temporolimbic structures is correlated with increased aggressiveness (Hallera and Kruk, 2006). A number of simple tests are available to assess aggressiveness and hyperexcitability in rodents following SE (Rice et al., 1998; Heinrichs and Seyfried, 2006). Rice et al., (1998) demonstrated that rats following pilocarpine-induced SE behaved more aggressively toward the experimenter in the handling (or pickup) test (e.g., involves picking the rat up around its midsection and ranking its behavioural response), and showed increased excitability in the touch-response test (e.g., ranking the startled response when the animal is prodded from behind). Kainic acid also resulted in increased aggression in rats when handled (Milgram et al., 1988; Stafstrom et al., 1993). Increased hyperexcitability and aggression in rats were detected within the first day following SE, and the behavioural alterations were long-lasting (Milgram et al., 1988; Rice et al., 1998) The effect of SE on spatial learning and memory Impaired learning and memory has been reported in adults and children following SE (Helmstadter et al., 2007), and in patients with TLE (Motamedi and Meador, 2003; Vingerhoets, 2006). More specifically, patients with TLE frequently showed deficits in declarative memory (ability to acquire facts and events related to one s personal past, which is often compared with visual-spatial learning in rats (Guerreiro et al., 2001; Heinrichs and Seyfried, 2006) and in the performance of visuospatial tasks ( Hermann et al., 1997; Gleissner et al., 1998; Abrahams et al.,

61 ). Cognitive impairment in human epilepsies is one aspect that can be modeled quite effectively and reliably in post-status epilepticus animal models (Heinrichs and Seyfried, 2006). The MWM and the radial arm maze are the most commonly used methods to assess spatial learning and memory in rodents, and both have been used frequently in epilepsy research (Stafstrom, 2006). Because the MWM was used to assess spatial learning and memory in the present thesis, this method and the effect of SE on MWM performance were discussed in detail in the subsequent sections The Morris water maze as a test of visual-spatial learning and memory The majority of research assessing the effects of SE on cognitive functioning is based on data from rats being tested on the hidden platform version of the MWM (Stafstrom, 2006). Briefly, the MWM consists of a large circular pool filled with opaque water in which a small escape platform is hidden (Morris, 1984; Brandeis et al., 1989; Vorhees and Williams, 2006). During a number of training trials, the animals learn to navigate to the submerged platform using distal, visual cues surrounding the pool. Rats are proficient swimmers, and the water maze utilizes negative reinforcement (water immersion) to motivate the animal to learn and recall the platform location. Performance is primarily assessed by the latency (or distance travelled) for the animal to locate and escape onto the platform. It is now recognized that acquisition of the MWM task has two main components: (1) behavioural-strategies learning and (2) spatial learning (McDonald and White, 1994; Morris and Frey, 1997; Inglish and Morris, 2004). The first component involves learning behavioural strategies that allow the rat to move around in its spatial environment and to learn the most effective strategies for locating and reaching its target (e.g., the submerged platform). Acquisition of behavioural strategies involves selection from a variety of instinctive behaviours, such as suppression of the rat s normal response of thigmotaxic swimming in close proximity to the pool wall, in favour of searching the inner portion of the pool where the platform is located, and recognizing and consistently using the submerged platform as an escape (McDonald and White, 1994; Whishaw, 1989). The second component is the spatial learning component, whereby the rat builds a spatial cognitive map correlating context information (extramaze cues) with platform location (Morris, 1984; DiMattia and Kesner, 1988; Fenton and Bures, 1993). Once this map is created, the rat is able to swim directly to the escape platform from any point of

62 40 the circumference of the tank. Success in the spatial learning component requires prior learning of the behavioural-strategies component (Whishaw, 1989; Morris, 1989; Bannerman et al., 1995; Saucier and Cain, 1995; Baldi et al., 2003). Independent memory systems appear to be required for learning of behavioural-strategies and for spatial learning (McDonald and White, 1994; McDonald and White, 1993; Miranda et al., 2006). Evidence strongly supports the role of the hippocampus and posterior parietal cortex in spatial mapping and memory (Cain et al., 2006). The prefrontal cortex, striatum, cerebellum and medial thalamus appear to be involved in acquisition of behavioural strategies (Cain et al., 2006; Packard and McGaugh, 1996; Leggio et al., 1999). For instance, rats with hippocampal damage acquired the visible platform version of the MWM normally but were impaired on acquisition of the hidden platform version of the task (Fenton and Bures, 1993; Broadbent et al., 2004; Leggio et al., 2006). In the visible (or cued) platform task, animals only require acquisition of behavioural strategies to locate the platform made visible either with a flag and/or with the platform elevated above the water surface. On the other hand, rats with damage to the striatum or cerebellum exhibited a characteristic behavioural pattern in which they did not swim towards the center of the pool, but continually scratched at and/or swam around the periphery of the walls (McDonald and White, 1994; Whishaw et al., 1987; Federico et al., 2006). This behaviour was interpreted as the animal s inability to suppress their thigmotaxic response to the task. In general, performance in the hidden platform version of the MWM task involves acquisition of behavioural strategies and spatial learning, with each component dependent on separate memory systems that work in concert to achieve optimal performance in the task (McDonald and White, 1994; McDonald and White, 1993; Miranda et al., 2006) The effect of SE on performance in the MWM task Numerous studies have demonstrated impaired MWM performance as assessed by the hidden platform MWM task in immature (Hung et al., 2002; Stafstrom, 2002; Kubova et al., 2004; Sayin et al., 2004) and adult (Milgram et al., 1988; Rice et al., 1998; Hort et al., 1999; Mikati et al., 2001;; Hort et al., 2000; Nissinen et al., 2001; McKay and Persinger, 2004; dos Santos et al., 2005; Frisch et al., 2007; Zhou et al., 2007; Cunha et al., 2009; Jun et al., 2009; Sun et al., 2009) rats and in mice (Mohajeri et al., 2003; Groticke et al., 2008; Müller et al., 2009) following SE. Impaired MWM performance has been detected as early as 3 to 7 days after SE ( Rice et al.,

63 ; Hort et al., 1999; Hort et al., 2000; Cunha et al., 2009) and appeared to be long-lasting (testing conducted 1 to 5 months after SE (Rice et al., 1998; Hort et al., 2000; Kubova et al., 2004; McKay and Persinger, 2004; dos Santos et al., 2005; Frisch et al., 2007; Groticke et al., 2008; Müller et al., 2009). Because animals are in poor physical condition for 3 days after SE, testing in the MWM has never been conducted earlier than 3 days (Rice et al., 1998; Hort et al., 1999; Hort et al., 2000; Cunha et al., 2009). Immature rats exhibited less severe cognitive deficits when compared to adult rats, and this difference is attributed to less severe neuronal loss occurring in immature brains (Stafstrom et al., 1993; Kubova et al., 2004; Stafstrom, 2006). Milgram et al., (1998) reported that MWM performance is improved when the task is repeated. However, this finding may depend on the SE-induction protocol or the rodent strain/species used. For instance, Hort et al., (2000) found that although LEH rats showed improved performance when the task was repeated, no improvement was detected in Wistar rats. Although SE is often suggested as being the primary cause of impaired MWM performance (Rice et al., 1998; Hort et al., 1999; Hort et al., 2000; dos Santos et al., 2005), two studies showed that development of SRSs can also contribute to cognitive decline (Hort et al., 1999; Sun et al., 2009). Of particular interest, Mohajeri et al., (2003) found that the severity of SE (i.e., intensity of seizures) and the extent of brain damage correlated with the severity of impairment in MWM performance (Mohajeri et al., 2003). Cognitive deficits in rats after SE have been reported in other behavioural tasks, including the radial arm maze (Letty et al., 1995; Wu et al., 2001; Sayin et al., 2004; Detour et al., 2005) and the displaced object-recognition task (Chauviere et al., 2009). Results are summarized in appendix I, Table A1-3. Although the MWM task has revealed impaired performance in rats following SE, the exact nature of this impairment is uncertain. This is because the commonly reported dependent measure, escape latency to locate the submerged platform, cannot distinguish between deficits in spatial learning and deficits in behavioural strategies. Most studies attribute poor MWM performance to impaired spatial learning and memory (see appendix I, Table A1-3). However, several studies have additionally reported a thigmotaxic response in rats after SE, and that this behaviour was not accounted for by the presence of sensory or motor impairment (results described in appendix I, Table A1-3; Milgram et al., 1988; Hort et al., 1999; Kubova et al., 2004; McKay and Persinger, 2004; Groticke et al., 2008; Jun et al., 2009); these findings indicate deficits in behavioural strategies. In the present thesis, a detailed analysis on search strategy use

64 42 during hidden platform testing in the MWM was completed to determine: (1) the effect of SE on search strategy selection, and (2) whether impaired search strategy use, along with deficits in spatial learning and memory, contribute to poor MWM performance in rats after SE. 1.7 The goals of the thesis The goals of the present thesis were threefold: 1. The first aim was to optimize our use of the lithium/pilocarpine model. This was accomplished by comparing the effects of different rat strains and procedural protocols on SE induction and mortality rates, seizure severity, and neuropathology (see chapter 3). 2. Our second goal was to characterize neurodegeneration and behavioural alterations in rats after SE induced by the RLDP lithium/pilocarpine procedure. Briefly, this was achieved by (1) completing a detailed timecourse analysis of neuronal loss following SE (see chapter 4), (2) determining the effect of SE on behavioural alterations as assessed by the open field test, elevated plus maze test, and four hyperexcitability tests (see chapter 6), and (3) assessing the effect of SE on MWM performance and on use of behavioural search strategies (see chapter 7). 3. Finally, our third goal was to test the effectiveness of a neuroprotective strategy using the RLDP lithium/pilocarpine procedure we developed and characterized. Briefly, this was achieved by assessing the ability of tat-nr2b9c to provide neuroprotection (see chapter 5) and mitigate behavioural alterations (see chapters 6 and 7) following lithium/pilocarpine-induced SE in rats. Tat-NR2B9c is a synthetic peptide contructed to disrupt the NMDAR signaling complex, and has been previously described in section The general hypothesis, specific hypotheses and specific objectives are discussed in chapter 2.

65 43 Chapter 2 Hypotheses and Specific Objectives Patients with MTLE-HS frequently have a history of an IPI such as febrile convulsions, SE, encephalitis, stroke or traumatic brain injury (Mathern et al., 1996; Mathern et al., 2002). The lithium/pilocarpine model reproduces most clinical and neuropathologic features of MTLE-HS (see section 1.1). In adult rats, pilocarpine leads to an acute episode of SE which serves as the IPI (see section 1.3). This is followed by a latent period and subsequent development of SRSs (see section 1.4). Data from humans and post-status epilepticus rodent models suggest that both the risk of epilepsy (Hesdorffer et al., 1998; Zhang et al., 2002; Persinger and Dupont, 2004) and the severity of behavioural alterations (Shorvon, 1994; Loscher, 2002; Devinsky, 2004a; Pitkanen and Kubova, 2004; Naegele, 2007; Acharya et al., 2008) are associated with neuropathological changes caused by the IPI (see section 1.5). In both conditions, neuronal death is observed in the hippocampus, amygdala, thalamus, and piriform and entorhinal cortices (see section 1.5.1). The relationship between neuronal death, epileptogenesis, and cognitive/behavioural alterations remains unclear. While some studies report a positive relationship between neuronal death and the severity of epileptic seizures, others have shown that epileptogenesis can occur in the absence of neuronal loss (see section ). Some (Bolanos et al., 1998; Rice et al., 1998; dos Santos et al., 2005; Brandt et al., 2006; Cunha et al., 2009; Jun et al., 2009), but not all (Cilio et al., 2001; Halonen et al., 2001; Narkilahti et al., 2003b; Pitkänen et al., 2004; Zhou et al., 2007), studies have demonstrated that reducing neuronal death can also alleviate SE-induced behavioural and cognitive morbidity in rats (see Table 8.1). Our general hypothesis is that genesis of SRSs, cognitive impairment and behavioural alterations are associated with neuronal death caused by SE. The causal relationship between neuronal death, epileptogenesis and cognitive and behavioural morbidity is discussed in chapter 8. This thesis was centered on 5 specific hypotheses and associated objectives that examined SE-induced neurodegeneration and behavioural alterations in the lithium/pilocarpine model; each of these is presented in the subsequent chapters (3 8).

66 Comparison of procedures for the induction of SE High mortality is a major drawback in using the lithium/pilocarpine model (section 1.3). Mortality can be abated, however, by administering lower doses of pilocarpine. Glien et al., (2001) demonstrated that when compared to a single dose of pilocarpine (30 mg/kg, LDP protocol), repeated administration with lower doses of pilocarpine (10 mg/kg, RLDP protocol) every ½ hour until SE onset significantly reduced mortality rates in Wistar rats (see section 1.3.5). Whether the RLDP protocol can similarly reduce mortality in other commonly used rat strains has not been determined. Previous studies have demonstrated interstrain differences with respect to seizure intensity, mortality rates and SE-induced neuropathology in various models of epilepsy, which includes kindling (Loscher, 2002; Brandt et al., 2003a), and chemically-induced SE produced by kainic acid (Sanberg and Fibiger, 1979; Golden et al., 1995; Xu et al., 2004), pentylenetetrazol (Becker et al., 1997a) or pilocarpine (Hort et al., 2000; Xu et al., 2004). Because interstrain differences exist with other models of SE (section 3.1), the ability of the RLDP procedure to reduce mortality rates in Wistar rats may not similarly reduce mortality rates in other rat strains. Specific hypothesis: The ability of the RLDP procedure to lower mortality and increase the incidence of SE in Wistar rats will not generalize to other rat strains. Specific objectives: To compare the effect of the LDP and RLDP procedures on the induction of SE and on mortality in Wistar and LEH rats (see chapter 3). 2.2 The effect of recovery time on SE-induced neurodegeneration Presently, it remains unclear how SE-induced neurodegeneration in different brain regions evolves over time (section ). Previous studies have described early (<24 hrs) and delayed (>24 hrs) neuronal death following prolonged seizures, and suggested that they may be attributed to necrotic (Fujikawa et al., 1999; Fujikawa et al., 2000b; Fujikawa, 2005; Kotariya et al., 2010) and apoptotic (Narkilahti et al., 2003a; Weise et al., 2005) processes, respectively. Furthermore, early necrotic and delayed neuronal death with apoptotic features may occur in different brain regions (Sloviter et al., 1996; Lopez-Meraz et al., 2010), or even within the same neuronal population (e.g., pyramidal cell layer of the hippocampus) (Narkilahti et al., 2003a; Weise et al., 2005). Neurodegeneration has not been previously investigated with the RLDP procedure.

67 45 Specific hypothesis: SE induced by the RLDP procedure results in differential rates of neuronal death in different brain regions. Specific objectives: To determine in which brain regions neuronal cell death first appears, how neuronal loss evolves over time, and the extent of neuronal death that occurs early (<24hrs) or is delayed (>24 hrs) (see chapter 4). 2.3 The effect of tat-nr2b9c on SE-induced neuropathology and cognitive impairment NMDA receptors mediate seizure-induced neuronal death by excitotoxicity (section ). Binding of the NMDA receptor to the postsynaptic density protein-95 (PSD95) links the receptor to downstream signaling molecules (section 5.1). During SE, excessive synaptic release of glutamate activates NMDA receptors resulting in the influx of Ca 2+ which, in turn, can activate signaling molecules located proximal to the NMDA receptor ion channel by virtue of their association with PSD-95, and initiate neurotoxic downstream signaling cascades. Tat-NR2B9c was constructed to interfere with the binding of the NR2B subunit of NMDA receptors with the PDZ1 and PDZ2 domains of PSD-95 (described in Methods 5.2.3). Administration of tat- NR2B9c protected cultured neurons from excitotoxicity, and markedly reduced focal ischemic brain damage and preserved cognitive function in rats when applied before or up to 3 hours after insult (Aarts et al., 2002; Sun et al., 2008). Specific hypothesis: Tat-NR2B9c will provide neuroprotection and mitigate cognitive outcome in rats following SE. Specific objectives: To determine the effects of tat-nr2b9c on (1) the neuronal death induced by SE and (2) the behavioural/cognitive deficits associated with SE (see chapter 5). 2.4 The effect of SE on behavioural performance in tasks assessing anxiety, exploration and aggression SE leads to development of behavioural alterations in the EPM test, the open field test, and the hyperexcitability tests (section 1.6.6). When these changes first occur and whether or not they are long lasting remains unclear. Previous studies have shown that SE-induced neurodegeneration occurs within days following SE (section ). Because the majority of

68 46 neuronal death occurs within the first several days (section ), and is associated with behavioural morbidity (sections and 1.6.3), it is likely that behavioural alterations in anxiety, exploration and aggression similarly develop within the first several days after SE. Furthermore, since the bulk of SE-induced neuronal death precedes the development of epilepsy, SRSs are unlikely to contribute to these behaviours. Specific hypothesis: SE will result in early and long-lasting behavioural changes in tasks assessing anxiety, exploration and aggression and will not be affected by the development of SRSs. Specific objectives: To determine the effect of SE on the behaviour of rats in the elevated plus maze, open field, and four hyperexcitability tests at early and late times following SE and following the development of SRSs (see chapter 6). 2.5 The effect of SE on search strategy use in the Morris water maze Although it has been shown that SE results in impaired performance in the hidden platform version of the MWM task (section 1.6.7), the effect of SE on search strategy use has not been previously assessed. SE-induced neuronal death affects the neural systems that underlie spatial learning and behavioural strategies learning, both components which are critical in MWM performance (section ). Since behavioural strategies are acquired before spatial learning in rats during testing (section ), impaired search strategy use may underlie the impaired MWM performance in epileptic rats. Specific hypothesis: SE results in the impaired development of search strategies that in turn contributes to suboptimal performance in the MWM task. Specific objectives: To determine the effect of SE on the development of search strategies in the MWM task, and to assess the relationship between search strategies and performance in the MWM (see chapter 7).

69 47 Chapter 3 A comparison between Long-Evans hooded and Wistar rats related to the induction and severity of status epilepticus in the low-dose and repeated lowdose lithium/pilocarpine procedures 3.1 Introduction Treatment with pilocarpine, alone or in combination with lithium, is widely used to produce seizures in animal models of both status epilepticus (SE) and latent seizure development (Turski et al., 1989; Pitkänen and McIntosh, 2006). Pilocarpine when used alone requires high doses ( mg/kg i.p or s.c.), and is associated with high mortality rates and low rates of seizure induction (Turski et al., 1983b; Turski et al., 1989; Cavalheiro et al., 2001). Pretreatment of rats with lithium chloride permits the dose of pilocarpine to be decreased approximately 10-fold (30 mg/kg) and results in both lower mortality rates and higher rates of seizure induction (Clifford et al., 1987). The repeated administration of pilocarpine to lithium pretreated rats allows the dosage of pilocarpine to be even further reduced (10 mg/kg) and is effective in reducing mortality, while producing SE and chronic epilepsy in a high proportion of animals (Glien et al., 2001). Interstrain difference with respect to seizure intensity, mortality rates and SE-induced neuropathology has been reported in various models of epilepsy, including kindling (Loscher, 2002; Brandt et al., 2003a), and chemically-induced SE produced by kainic acid (Sanberg and Fibiger, 1979; Golden et al., 1995; Xu et al., 2004), pentylenetetrazol (Becker et al., 1997a) or pilocarpine (Hort et al., 2000; Xu et al., 2004). For instance, Wistar rats showed greater sensitivity and exhibited more reliable convulsant responses to kainic acid than Sprague Dawley and Long-Evans hooded (LEH) rats (Golden et al., 1991; Golden et al., 1995). Such strain differences, however, may not be consistent across models. Hort et al., (2000) reported that pilocarpine effects were more pronounced in LEH rats that exhibited a higher rate of SE induction, higher mortality rate, more severe neuropathology and a behavioural outcome worse than Wistar rats.

70 48 In view of the reported advantages of using repeated administration of low-doses of pilocarpine in combination with lithium for inducing SE in Wistar rats (Glein et al., 2001), it was important to establish whether this effect generalizes across other strains. Accordingly, our first objective was to compare the rates of seizure induction, intensity of behavioural seizures, and mortality rates of Wistar and LEH rats subjected to either the single-dose injection of 30 mg/kg pilocarpine, or the repeated injections of 10 mg/kg pilocarpine, in combination with lithium. The results identified several differences between the responses of the two strains to the SE-inducing protocols, and demonstrated that the repeated administration of low-doses of pilocarpine significantly reduced mortality rates in Wistar, but not in LEH, rats. The relationship between convulsive seizure intensity and neuropathology remains unclear. Although a few studies report a correlation between seizure severity and neuronal injury (Brandt et al., 2003a; Tilelli et al., 2005), others have failed to detect such a relationship (McKhann et al., 2003; Schauwecker et al., 2004; McLin and Steward, 2006; Lorenzana et al., 2007). Because seizure severity during SE varied with rat strain and seizure-induction protocol, we also examined the relationship between seizure intensity and neuropathology. 3.2 Methods Animals All procedures were approved by the University of Toronto Scarborough Animal Care Committee and were in accordance with the guidelines established by the Canadian Council on Animal Care. Male LEH and Wistar rats (Charles River, Montreal) weighing between 300 and 350 g were individually housed with free access to food and water for at least 7 days in 12 h light/dark cycles before experimental use. A total of 89 Wistar rats and 88 LEH rats were used in the analyses of the behavioural responses to convulsant treatment Induction of status epilepticus Two procedures were used for the induction of SE. In the low-dose lithium/pilocarpine ( LDP) procedure rats were pretreated with lithium chloride (3mEq/kg, i.p.) 24 hours before the injection of pilocarpine, and received methylatropine nitrate (10 mg/kg, i.p) 30 min prior to pilocarpine. LEH (n=42) or Wistar (n=25) rats were then administered an initial injection of pilocarpine (30 mg/kg, i.p.). If SE did not develop within 60 min, a second pilocarpine injection (15 mg/kg) was

71 49 administered. A total of 5 LEH and 2 Wistar rats required the second injection. Diazepam (4 mg/kg, i.p.) was administered at 1, 3 and 5 hours following the onset of SE to control the duration of seizure activity. For the repeated low-dose lithium/pilocarpine (RLDP) procedure, rats were pretreated with lithium chloride (3mEq/kg, i.p.) 24 hours before and methylatropine nitrate (10 mg/kg, i.p) 30 min before the initial injection of pilocarpine. Pilocarpine (10 mg/kg, i.p.) was then administered to LEH (n=46) or Wistar (n=64) rats every 30 min as described by Glien et al., (2001) until the rat experienced a generalized, class IV/V seizure (see section 3.2.3), since rats generally developed SE shortly thereafter. Animals that did not develop SE within 30 min of the first class IV/V seizure received additional pilocarpine injections at 30-min intervals up to a maximum of 6 injections. Diazepam (4 mg/kg, i.p.) was administered at 1, 3 and 5 hours following onset of SE to terminate seizure activity Monitoring of seizure activity The behavioural progression of pilocarpine-induced seizures was assessed using a modified Racine scale (1972) as described previously in Cammisuli et al., (1997) and in Veliskova (2006). Seizure activity was recorded as: stage I, staring with mouth clonus; stage II, head nodding, automatisms (e.g., scratching, sniffing orientation); stage III, unilateral forelimb clonus; stage IV, bilateral forelimb clonus (rearing); stage V, forelimb clonus with rearing and one fall; stage VI, forelimb clonus with rearing and multiple successive falls; stage VII, tonic/clonic seizures (e.g., running and jumping). Animals were continuously monitored following the first injection of pilocarpine, and the maximum stage of seizure activity occurring in each 5 minute interval following SE onset was analyzed Post-seizure animal care: Immediately following the first injection of diazepam to terminate seizures, animals received 5 ml 0.9% saline (3ml i.p. and 2ml s.c). Additional saline (5ml i.p) was given on the morning and evening of the next day (Glien et al., 2001). Starting on the second day after SE, animals were tube-fed with softened rat chow mixed with applesauce for 3 days on average. Softened rat chow was also provided in dishes until the rats commenced to eat hard pellets.

72 Histology and Stereological analysis: The extent of neuronal injury after SE was assessed by staining brain sections with NeuN antibodies, a specific neuronal marker (see section 4.2.5). Because of high mortality rates in LEH rats, the comparison of neuropathology between rat strains was conducted at 24 hours after SE induced with the RLDP protocol. In contrast, comparison of neuropathology between the LDP and RLDP procedures was assessed in Wistar rats at 3 months, subsequent to behavioural tests conducted for a separate study. Animals were anaesthetized with a mixture of xyaline (26 mg/kg i.p.; CDMV, Saint-Hyacinthe, Quebec) and ketamine (174 mg/kg i.p., CDMV, Saint- Hyacinthe, Quebec), and sequentially perfused transcardially with 90 ml of 0.1 M phosphatebuffered saline, ph 7.4 (PBS), followed by 400 to 500 ml of 4% (w/v) paraformaldehyde, ph 7.4 in PBS. Brains were removed and left overnight at 4 C in the paraformaldehyde solution. The following day, brains were soaked for cryoprotection in 30% (w/v) sucrose in PBS until they sank at room temperature. Next, brains were frozen in 2-methylbutane at -35 C, and stored at - 80 C until further use. Forty µm coronal sections were prepared with a freezing microtome. Sections were placed in 24-well culture plates containing antifreeze solution (50 mm of ph 7.4 phosphate buffer, 30% (v/v) ethylene glycol, 15% (w/v) glucose), and stored at -20 C. For each animal, 3 coronal brain sections containing the dorsal hippocampus were selected between Bregma -3.2 mm through mm (Paxinos and Watsons rat brain atlas, 5 th edition), with the first section presenting all hippocampal subfields, exhibiting only the dorsal portion of the lateral ventricle, and containing the most ventral portions of the capsular division (CeC) and lateral division (CeL) of the central amygdala. The subsequent sections were selected ventrally at 240 µm intervals, and exhibited the lateral ventricle extending to the ventral portion of the brain, replacing the structures CeC and CeL detected in the initial section. Section were rinsed in PBS (3 x 5 min washes), reacted overnight at 4 C with NeuN antibody (1:1000; Chemicon, Billerica, MA, USA) in 0.2% (v/v) goat serum, 0.3% (v/v) Triton X-100 in PBS followed by 2 hours at room temperature with Cy3 conjugated secondary antibody (1:200; Chemicon, Billerica, MA, USA) in 0.2% (v/v) goat serum, 0.3% (v/v) Triton X-100 in PBS. Neurons were imaged using a Zeiss LSM510 Laser Scanning Confocal microscope equipped with a 40x/1.3 oil-immersion objective lens, and neuronal density determined using the unbiased optical dissector technique as described by West and Gunderson, Briefly, three counting

73 51 frames (120 X 60 µm) were positioned in the CA1, CA3, hilus, and posterior piriform cortex (PPC), two counting frames were positioned in the CA4 region, and 1 counting frame was placed in CA2 of the left brain hemisphere as shown in Figure 3.1, A-C. Right brain structures were visually inspected and found to be comparable to the left side. Neurons were identified as NeuN-positive cells that contained a relatively large (>8µm) soma (Shi et al., 2004). Chromophilic somas contained within each counting frame, or touching the inclusion borders of the frame (upper and right borders) were counted (West and Gunderson, 1990). The dissector height equivalent to the known tissue height prior to staining (40 µm) was used in all calculations (Hatton and Von Bartheld, 1999), and upper or lower exclusion borders in the z plane were excluded as previous studies demonstrated that no significant variation in neuronal density occur whether such borders were used or not (Harding et al., 1994; Gardella et al., 2003; Azcoitia et al., 2005). Unless otherwise specified, cell densities for individual animals represent the average densities of all counting frames in a particular region for 3 brain sections. All results are expressed as neurons per mm 3.

74 52 Figure 3.1 Placement of counting frames within the hippocampus, hilus and piriform cortex. Representative images of NeuN stained coronal sections of a sham animal, depicting the left dorsal hippocampus (A), hilus (B) and posterior piriform cortex (PPC) (C). Boxes represent placement of counting frames in the pyramidal cell layer (pyr) of the CA1 (1a-c), CA2 (2a), CA3 (3a-c) and CA4 (4a-b), in the hilus (5a-c), or also referred to as the polymorph layer of the dentate gyrus (PoDG), and in layer II of the PPC (a-c).

75 53

76 Drugs: Pilocarpine, lithium chloride and methylatropine nitrate were purchased from Sigma (St Louis, Missouri) and dissolved in 0.9% (w/v) saline prior to administration. Diazepam was purchased from CDMV (Saint-Hyacinthe, Quebec) and used as the commercial solution (5 mg/ml) Statistical Analysis: Analysis of variance (ANOVA) and chi-square analysis were performed using Statistica 9.0 software. Non-parametric testing was performed using GraphPad Prism 5 software. Significance was set at a p-value of 0.05 or less. 3.3 Results Induction of SE in Wistars and Long Evans Hooded rats We initially compared the effectiveness of the RLDP and LDP procedures in inducing SE in Wistar and LEH rats. SE induction rates between groups were assessed using chi-square analysis. The number of pilocarpine injections administered in the RLDP protocol and the latency to SE onset were assessed by the Student s t-test. The results are summarized in Table 3.1, and indicate that the LEH rats are more sensitive to the convulsive effects of pilocarpine compared to Wistar rats. The average number of pilocarpine injections required to induce SE using the RLDP procedure was significantly lower for LEH than for Wistar rats. With the LDP procedure, Wistar rats had a significantly longer latency to SE development than the LEH rats. Although both the RLDP and LDP procedures resulted in similar rates of SE induction within strains (LEH: RLDP - 80% induction, LDP 88%; Wistars: RLDP 59%, LDP 68%), a greater percentage of LEH rats, as compared to Wistar rats, entered SE with both procedures (see Table 3.1) The effect of SE on mortality Mortality rates between groups were assessed using chi-square analysis. Table 3.1 summarizes mortality data as a function of strain and protocol. LEH rats exhibited a higher rate of mortality than Wistar rats with both the LDP and RLDP induction protocols. Wistars showed a significant reduction in mortality from 41% with the LDP protocol to 16% with the RLDP procedure.

77 55 Although more Long Evans rats died following the LDP procedure than the RLDP procedure (80% as compared to 65%), this difference was not significant (p = 0.09). Table 3.1: Comparison of rat strain and SE-inducing protocols between SE induction and mortality rates at 3 days following SE. Treatment Total # of rats per group # of pilocarpine injections for RLDP protocol 1 Latency to SE onset for LDP protocol 2 SE Survived 4 Died 4 Induction 3 LEH RLDP ± (80%) (65%) LEH LDP ± 16 min 37 (88%) 7 30 (80%) Wistar RLDP Wistar LDP ± 1.4 * (59%)* 32 6 (16%)*^ ± 16 min* 17 (68%)* 10 7 (41%)* 1. Number of repeated low-dose pilocarpine injections (10 mg/kg) administered with the RLDP protocol. * Wistars required a greater # of pilocarpine injections compared to LEH rats (p<0.05). 2. Latency to SE onset in the LDP protocol (average ± SD). * Wistars had longer latency to SE onset compared to LEH rats (p<0.05). 3. Number of animals that entered SE for 60 min. Numbers in () represent % of animal that developed SE. * Strain difference in SE induction rates detected across LDP and RLDP protocols (p<0.05). 4. Number of animals that survived or died within 3 days following SE. Numbers in ( ) represent the % of animals that died. * Reduced mortality rates detected in Wistar compared to LEH rats across LDP and RLDP protocols (p<0.05). ^ Wistar RLDP has a lower mortality rate than Wistar LDP (p<0.05). Data analyzed by Chi-square analysis or Student s t-test Severity of seizures in LEH and Wistar rats We next compared the effect of SE induction protocol and rat strain on the intensity of behavioural seizures during 60 min of SE (see Methods 3.2.3). Because behavioural seizures was assessed using a I- to VII-grade ordinal scale, data from the initial 10 min of SE and from the subsequent 50 min of SE was first analyzed using the non-parametric Kruskall-Wallis

78 56 ANOVA by ranks, followed by the Dunn s test to determine individual group differences. With both SE induction protocols, the severity of seizures declined during the initial 10 min of SE, with initial seizures scores of 4 to 5 decreasing to 2.5 to 3.5 over time for both LEH and Wistar rats (Figure 3.2). No group differences in seizure severity were detected within the initial 10 min of SE (Fig 3.2, black bars). During the subsequent 50 min of SE, LEH rats exhibited significantly more severe seizure activity than Wistar rats with both procedures (Figure 3.2, grey bars; p<0.05). Furthermore, Wistar rats treated with the RLDP protocol consistently exhibited significantly less severe convulsive seizures than Wistar rats treated with the LDP protocol, or LEH rats treated with either seizure-inducing protocol (Figure 3.2 B, grey bars; p<0.05).

79 57 Figure 3.2 Comparison of behavioural seizure activity between rat strain and SE-inducing protocol. (A) Average seizure activity was determined for the initial 10 min (black bars) and for the subsequent 50 min (grey bars) of SE as described in Methods (mean ± SEM). * Behavioural seizure activity less severe than the corresponding 10 min value (p<0.05). ** Behavioural seizure activity less severe than the values for LEH rats treated with either the LDP or RLDP procedures, and Wistar rats treated with the LDP procedure (p<0.05). *** Behavioural seizure activity less severe than the value for LEH rats with the LDP procedure (p<0.05). LEH RLDP (n=37), LEH LDP (n=37), Wistar RLDP (n=38), Wistar LDP (n=17). Data analyzed using the non-parametric Kruskall-Wallis ANOVA by ranks, followed by the Dunn s test to determine individual group differences.

80 58

81 SE-induced neuropathology in LEH and Wistar rats following SE induced with the RLDP procedure The effect of the RLDP procedure on SE-induced neuronal death was compared between LEH and Wistar rats to determine if there were any strain differences in neuropathology. Because of difficulty in keeping LEH rats alive for prolonged recovery times, neuron density was assessed 24 hours following termination of SE. Data were first analyzed using one-way ANOVA, followed by the Newman-Keuls post-hoc test to detect group differences. As illustrated in Figure 3.3 (A, B), SE induced with the RLDP procedure resulted in significant neuron loss in the CA1, CA3, and CA4 pyramidal cell layers in both LEH and Wistar rats. Neuronal damage was more severe in LEH rats for each of these regions. Extensive neurodegeneration was observed in the PPC in both strains, whereas neuron loss in the hilus was only observed in Wistar rats. No neuron loss was detected within the CA2 subfield with either rat strain Comparison of SE-induced neuropathology resulting from the LDP and RLDP protocols in Wistar rats Since the RLDP protocol reduced the intensity of convulsive seizures in Wistar rats compared to the LDP protocol, the extent of SE-induced neuronal loss resulting from each procedure was examined. Brain sections were stained with anti-neun antibodies and quantified as in Methods (section 3.2.5) 3 months after SE, subsequent to behavioural testing conducted as a separate study. Data were first analyzed using one-way ANOVA, followed by the Newman-Keuls posthoc test to detect group differences. As illustrated in Figure 3.4 (A, B), significant neuron loss was detected in the CA1, CA3, CA4, hilus and PPC, but not in the CA2. No difference in neuron densities between LDP and RLDP protocols was detected.

82 60 Figure 3.3 Comparison of neuron cell densities in the hippocampus and piriform cortex of LEH and Wistar rats following SE. SE was induced with the RLDP procedure, terminated after 60 minutes, and animals sacrificed 24 hours later. (A) Confocal micrographs (400X) of NeuN stained cells in the pyramidal cell layer of the CA1-CA4 subfields of the hippocampus, hilus, and in layer II of the PPC. Scale bar = 20 µm. (B) Stereological analysis of NeuNpositive cells was determined as in Methods (section 3.2.5), and neuron densities for individual animals plotted as open circles. Mean cell density for each group is represented as a horizontal bar. * Reduced neuron density in animals after SE compared to sham animals (p<0.05). ** Reduced neuron density in LEH SE compared to Wistar SE animals (p<0.05). Data analyzed by on-way ANOVA followed by Newman-Keuls multiple comparison procedure.

83 61

84 62 Figure 3.4 Comparison of neuronal cell densities in the hippocampus and piriform cortex of Wistar rats following SE. SE induced with the RLDP or LDP procedure was terminated after 60 minutes, and animals sacrificed 3 months later. (A) Confocal micrographs (400X) of NeuN stained cells in the pyramidal cell layer of the CA1-CA4 subfields of the hippocampus, hilus, and in layer II of the PPC. Scale bar = 20 µm. (B) Stereological analysis of NeuNpositive cells was determined as in Methods (section 3.2.5), and neuron densities for individual animals plotted as open circles. Mean cell density for each group is represented by a horizontal bar. * Reduced neuron density in animals after SE compared to sham animals (p<0.05). Data analyzed by one-way ANOVA followed by Newman-Keuls multiple comparison procedure.

85 63

86 Discussion High mortality rates have been a major drawback in chemoconvulsant models of seizure development. Previous studies have suggested two possible means of reducing mortality: modification of the SE induction procedure and/or selection of rat strains (Hellier et al., 1998; Hort et al., 2000; Glien et al., 2001). In the lithium/pilocarpine model, Glien et al., (2001) demonstrated that repeated low-doses of pilocarpine (10 mg/kg) increased survival rates in Wistar rats. However, the effectiveness of this procedure in other rat strains was not determined. We therefore compared the RLDP protocol with the LDP protocol in LEH and Wistar rats, and identified several interstrain differences in response to the two seizure-inducing procedures. The main findings were: 1) Wistar rats showed greater sensitivity to the convulsive effects of pilocarpine and exhibited less severe status in the LDP and RLDP procedures than LEH rats, 2) the RLDP protocol reduced mortality and severity of status in the Wistar rats when compared to the LDP protocol, but had no significant effect in LEH rats, 3) with the RLDP procedure, LEH rats exhibited more severe SE-induced damage in CA1, CA3 and CA4 than Wistar rats, while hilar neuron loss was only detected in Wistar rats, and 4) the differences in seizure severity between the LDP and RLDP procedures in Wistar rats did not result in detectable differences in neuronal death Differential effects of induction procedure in LEH and Wistar rats Differences in the responses of LEH and Wistar rats with respect to seizure susceptibility and mortality were found. Wistar rats were more resistant to the convulsive effects of pilocarpine than LEH rats, requiring a greater number of pilocarpine injections to induce SE with the RLDP protocol, displaying a longer latency to SE development with the LDP protocol, and exhibiting a lower rate of SE induction with both seizure-inducing procedures. Wistar rats also exhibited less severe behavioural seizures and lower mortality rates when compared to LEH rats with the LDP and RLDP procedures. Strain differences in response to pilocarpine have previously been reported. Consistent with our results, Hort et al., (2000) reported that LEH rats exhibit more severe convulsive SE and sustained higher mortality rates when compared to Wistars following a single high-dose injection of pilocarpine ( mg/kg). In contrast, Xu et al., (2004) failed to detect any interstrain differences in status severity or mortality after the administration of highdoses of pilocarpine (300 mg/kg). Since the length of SE affects mortality (Curia et al., 2008),

87 65 progression of behavioural seizures (Turski et al., 1983b) and ensuing neuropathology (Fujikawa, 1996), the shorter duration of SE (approximately 20 min) used by Xu et al., (2004) may not have been long enough to detect interstrain differences reported after 3 hrs of SE by Hort et al., (2000), or after 1 hr of SE in the present study. The present findings also indicate that the beneficial effects of the RLDP protocol are strain dependent. Compared to the LDP procedure, the RLDP protocol reduced mortality and severity of SE in Wistar rats, but had no significant effect in LEH rats. Presently, the molecular or genetic basis of the difference in response to pilocarpine of LEH and Wistar rats is not known Comparison of SE-induced neuropathology in LEH and Wistar rats following SE induction with the RLDP procedure Previous studies using electrical stimulation of the basolateral amygdala to induce SE have demonstrated that the severity of SE affects the subsequent extent of neuronal loss (Brandt et al., 2003a; Tilelli et al., 2005). In the present study, we investigated whether a similar relationship can be detected in the lithium/pilocarpine model. Because LEH rats experienced more severe seizures than Wistars following SE induced with the RLDP protocol, we compared SE-induced neuropathology between these strains. Since LEH rats exhibited high mortality rates following pilocarpine treatment, quantitative stereological analysis was conducted 24 hrs after SE. Consistent with the occurrence of more severe seizures, LEH rats exhibited greater pyramidal cell loss in the CA1, CA3 and CA4 regions of the hippocampus than Wistar rats. In contrast, however, the loss of hilar neurons only occurred in Wistar rats. Both strains exhibited similar and severe neurodegeneration within layer II of the piriform cortex and preservation of pyramidal cells in CA2. As described in the subsequent chapter, maximum pyramidal cell loss in Wistar rats occurs in the CA3, CA4 and piriform cortex within 24 hours after SE; the majority of CA1 pyramidal cell loss, however, is delayed, with only a 29% decrease by 24 hours, and an 80% decrease within 3 days. In contrast to this delay in Wistar rats, CA1 pyramidal cells in LEH rats had decreased by 80% within 24 hours, indicating that the initial loss of CA1 pyramidal cells is more rapid in the LEH strain. In general agreement with our results, Hort et al., (2000) also found less pathology in Wistar rats than in LEH rats. The different pattern of neuronal injury between the two strains may be caused by specific factors that affect the phenotype of cell death. For instance, the latency to onset of SE and intensity of convulsive seizures have been reported to be correlated with the type of cell death observed; shorter latencies or more severe seizures

88 66 correlated with necrotic cell death, whereas longer latencies or milder seizures resulted in delayed cell death with apoptotic features (Kondratyev and Gale, 2004; Tokuhara et al., 2007). Although the phenotype of cell death in the present study cannot be confirmed without ultrastructural analysis, the rapid pyramidal cell loss in the CA1 of LEH rats may be attributed to the more severe seizure intensity and shorter latency to SE development compared to Wistar rats Comparison of the effect of the LDP and RLDP protocols on SE-induced neurodegeneration in Wistar rats The differences in neuronal death between LEH (more severe) and Wistar (less severe) rats correlated with the relative severity of SE exhibited by these two strains. The relationship between seizure severity and pathology suggested by these results is, however, confounded by possible differences in genetic influences between the strains. To remove the possible influence of strain difference in assessing the relationship between seizure severity and neuropathology, we exploited our finding that Wistar rats treated with the RLDP protocol experienced less severe seizures than Wistars treated with the LDP protocol. The extent of neurodegeneration under both conditions was examined 3 months after SE. In the subsequent chapter, we demonstrated that by 3 months, maximum cell loss occurs within the regions assessed and is unaffected by development of SRSs. Despite the differences in seizure intensities and mortality rates between the two procedures, the pattern of neuron loss in the hippocampus and piriform cortex was similar in both cases. Although some studies have demonstrated a link between seizure severity and neuronal death, others have failed to detect this relationship. For example, in comparing five different strains of mice, McKhann et al., (2003) failed to detect a relationship of kainic acid susceptibility, behavioural seizure severity, and loss of hippocampal pyramidal cells. Subsequent studies confirmed these results, indicating that seizure susceptibility, convulsive seizure intensity, and SE-induced neuropathology are controlled independently by genetic factors (McKhann et al., 2003; Schauwecker et al., 2004; McLin and Steward, 2006; Lorenzana et al., 2007). In contrast, studies that involved electrical stimulation in the left basolateral amygdala of rats to induce different types of SE supported a positive correlation between behavioural seizure intensity and severity of neurodegeneration in the hippocampus and piriform cortex (Brandt et al., 2003; Tilelli et al., 2005). Although it is possible that the difference in seizure severity between the LDP and RLDP procedures in the present study was insufficient to result in detectable

89 67 differences in neuropathology, our findings support the conclusion that the extent of neuropathology is not directly related to seizure severity Conclusion In summary, the present study identifies several differences between the response of LEH and Wistar rats to SE-inducing protocols, and demonstrates that the repeated administration of lowdoses of pilocarpine is most effective in reducing mortality in Wistar rats. Furthermore, in spite of eliciting less severe seizures during SE, the repeated low-dose injections of pilocarpine did not reduce the extent of SE-induced neurodegeneration in the hippocampus and piriform cortex. Based on these findings, we investigated SE-induced neuropathology and the effects of neuroprotective peptides in Wistar rats treated with the RLDP protocol.

90 68 Chapter 4 Temporal profile of neuronal death following lithium/pilocarpine-induced status epilepticus 4.1 Introduction Status epilepticus (SE) is a prolonged seizure condition representing a major medical and neurological emergency (Delorenzo, 2006). The incidence rates of SE in epidemiological studies range from 9.9 to 41 per 100,000 population annually (Chin et al., 2004; Rosenow et al., 2007). SE is associated with substantial mortality and morbidity (Lowenstein, 1999), including epileptogenesis (Hesdorffer et al., 1998) and cognitive decline (Shorvon, 1994; Devinsky, 2004a). Data from humans and post-status epilepticus animal models suggest that both the risk of epilepsy and the severity of cognitive impairments are associated with brain damage caused by prolonged seizure activity (Shorvon, 1994; Lowenstein, 1999; Fountain, 2000; Devinsky, 2004b; Delorenzo, 2006; Helmstaedter, 2007). Histological studies in experimental models indicate that 30 to 60 minutes of SE is required to initiate neurodegeneration (Nevander et al., 1985; Fujikawa, 1996). In 1983, Turski and colleagues described a rodent model of seizure-induced brain damage produced by the systemic administration of pilocarpine, a muscarinic cholinergic agonist. The same year Honchar et al., (1983) reported that if rats were pretreated with lithium-chloride, seizure-induced brain damage could be produced with a substantially lower dose of pilocarpine. In humans, SE results in neurodegeneration in the hippocampus, neocortex, piriform and entorhinal cortices, septum, amygdaloid and thalamic structures (Fujikawa et al., 2000a). This pattern of neuronal loss is replicated in the pilocarpine and lithium/pilocarpine models of SE, and is generalized in other post-status epilepticus rodent models including kainic acid, bicuculline, picrotoxic and pentetrazole (Ben-Ari et al., 1981; Lothman and Collins, 1981; Ben-Ari, 1985; Turski et al., 1985). Whereas others have described the extent and severity of SE-induced neurodegeneration (Turski et al., 1983b; Honchar et al., 1983; Fujikawa, 1996; Motte et al., 1998; Covolan and Mello, 2000;

91 69 Peredery et al., 2000; Poirier et al., 2000), a detailed analysis comparing the temporal pattern of neuronal death in different brain regions has not been reported. Previous studies using the lithium or lithium/pilocarpine models have been limited by the semi-quantitative assessment of neuronal damage, and/or by the restricted recovery times examined (see appendix I, Table A1-1). For instance, times are restricted to less than 72 hours after SE (Fujikawa, 1996; Covolan and Mello, 2000) or times examined are spaced far apart (Motte et al., 1998; Peredery et al., 2000; Poirier et al., 2000). SE activates different cell death mechanisms that result in early necrotic ( 1 day after SE) (Fujikawa et al., 1999; Araújo et al., 2008; Wang et al., 2008) and delayed apoptotic (3 to 7 days after SE) (Narkilahti et al., 2003a; Weise et al., 2005; Wang et al., 2008) degenerative morphologies. These different cell-death morphologies can occur in different brain regions (Sloviter et al., 1996; Lopez-Meraz et al., 2010) or within the same population of neurons (e.g. pyramidal cell layer of the hippocampus) (Narkilahti et al., 2003a; Weise et al., 2005). Thus, we hypothesized that regions in the hippocampus, thalamus, amygdala and piriform cortex would exhibit different temporal profiles of neuronal death following SE. In the present study, our objective was to provide a comparative, quantitative analysis of SEinduced neurodegeneration in several brain structures. Specifically, we sought to determine: (1) in which brain regions neuronal death first appeared, (2) how neuronal loss evolved over time, and (3) the extent of brain damage attributed to early (<24hrs) and to delayed (>24 hrs) neuronal loss. We therefore analyzed neurodegeneration in 19 brain regions following 60 minutes of SE induced by the repeated low-dose lithium/pilocarpine (RLDP) procedure (Glien et al., 2001). Because neurodegeneration has not been previously investigated with the RLDP procedure, we also sought to determine the effect of SE induced with this procedure on neuronal loss and to compare this to other models. Neuronal death was assessed by stereological analysis of neurons (stained for the neuronal specific marker [NeuN]) at times ranging from 1 hr to 90 days after SE. The results demonstrate that depending upon the brain region, neuronal death occurred as early as 1 hour following SE, and that different brain regions exhibit differential rates of neuronal loss, with the majority of SE-induced neuronal death present by 24 hours.

92 Methods Animals All procedures were approved by the University of Toronto Animal Care Committee and were in accordance with the guidelines established by the Canadian Council on Animal Care. Male Wistar rats (Charles River Laboratories, Sherbrooke, Quebec, Canada) weighing between 300 and 350 g were individually housed with free access to food and water for at least 7 days in 12 h light/dark cycles (i.e., lights on between 7 a.m. and 7 p.m.) before experimental use. A total of 64 Wistar rats were used in the present study Induction of status epilepticus SE was induced using the RLDP procedure exactly as described in Methods section Duration of seizure activity was controlled by the administration of diazepam (4 mg/kg, i.p.) 1, 3 and 5 hours following the onset of SE and animals were allowed to survive for various times up to 3 months following SE. Sham animals underwent identical procedures but received saline in place of pilocarpine, and did not exhibit any seizures Post-seizure care Following the termination of SE, animals were kept in a quiet room for 3 days. Immediately following the first injection of diazepam, animals received 5 ml 0.9% saline (3ml i.p. and 2ml s.c.), and this was repeated in the morning and evening of the following day (5ml i.p.) as described by Glien et al., (2001). Starting on the second day after SE, animals were tube-fed softened rat chow mixed with applesauce for 3 days on average. Softened rat chow was also provided in dishes until the rats commenced to eat hard pellets Detection of SRSs Glein et al., (2001) previously demonstrated that the average latency onset to SRSs in rats treated with the RLDP protocol was 6 weeks. In the present study, two methods for recording of spontaneous seizures were used. First, all seizures observed during handling, or by direct observation of the rats in their home cages were noted. The rats were continuously monitored during the first 3 days following SE in their home cages, and checked on daily thereafter. Second, a group of 8 rats was videotaped between 6 and 8 weeks after SE to confirm that rats

93 71 used for histological analyses at the 3 months recovery time were epileptic. The group of 8 rats was videotaped for 4 hours per day, 6 days each week. Because the frequency of SRSs in rats after pilocarpine-induced SE is much higher during the light (diurnal) compared to the dark (nocturnal) period (Arida et al., 1999; Goffin et al., 2007), all recordings for spontaneous seizures were done during the light period (7 a.m. 6 p.m.). All 4-hr video recordings were analyzed for seizures by using the fast-forward speed (six times the normal speed) of the video recorder. Once a seizure-like activity was observed, the videotape was rewound to the beginning of the behavior and examined at real-time speed. Since most SRSs following pilocarpineinduced SE are generalized (Cavalheiro et al., 2006), only the occurrence of class 4/5 behavioural seizures was recorded. A rat was considered epileptic after exhibiting one or more SRSs Histology and Stereological analysis The extent of neuronal injury at time points ranging from 1 hour to 3 months after SE was compared by staining brain sections with NeuN antibodies, a specific neuronal marker. NeuN antibody recognizes the DNA-binding, neuron-specific protein NeuN, which is present in most neuronal cell types of vertebrates tested (i.e., rats, humans, chicks, salamander) (Mullen et al., 1992; Wolf et al., 1996). Developmentally, immunoreactivity of NeuN antibodies appears initially after neurons become post-mitotic, and remains present throughout the life of these cells (Mullen et al., 1992; Wolf et al., 1996). The immunohistochemical staining is primarily localized in the nucleus of the neurons with lighter staining in the cytoplasm. The strong nuclear staining suggests a nuclear regulatory protein function; however, no evidence currently exists as to whether the NeuN protein antigen has a function in the cytoplasm, or whether it is simply synthesized there before being transported into the nucleus (Mullen et al., 1992). Animals were anaesthetized with a mixture of xyline (26 mg/kg i.p., Rompun, CDMV, Saint-Hyacinthe, Quebec, Canada) and ketamine (174 mg/kg i.p., Ketalar, CDMV, Saint-Hyacinthe, Quebec, Canada), and perfused transcardially with 90 ml of 0.1 M phosphate-buffered saline, ph 7.4 (PBS) followed by ml of 4% (w/v) paraformaldehyde in PBS. Brains were removed and left overnight at 4 C in the paraformaldehyde solution. The following day, brains were soaked for cryoprotection in 30% (w/v) sucrose in PBS until they sank at room temperature. Next, brains were frozen in 2-methylbutane at -35 C and stored at -80 C. Forty µm coronal sections were prepared with a freezing microtome. Sections were placed in 24-well culture

94 72 plates containing antifreeze solution (0.05 M sodium phosphate buffer, ph 7.4, 30% (w/v) ethylene glycol, 15% (w/v) glucose), and stored at -20 C. For each animal, 3 coronal brain sections containing the dorsal hippocampus were selected between Bregma -3.2 mm through mm (Paxinos and Watsons rat brain atlas, 5 th edition), with the first section presenting all hippocampal subfields, exhibiting only the dorsal portion of the lateral ventricle, and containing the most ventral portions of the capsular division (CeC) and lateral division (CeL) of the central amygdala. The subsequent sections were selected ventrally at 240 µm intervals, and exhibited the lateral ventricle extending to the ventral portion of the brain, replacing the structures CeC and CeL detected in the initial section. Coronal brain section containing the ventral hippocampus were selected between Bregma mm through 5.4, with the first section clearly presenting a continuous pyramidal cell layer connecting the dorsal and ventral CA1 and CA3 regions. Section were rinsed in PBS (3 x 5 min washes), reacted overnight at 4 C with NeuN antibody (1:1000; Chemicon, Billerica, MA, USA) in 0.2% (v/v) goat serum, 0.3% (v/v) Triton X-100 in PBS followed by 2 hours at room temperature with Cy3 conjugated secondary antibody (1:200; Chemicon, Billerica, MA, USA) in 0.2% (v/v) goat serum, 0.3% (v/v) Triton X-100 in PBS. Neurons were imaged using a Zeiss LSM510 Laser Scanning Confocal microscope (Carl Zeiss) equipped with a 40x/1.3 oil-immersion objective lens. Digital images were acquired with the Zeiss LSM 510 software. Neuronal density was determined using the unbiased optical dissector technique as described by West and Gundersen (1990). As illustrated in Figure 4.1, images were captured in the left brain hemisphere of specific regions within the hippocampus, thalamus, amygdala and piriform cortex. In the dorsal hippocampus (Fig 4.1 A, B), images were taken in the pyramidal cell layer of CA1 (boxes 1a-c), CA2 (2a), CA3 (3a-c), and CA4 (4a-b) and in the polymorphic layer of the hilus (5a-f). In the ventral hippocampus (Fig 4.1 C), images were taken in the pyramidal cell layer of CA1 (6a-c) and CA3 (7a-c). For the thalamus, images were captured in the following regions (Figure 4.1 D): laterodorsal thalamic nucleus, dorsomedial part (LDDM, 8a-b); laterodorsal thalamic nucleus, ventrolateral part (LDVL, 9a-b); posterior thalamic nuclei (Po, 10a-b); ventral posteromedial thalamic nucleus (VPM, 11a-b); ventral posterolateral thalamic nucleus (VPL, 12a-b); reticular thalamus nucleus (Rt, 13a-b). Likewise, images were captured in the following regions of the amygdala (Figure 4.1 E): lateral amygdaloid nucleus, dorsolateral part (LaDL, 14a-b); lateral amygdaloid nucleus, ventrolateral

95 73 part (LaVM, 15a-b); basolateral amygdaloid nucleus, posterior part (BLP, 16a-b); basomedial amygdaloid nucleus, posterior part (BMP, 17a-b); posteromedial cortical amygdaloid nucleus (PMCo, 18a-b). Finally, images were also taken within layer II of the posterior piriform cortex (Figure 4 E, 19a-c). Since the pyramidal cell layer of the CA1, CA2, CA3, and CA4 and layer II of the piriform cortex formed a thick row of densely packed neurons, a single counting frame (120 X 60 µm) was positioned within the images captured (depicted as green boxes in Figure 4.1). Two counting frames (120 X 60 µm) were positioned within the images captured for the thalamus, amygdala and hilus, since neurons were less numerous and uniformly scattered within these structures (depicted as small white boxes in Figure 4.1). Right brain structures were visually inspected and found to be comparable to the left side. Neurons were identified as NeuN-positive cells that contained a relatively large ( 8µm) soma (Shi et al., 2004). Chromophilic somas contained within each counting frame, or touching the inclusion borders of the frame (upper and right borders) were counted (West and Gunderson, 1990). The dissector height equivalent to the known tissue height prior to staining (40 µm) was used in all calculations (Hatton and Von Bartheld, 1999), and upper or lower exclusion borders in the z plane were excluded as previous studies demonstrated that no significant variation in neuronal density occur whether such borders were used or not (Harding et al., 1994; Gardella et al., 2003; Azcoitia et al., 2005). Unless otherwise specified, cell densities for individual animals represent the average densities of all counting frames in a particular region for 3 brain sections. All results are expressed as neurons per mm 3. To assess whether tissue shrinkage occurred at specific times after SE, the area size (expressed as mm 2 ) of the dorsal and ventral hippocampus, thalamus and amygdala in the left brain hemisphere was determined. Images were captured by the fluorescent microscope equipped with a 2.5x objective lens. The AxioVision LE 4.0 analysis software was used to measure area size by drawing an outline around the region of interest. As illustrated in Figure 4.2, the brain areas assessed were based on regional boundaries defined in the Paxinos and Watson s rat brain atlas, 5 th edition. For the dorsal (Figure 4.2A, grey region) and ventral (Figure 4.2B, grey region) hippocampus, an outline was drawn around the hippocampal pyramidal cell layer (pyr) and included the dentate gyrus (DG). The area size of the thalamus and nearby structures (Figure 4.2A, green region) included an outline encircling the perimeter of the lateral posterior thalamic nuclei (LPMR, LPLR), the reticular thalamic nucleus (Rt), the subthalamic nucleus (STh), the

96 74 medial tuberal nucleus (MTu), the dorsomedial hypothalamic nucleus (DMV, DMD), the central medial thalamic nucleus (CM), and the mediodorsal thalamic nucleus (MD). The area excluded the dorsal and ventral 3 rd ventricular spaces (d3v, 3V). Finally, for the area size of the amygdala and adjoining structures (Figure 4.2, blue region), an outline was traced starting at the rhinal fissure, and continued along the perimeter of the piriform cortex (Pir), the posteromedial cortical amygdaloid nucleus (PMCo), the medial amygdaloid nuclei (MePV, MePD), the bed nucleus of the stria terminalis, intraamygdaloid division (STIA), and the lateral amygdaloid nucleus (LaVM, LaDL). The area excluded the lateral ventricular space (LV). The area sizes of the brain structures assessed for individual animals represent the average area sizes of the corresponding structure for 3 brain sections (the same sections used for stereological analysis of neurons described above) Fluoro-Jade B staining For each animal, coronal brain sections were selected as described in section 4.2.5, and stained with Fluoro-Jade B (FJB). FJB is an anionic fluorochrome reported to selectively stain degenerating neurons (Poirier et al., 2000; Schmued and Hopkins, 2000). Briefly, brain sections were immersed in 100% ethanol for 5 min, followed by 2 min in 70% ethanol and two 1-minlong rinses in distilled water. Slides were then transferred to a solution of 0.06% potassium permanganate for 10 min, gently shaken, to minimize background staining. After two more rinses, slides were placed in FJB staining solution for 30 min at room temperature. The staining solution was prepared from a 0.01% stock solution of FJB (Histo-Chem Inc., Jefferson AR, USA) that was made by adding 10 mg of the dye powder to 100 ml of distilled water. To make up 100 ml of staining solution, 4 ml of stock solution was added to 96 ml of 0.1% acetic acid vehicle. This results in a final dye concentration of %. Following staining, the sections were rinsed three times with distilled water. The slides were dried, immersed in xylene, and mounted. Slides were examined on a Zeiss LSM510 Laser Scanning Confocal microscope (Carl Zeiss) equipped with an Argon laser. Digital images were acquired using the Zeiss LSM 510 software.

97 Statistical Analysis: Statistical analysis was performed using Statistica 6.0 software. Significant differences were determined using one-way analysis of variance (ANOVA). The Newman-Keuls post-hoc test was used to determine differences between treatment groups. Significance was set at a p-value of 0.05 or less. Moving average trendlines were drawn for the temporal profiles of neuronal densities of each brain region (shown in Figures 4.6, 4.9, and 4.12) using the Excel Microsoft 2007 software program. Moving average trendlines are used to smooth out fluctuations in data to show a trend (i.e., reduction in neuronal densities over a period of time) more clearly. In the treadlines, two data points were averaged, and the average value was used as a point in the line.

98 76 Figure 4.1: Placement of counting frames in the dorsal hippocampus (A): pyramidal cell layer of CA1 (boxes 1a-c), CA2 (2a), CA3 (3a-c), and CA4 (4a-b). Hilus (B): polymorphic layer of the hilus (5a-f). Ventral hippocampus (C): pyramidal cell layer of CA1 (6a-c) and CA3 (7a-c). Thalamus (D): laterodorsal thalamic nucleus, dorsomedial part (LDDM, 8a-b); laterodorsal thalamic nucleus, ventrolateral part (LDVL, 9a-b); posterior thalamic nuclei (Po, 10a-b); ventral posteromedial thalamic nucleus (VPM, 11a-b); ventral posterolateral thalamic nucleus (VPL, 12a-b); reticular thalamus nucleus (Rt, 13a-b). Amygdala (E): lateral amygdaloid nucleus, dorsolateral part (LaDL, 14a-b); lateral amygdaloid nucleus, ventrolateral part (LaVM, 15a-b); basolateral amygdaloid nucleus, posterior part (BLP, 16a-b); basomedial amygdaloid nucleus, posterior part (BMP, 17a-b); posteromedial cortical amygdaloid nucleus (PMCo, 18a-b). Piriform cortex (E): layer II of posterior piriform cortex (PPC, 19a-c). See Methods section for details on stereological analysis of neurons.

99 77

100 78 Figure 4.2: The area size of different brain regions assessed. A: Dorsal hippocampus (indicated by grey region), thalamus and adjoining structures (green region), amygdala and adjoining structures (blue region). Image depicted: Bregma mm (Paxinos and Watson, 2005). B: Ventral hippocampus (indicated by grey region). Image depicted: Bregma mm (Paxinos and Watson, 2005). Area size determined as described in Methods section

101 79

102 Results SE induction and survival rates: Sixty-four lithium-pretreated rats were administered repeated doses of pilocarpine (10 mg/kg), of which 44 (69%) developed SE. Eight rats died between recovery time points and were not used in the present study. Four rats were analyzed for each of the 9 recovery times. An additional 4 rats were used as sham animals Spontaneous seizures after lithium/pilocarpine induced SE A group of eight rats were videotaped between 6 and 8 weeks after SE to determine whether they were epileptic. All 8 of the rats exhibited one or more SRSs within this timeframe. No difference in the frequency of SRSs between the weeks assessed were found. The average frequency in the eight rats was 0.75 ± 0.3 seizures per 24 hours of recording during week 6, 1.5 ± 0.5 during week 7, and 0.9 ± 0.3 during week 8. Four of these animals randomly selected were later used for histological analyses at the 3 month recovery time. Although we cannot rule out the presence of SRSs at earlier times, no SRSs were noted during handling of the animals, or by direct observation of the animals in their home cages, during the first 2 weeks following SE Neuropathology following status epilepticus: Overview Because the hippocampal formation, thalamus, amygdala and piriform cortex are highly susceptible to SE-induced neurodegeneration (Honchar et al., 1983; Clifford et al., 1987), we concentrated our analyses on 19 regions within these structures. With the exception of hippocampal subfield CA2, significant neuronal loss was observed in all other regions assessed. The severity and temporal profile of neuronal loss for regions within each brain structure are described in detail below. Results for each brain structure assessed are presented as follows: (1) low-powered images of NeuN immunohistochemical staining providing an overall view of damage in each structure, (2) representative high-powered images depicting NeuN positive cells (neurons) that were quantified as in Methods (section 4.2.5), and (3) graphs displaying changes in neuron density, expressed as percent of controls, over time. With the exception of the amygdala at 90 days following SE (see section 4.3.5), no significant tissue shrinkage at times after SE occurred in the dorsal and ventral hippocampus, thalamus, or amygdala (Table 4.1),

103 81 permitting the direct comparison of neuron densities in SE and sham animals. Actual neuron densities are given in appendix II (Tables AII-1, AII-2 and AII-3). Table 4.1: The effect of SE on the area size (mm 2 ) of the hippocampus, thalamus and amygdala Recovery after SE 1 Dorsal hippocampus Ventral hippocampus Thalamus Amygdala Sham 3.55 ± ± ± ± hours 3.53 ± ± ± ± days 3.52 ± ± ± ± days 3.62 ± ± ± ± months 3.54 ± ± ± ± 0.15 * Area size of the left brain hemisphere in various brain regions determined at specific times (1) following SE (see Methods 4.2.5). Data expressed as mm 2 ± SEM. * Area size of the amygdala decreased between 14 days and 3 months following SE. No change in area size of the dorsal and ventral hippocampus and thalamus was detected. A total of 4 animals were analyzed at each recovery time.

104 SE-induced neurodegeneration in the hippocampus: The effect of SE on the hippocampus is shown in Figures Neuronal edema was detected by 3 hours in CA1, CA3 and CA4 (Fig 4.4, indicated by solid arrows). Enlargement of the 3 rd and lateral ventricles was visible by 3 months after SE (Fig 4.3B and D, indicated by φ). SE resulted in the loss of neurons in all hippocampal regions except CA2 (Fig 4.4). Likewise, with the exception of 3 animals that exhibited partial or complete destruction of the suprapyramidal (or enclosed) blade of the granule cell layer (<8% of SE animals), no loss of dentate granule cells, defined indirectly by a shortening of the total length of the dentate gyrus (Bertram et al., 1990), was detected (Fig 4.5). The three animals that exhibited damage within the dentate gyrus were assessed at the 24-hours, 7 days, and 3 months recovery times, respectively. Quantification of NeuN-stained neurons showed that the temporal profile of neuronal density varied between hippocampal regions (Fig 4.6). SE-induced loss of pyramidal cells in the dorsal CA1 occurred within two distinct times following SE: a 28% decrease in cell density occurred within 1 hour, followed by a further 53% decrease in cell density between 1 and 3 days (Fig 4.6A). By 3 days after SE, 27 ± 8% (avg ± SEM) of pyramidal cells remained in dorsal CA1, with no further decrease after this time. In contrast to the rapid initial loss of pyramidal cells in the dorsal CA1, neuronal loss in ventral CA1 was delayed for at least 12 hrs, after which time the density of neurons decreased to a minimum of 33 ± 4% of shams by 7 days (Fig 4.6A). Dorsal and ventral CA1 were the most severely damaged hippocampal regions, while less extensive damage occurred in CA3, CA4 and hilus. Although neuronal loss in dorsal CA3 was initially slower than in ventral CA3, the ultimate severity of damage in these regions was not significantly different, with 57 ± 10% and 42 ± 2% of pyramidal cells remaining after 3 months, respectively (Fig 4.6B). Significant neuronal loss was detected in the CA4 and hilus within 3-6 hrs and 1-3 hrs after SE, respectively. No further damage evolved after the initial loss in the CA4 and hilus, with 43 ± 14% and 57 ± 7% of neurons remaining at 3 months, respectively (Fig 4.6A, B).

105 83 Figure 4.3: Anti-NeuN immunohistochemical staining decreases following SE in the dorsal and ventral hippocampus. Representative images of the left dorsal (A, B) and ventral (C, D) hippocampus from a sham rat (A, C), and from a rat 3 months after SE (B, D). Dotted line indicates the border between dorsal CA3 and CA4 (A). Arrows depicts decreased NeuN staining in the pyramidal cell layer: more severe damage is detected in CA1 (unfilled arrows) compared to CA3 and CA4 (solid arrows). Φ Ventricular enlargement detected within 3 months after SE. Scale bar = 500 µm. 25X magnification. Abbreviations: dorsal 3 rd ventricle (DV3); lateral ventricle (LV); polymorphic layer of the hilus (PoDG); pyramidal cell layer (pyr).

106 84

107 85 Figure 4.4: Confocal micrographs (400X) of NeuN stained cells in hippocampal subfields: dorsal CA1 (A), CA2 (B), CA3 (C) and CA4 (D) in a sham rat, and in rats at different recovery times after SE. Neuronal edema detected in CA1, CA3, and CA4 by 3 hrs after SE (solid arrows). No damage detected in CA2 (B). Scale bar = 50 µm.

108 86

109 87 Figure 4.5 Total length of dentate gyrus remains constant following lithium/pilocarpine induced SE. The length of the dentate gryus in the left (black bars) and right (grey bars) brain hemispheres were determined at different time points (1 hr 3 months) after SE. Values represent length of dentate gyrus (µm ± SEM). Four animals were analyzed at each time point.

110 88

111 89 Figure 4.6: Temporal profiles of neuronal loss in hippocampal subfields following lithium/pilocarpine induced SE. Coronal brain sections prepared at various times following SE were stained with anti-neun antibodies and neurons counted as in Methods (section 4.2.5). Neuron density was graphed as percentage of control (± SEM) values. A: * Dorsal CA1 at 1 hr different from shams (p<0.05), ** hilus at 3 hrs different from shams (p<0.05), *** ventral CA1 at 1 day different from shams (p<0.05), ^ dorsal CA1 at 3 days different from 1 day after SE (p<0.05), ^^ ventral CA1 at 7days different from 1 day after SE, # neuronal loss more severe in CA1 compared to all other regions with combined analyses at 14 and 90 days (p<0.05). B: * Ventral CA3 at 1 hour different from shams (p<0.05), ** CA4 at 6 hours different from shams (p<0.05), *** dorsal CA3 at 12 hours different from shams (p<0.05).

112 90

113 SE-induced neurodegeneration in thalamic nuclei: All thalamic regions exhibited SE-induced neurodegeneration as shown in Figures However, there were differences between the thalamic nuclei in the temporal profiles and severity of neuronal loss. Neuronal death in the VPM and VPL occurred within two separate times following SE: an initial phase within the first 12 hours after SE, and a second phase between 2 weeks and 3 months. While the initial loss of neurons was similar in both regions, by 3 months damage to the VPL was more severe than to the VPM, with 24± 3% and 40 ± 1% of neurons remaining, respectively (Fig 4.9A). Neuronal loss occurred more rapidly in RT than in either VPM or VPL, with only 57 ± 3% of RT neurons remaining 6 hours after SE (Fig 4.9A). Compared to RT and VPM at 3 months, more severe damage occurred in the LDDM, LDVL and Po. In the LDDM, neuron density was reduced by 25 ± 3.7% of shams within the first hour after SE, with 26 ± 3% of neurons remaining after 24 hours (Fig 4.9B). Although the onset of damage was slightly delayed in the LDVL, the progression of neuronal loss was similar to LDDM thereafter, with 24 ± 7% of neurons remaining 24 hours after SE. In Po, a 45 ± 5.9% reduction in neuron density occurred between 1-3 hours after SE, with 33 ± 6% neurons remaining by 6 hours.

114 92 Figure 4.7: Anti-NeuN immunohistochemical staining decreases following SE in several thalamic nuclei. Representative images of the thalamus in a sham rat (A), and in rats at recovery time points 24 hours (B) and 3 months (C). Arrows depict decreased NeuN staining in the LDVL, LDDM, Po and Rt by 24 hours after SE. * Additional damage detected in the VPM and VPL by 3 months after SE. Φ Ventricular enlargement detected within 3 months after SE. Scale bar = 500 µm. 25X magnification. Abbreviations: laterodorsal thalamic nucleus, dorsomedial part (LDDM); laterodorsal thalamic nucleus, ventrolateral part (LDVL); posterior thalamic nuclei (Po); ventral posteromedial thalamic nucleus (VPM); ventral posterolateral thalamic nucleus (VPL); reticular thalamus nucleus (Rt); 3 rd ventricle (3V).

115 93

116 94 FIGURE 4.8: Confocal micrographs (400X) of NeuN stained cells in several thalamic nuclei: (A) Laterodorsal thalamic nucleus, dorsomedial part (LDDM), (B) posterior thalamic nuclei (Po), (C) ventral posterolateral thalamic nucleus (VPL), and (D) reticular thalamus nucleus (Rt) in a sham rat, and in rats at different recovery time points after SE. Scale bar = 50 µm.

117 95

118 96 FIGURE 4.9: Temporal profiles of neuronal loss in several thalamic nuclei following lithium/pilocarpine induced status epilepticus (SE). Coronal brain sections prepared at various time points following SE were stained with anti-neun antibodies and neurons counted as in Methods (section 4.2.5). Neuron density graphed as percentage of control (± SEM) values. A: * Rt at 3 hrs different from shams (p<0.05), ** VPM and VPL at 6 hrs different from shams (p<0.05), ^ VPM and VPL at 3 months different from 14 days (p<0.05), # VPL more severely damaged compared to VPM (p<0.05). B: * LDDM at 1 hr different from shams (p<0.05), **LDVL and Po at 3 hrs different from shams (p<0.05), ^ LDDM and LDVL at 1 day different from 12 hrs (p<0.05). Abbreviations: laterodorsal thalamic nucleus, dorsomedial part (LDDM), laterodorsal thalamic nucleus, ventrolateral part (LDVL), posterior thalamic nuclei (Po), ventral posteromedial thalamic nucleus (VPM), ventral posterolateral thalamic nucleus (VPL), reticular thalamus nucleus (Rt).

119 97

120 SE-induced neurodegeneration in amygdaloid nuclei: Extensive damage was observed in the amygdala, with necrotic lesions (Fig 4.10, indicated by *) and enlargement of the lateral ventricles (Fig 4.10, indicated by φ) present by 3 months after SE. A significant decrease in the area size of the amygdala was also detected between 2 weeks and 3 months following SE (Table 4.1). As illustrated in Figures 4.11 and 4.12, differences in the temporal profiles and severity of neuronal loss were observed between amygdaloid nuclei. Neuronal loss in the LaDL and LaVM was similar, and occurred within two separate times: following an initial decrease within 12 hours after SE, neuron densities remained constant until additional losses occurred between 1 and 2 weeks, with 33 ± 5% and 24 ± 6% of neurons remaining, respectively (Fig 4.12A). In contrast to the pattern of neuronal loss observed in the lateral nucleus, neuronal loss in the BLP and BMP occurred during the first week after SE, reaching minimum neuron densities of 9 ± 4% and 7 ± 6% of shams, respectively (Fig 4.12B). Although severity of damage by 3 months in the BLP and BMP was similar, the progression of neuronal loss in the first 24-hours after SE was different between these regions. While a decrease of 53 ± 3.1% occurred between 3 and 6 hours in the BMP, neuronal loss was delayed in BLP, with only an initial 13 ± 4.7% reduction occurring within 12 hours. The PMCo was the least severely damaged region within the amygdala, with neuron density decreasing to a minimum of 72 ± 5% of shams between 1 and 3 hours after SE (Fig 4.12A) SE-induced neurodegeneration in the piriform cortex: Severe damage was detected in layer II of the posterior piriform cortex (PPC), with necrotic lesions visible within this region by 3 months after SE (Fig 4.10C). In PPC, initial neuronal loss occurred between 3 and 6 hours after SE, with a minimum of 35 ± 18% of pyramidal cells remaining by 12 hours (Fig 4.11D and Fig 4.12B) Detection of Fluoro-jade B stained neurons As shown in Figure 4.13, FJB stained neurons were present by 24 hours following SE in the hippocampus, thalamus, and amygdala. In contrast, no FJB stained neurons were observed in epileptic rats at 3 months following SE, or in shams.

121 99 Figure 4.10: Anti-NeuN immunohistochemistry staining decreases following SE in several amygdaloid nuclei and in the posterior piriform cortex. Representative images of the amygdala and PPC from a sham rat (A), and from rats at recovery time points 24 hours (B) and 3 months (C) after SE. Arrows illustrate decreased NeuN staining in the lateral and basolateral amygdaloid nuclei and piriform cortex 24 hours after SE. * depicts necrotic lesion in amygdala by 3 months. Φ Ventricular enlargement detected at 3 months. Scale bar = 500 µm. 25X magnification. Lateral amygdaloid nucleus, dorsolateral part (LaDL); lateral amygdaloid nucleus, ventrolateral part (LaVM); basolateral amygdaloid nucleus, posterior part (BLP); basomedial amygdaloid nucleus, posterior part (BMP); posteromedial cortical amygdaloid nucleus (PMCo); posterior piriform cortex (Pir); lateral ventricle (LV).

122 100

123 101 FIGURE 4.11: Confocal micrographs (400X) of NeuN stained cells in several amygdaloid nuclei and in the piriform cortex: (A) Lateral amygdala nucleus, ventrolateral part (LaVM), (B) basolateral amygdaloid nucleus, posterior part (BLP), (C) posteromedial cortical amygdaloid nucleus (PMCo), and (D) posterior piriform cortex (PPC) in a sham rat, and in rats at different recovery times following SE. Scale bar = 50 µm.

124 102

125 103 FIGURE 4.12: Temporal profiles of neuronal loss in several amygdaloid nuclei and in the posterior piriform cortex. Coronal brain sections prepared at various time points following SE were stained with anti-neun antibodies and neurons counted as in Methods (Section 4.2.5). Neuron density graphed as percentage of control (± SEM) values. A: * LaDL at 1 hr different from shams (p<0.05), ** PMCo at 3 hrs different from shams (p<0.05), *** LaVM at 6 hrs different from shams (p<0.05), ^ LaDL and LaVM at 14 days different from 7 days (p<0.05). B: * BMP at 3 hrs different from shams (p<0.05), ** PPC at 6 hrs different from shams (p<0.05), *** BLP at 12 hrs different from shams (p<0.05), ^ PPC at 12 hrs different from 6 hrs (p<0.05), ^^ BLP and BMP at 7 days different from 3 days (p<0.05). Abbreviations: lateral amygdaloid nucleus, dorsolateral part (LaDL), lateral amygdaloid nucleus, ventrolateral part (LaVM), basolateral amygdaloid nucleus, posterior part (BLP), basomedial amygdaloid nucleus, posterior part (BMP), posteromedial cortical amygdaloid nucleus (PMCo), Posterior piriform cortex (PPC).

126 104

127 105 FIGURE 4.13: Confocal micrographs (400X) of Fluoro-jade B (FJB) stained cells present at 24 hours but not at 3 months after SE in the hippocampus, thalamus and amygdala. Coronal brain sections were stained with FJB, a marker for degenerationing neurons. (A) Dorsal CA1 subfield of the hippocampus, (B) ventral posteromedial thalamic nucleus (VPM), (C) basolateral amygdaloid nucleus, posterior part (BLP) in a sham rat, and in rats at 24 hours and 3 months recovery times following SE. Scale bar = 50 µm.

128 106

129 Discussion In the present study, we performed a detailed quantitative analysis of neuronal death in 19 brain regions at times ranging from 1 hr to 3 months following 60 minutes of SE. The major findings of this study were that: (1) the RLDP procedure resulted in widespread neuronal death that was generally similar to that found with the low-dose lithium/pilocarpine and the high-dose pilocarpine procedures, (2) in some brain regions, neuronal death appeared as early as 1 hr following SE, with the majority of neuronal death in all brain structures present by 24 hrs after SE, (3) while specific regions within the hippocampus (dorsal and ventral CA1) and amygdala (LaDL, LaVM, BLP, BMP) showed additional neuronal loss between 1 and 14 days after recovery, the somatosensory thalamic nuclei (VPM, VPL) were the only areas with additional neuronal death between 2 weeks and 3 months of recovery, and finally, (4) different regions within the hippocampus, thalamus, amygdala and piriform cortex exhibited differential rates of neuronal loss. These results are further discussed in the subsequent sections The neuropathological effect of increasing survival time following status epilepticus In the present study, we provided the first analysis of neuronal loss with the RLDP protocol for the induction of SE, and found that the extent of brain damage was similar to that previously described with both the low-dose lithium/pilocarpine (LDP) and the high-dose pilocarpine procedures (Honchar et al., 1983; Turski et al., 1983a; Turski et al., 1983b; Clifford et al., 1987; Fujikawa, 1996; Motte et al., 1998; Covolan and Mello, 2000; Peredery et al., 2000; Poirier et al., 2000). In chapter 3, we showed that the severity of neuronal death between rats treated with the LDP and the RLDP protocols was similar at 3 months, despite procedural differences in the mortality rates and in the severity of seizures. Because we did not directly compare the 2 methods at all the different times analyzed here, however, we cannot determine whether the rates of neuronal death between brain regions also occurred similarly. In apparent contrast, it was previously reported that in rats without lithium-pretreatment, seizure severity, mortality rates and neuropathology was affected by different doses of pilocarpine administered (Clifford et al., 1987; Liu et al., 1994; Curia et al., 2008). With sub-convulsive doses of pilocarpine alone (100 mg/kg, i.p.), neuronal damage was confined to the piriform cortex and anterior olfactory nuclei (Clifford et al., 1987). Damage was extended to include the amygdala, cortical and basal nuclei

130 108 if the dosage was increased to 200 mg/kg. Rats exhibiting convulsive seizures after an injection of 200 mg/kg showed additional damage in the medio-thalamic nuclei and neocortex. Injection of pilocarpine at the highest dose of 400 mg/kg resulted in rats exhibiting the highest mortality rates, the most severe convulsive seizures, and extensive neuronal damage that were the most comparable to that observed in the lithum/pilocapine model (Clifford et al., 1987). Therefore, the RLDP protocol has the advantage of reducing mortality rates in rats while producing a similar pattern of neurodegeneration to both the low-dose lithium/pilocarpine and high-dose pilocarpine procedures. Because several recovery times ranging between 1 hr and 3 months were analyzed in the present study, we were able to provide a detailed description of the progression of neuronal death between brain regions following lithium/pilocarpine-induced SE. As illustrated in Figure 4.13, the number of brain regions first exhibiting SE-induced neuronal loss (Fig 4.14, grey bars), and brain regions sustaining maximum neuronal death (Fig 4.14, black bars), increased quickly with time following cessation of SE; a list of these brain regions can be found in Tables 4.2 and 4.3, respectively. A decrease in the density of neurons was apparent as early as 1 hr in 3 brain regions (dorsal CA1, dorsomedial part of the laterodorsal thalamic nucleus (LDDM) and basolateral amygdaloid nucleus (BLP)), and the number of brain regions exhibiting significant neuronal death increased to 10 by 3 hrs, 15 by 6 hrs, and all brain regions, with the exception of CA2, by 24 hrs (see Table 4.2). Eleven out of 19 brain regions exhibited no further neuronal loss after 24 hrs of recovery (see Table 4.3). Of the 8 brain regions exhibiting the loss of neurons after 24 hrs of recovery, 6 (dorsal and ventral CA1, dorsolateral (LaDL) and ventrolateral (LaVM) parts of the lateral amygdaloid nucleus, basolateral amygdaloid nucleus (BLP), and basomedial amygdaloid nucleus (BMP)) sustained additional neuronal loss between 1 and 14 days after SE. The thalamic somatosensory nuclei (ventral posteromedial (VPM) and ventral posterolateral (VPL) thalamic nuclei) were the only areas for which additional neuronal death occurred after 2 weeks. Although there was a delay between the times at which neuronal loss first became apparent (Table 4.2), and when maximal neurodegeneration occurred for most brain regions examined (Table 4.3), this period was brief since most of the damage was present by 24 hrs (depicted in Fig 4.14). Our findings demonstrated that with the RLDP model of SE, the majority of neuronal death in the hippocampus, thalamus, amygdala and piriform cortex occurred by 24 hrs after SE. Previous

131 109 studies using the LDP or the high-dose pilocarpine procedures and different staining procedures analyzed the same brain regions, and similarly showed that most of the neuronal damage appeared within the initial 24 hrs of recovery (Fujikawa, 1996; Motte et al., 1998; Covolan and Mello, 2000; Poirier et al., 2000) (see table A1-1 in appendix I). For example, Fujikawa et al., (1996) used hematoyxlin and eosin (H&E) staining to qualitatively assess for the presence of acidophilic neurons, which was used as a criterion of irreversible neuronal injury; the majority of neuronal damage was observed between the 0-4 hrs and 24 hrs time points following SE in the dorsal and ventral CA1 and CA3 subfields of the hippocampus, hilus, amygdala, thalamus, and piriform cortex. The mediodorsal and lateroposterior thalamic nuclei were the only regions to exhibit additional damage between the 24 hrs and 72 hrs recovery times (Fujikawa, 1996). Because injured neurons are also agryophillic (Horvath et al., 1997), Covolan and Mello (2000) used the Gallyas s silver impregnation method to qualitatively assess neuronal damage, and showed that the most intense staining appeared in regions within the hippocampus, amygdala and thalamus between the 8 hrs and 24 hrs recovery times. Finally, Poirier, Capek and Koninck (2000) showed that the most intense Dark Nuclear (silver) stain occurred at 3 hrs following SE in the hippocampus, and preceded the presence of Flouro-jade (FJ) staining (a marker for degenerating neurons), which maximally appeared between 24 hrs and 1 weeks after SE. While the Dark Nuclear stain was suggested to detect an early molecular event associated with neuronal stress (Poirier et al., 2000b), FJ staining was proposed to detect an unknown marker associated with the later stages of neuronal degeneration (Poirier et al., 2000b; Schmued and Hopkins, 2000). In the present study, Fluoro-jade B (FJB) was used and it produced results very similar to the staining pattern produced by its predecessor, FJ (i.e., share the affinity for the same biomolecule(s), but differ in relative strength of affinity) (Schmued et al., 2000). In pilot studies, we comparably found that the presence of FJB staining in the hippocampus slowly increased after the 3 hrs recovery time, and was maximally present by 24 hrs. FJB staining was still present within the pyramidal cell layer of the dorsal and ventral CA1 subfields at 1 week following SE, which is consistent with the additional reduction of neuronal densities we quantified for these regions. With this staining method, however, we found it difficult to determine whether the presence of FJB was attributed to ongoing degeneration, or debris from degenerated neurons that had not yet been removed. For instance, although maximal neuronal

132 110 loss in the CA3 and CA4 subfields were quantified to occur prior to the 24 hrs recovery time, some FJB staining was still present at 24 hrs, indicating that this staining was from debris of already degenerated cells. Overall, the spatio-temporal staining pattern of FJB provides additional evidence that the majority of neuronal damage occurs early and is attributed to SE (see section 4.4.2). Because we used an unbiased quantification method (optical dissector method) to assess neuronal death, and increased the number of early times analyzed, we were able to demonstrate, in a precise, quantitative manner, that maximum neuronal loss in several brain regions occurred before 24 hrs of recovery. In the present study, maximal neuronal loss was detected as early as 3 hrs in the hilus, ventral CA3, reticular thalamic nucleus (Rt) and posteromedial cortical amygdaloid nucleus (PMCo), by 6 hrs in the dorsal CA4 and posterior thalamic nuclei, and by 12 hrs in the dorsal CA3 and posterior piriform cortex (PPC) (see table 4.3). These findings represent the first detailed, quantitative time-course comparison of SE-induced neuronal death between brain regions at several times (1, 3, 6, and 12 hrs) preceding 24 hrs recovery The relationship between SE, SRSs and delayed neuronal death Presently, it is difficult to ascertain whether damage occurring after the 24 hr period is the consequence of SRSs, or the delayed damage occurring from SE as the IPI (Dudek et al., 2002). For this reason, the effect of SRSs on neurodegeneration remains unclear. While some studies report no correlation of neuronal death and the frequency of SRSs (Pitkänen et al., 2002; Gorter et al., 2004), others have demonstrated progressive neuronal loss in chronically epileptic rats (Roch et al., 2002). Although we did not determine the latency to onset of SRSs in rats following SE, our findings did demonstrate that by 6 weeks of recovery, all rats were epileptic. Glien et al., (2001) reported that following 60 minutes of SE induced by the RLDP protocol, the average latency to the first SRS was 40 days (range days). It is therefore reasonable to assume that any neuronal death prior to 40 days or so was likely the consequence of SE, and that any neuronal loss after this period might result from SE, SRSs or both. The only regions to exhibit additional loss of neurons after 2 weeks in the present study were the thalamic somatosensory nuclei (VPM, VPL), although we do not know if this death occurred prior to or after the initiation of SRSs. We further showed that FJB-stained neurons are present at 24 hrs

133 111 following SE, but not at the 3 months recovery time in epileptic rats. Overall, our results indicate that the vast majority of neuronal death is the result of SE and that little, if any additional death, occurs subsequent to the start of SRSs. In support of the suggestion that SE primarily contributes to neuronal death, Liu et al., (1994) used the high-dose pilocarpine procedure and showed a significant decrease in neuronal density and total neuron number at 3 weeks recovery in the dorsal CA1 and CA3. The authors reasoned that since SRSs appeared approximately 2 to 2 ½ weeks after SE in these animals, but did not contribute to any significant additional neuronal loss 6 to 12 weeks later, that neuronal death primarily resulted from the acute pilocarpine-induced seizures (Liu et al., 1994). At this time, we are unable to determine whether the delayed neuronal loss between 14 days and 3 months in the thalamic somatosensory nuclei (VPM, VPL) was caused by delayed damage from SE, or from damage caused by the development of SRSs. Although Peredery et al., (2000) similarly showed that the thalamus was the only structure to exhibit some additional loss after 20 days of recovery, further studies are necessary to determine the cause (e.g., SE and/or SRSs) of this damage Differences in the severity and spatial pattern of neuronal death following SE In the present study, differences in the severity of SE-induced neuronal death between regions were detected. For instance, while the dorsal and ventral CA1 were the most severely damaged regions within the hippocampus, with neuronal loss exceeding 70% of shams, no neuronal death was detected in the CA2 or dentate gyrus. In the thalamus, the LDDM, LDVL, Po, and VPL showed severe neuronal death exceeding 75% of shams, while less severe neuronal loss of 60% and 40% was detected in the VPM and Rt, respectively. Similarly, in the amygdala, severe neuronal death exceeding 75% of shams was detected in LaDL, LaVM, BLP and BMP, while only moderate neuronal loss of 28% was detected in the PMCo. Although the specific mechanisms responsible for these differences remain unclear, several observations regarding the spatial pattern in severity of SE-induced neuronal death have been made. Studies using 14 C-2- deoxyglucose functional mapping have shown that brain regions exhibiting the highest metabolic rates during SE also exhibit the most severe neuronal damage (Ingvar, 1986; Ingvar et al., 1987; Handforth and Ackermann, 1992; Handforth and Ackermann, 1995; Fernandes et al., 1999;

134 112 Bouilleret et al., 2000). Similar results were obtained in studies examining Fos expression, a marker for cellular hyperactivation (Motte et al., 1998; Fernandes et al., 1999). The regional distribution of glutamatergic receptors (Olney et al., 1986; Fernandes et al., 1999) and regions exhibiting Ca 2+ accumulation (Friedman et al., 2008) were other factors shown to coincide with the spatial pattern of SE-induced brain damage. These results indicate that the most heavily damaged brain regions are those that are most strongly activated during SE, and support the suggestion that SE-induced neuronal death is initiated by excitotoxicity (Fernandes et al., 1999). Excitotoxicity occurs when glutamate receptors are excessively stimulated, allowing high levels of Ca 2+ to enter the cell and activate cell-death signaling mechanisms (Fujikawa, 2005). Regions resistant to cell death are proposed to be rich in Ca 2+ -binding proteins, such as calbindin, and are therefore able to buffer the seizure-induced excessive calcium load (Chard et al., 1993). This idea is based on the findings that seizure-induced damage in the hippocampus displays a topographical profile that closely resembles the pattern of distribution of Ca 2+ -binding proteins in the hippocampal formation (Sloviter, 1989; Sloviter et al., 1991). In the hippocampus, pyramidal cells in the CA1 and CA3, which contain no or only small traces of calbindin, are most heavily damaged while the dentate granular cells and pyramidal cells in the CA2, which are rich in calbindin, remain resistant to seizure-induced damage (Sloviter, 1989; Sloviter et al., 1991). This supports the role of Ca 2+ in seizure-mediated neuronal death, and is consistent with our own findings and results from other studies (Turski et al., 1983a; Clifford et al., 1987; Liu et al., 1994) that showed no overt damage in the dentate gyrus and CA The type of cell death produced by SE The present data showed that regions within the hippocampus, thalamus, amygdala and piriform cortex have different temporal patterns of neuronal death following SE. Although our results do not allow the specific mechanisms responsible for neuronal death to be identified, other studies indicate that variation in the temporal profile of neuronal loss may reflect varying combinations of apoptotic and necrotic processes. Several studies demonstrated that dying neurons present by 24 hrs after SE predominately exhibited a necrotic morphology (Fujikawa, 2005; Fujikawa et al., 1999; Fujikawa et al., 2000b; Kotariya et al., 2010). On the other hand, delayed neuronal death (3 to 7 days) exhibited apoptotic features (Narkilahti et al., 2003a; Weise et al., 2005). Several regions in the present study exhibited early and delayed stages of neuronal death, and this may reflect different forms of cell death in the same neuronal population. For instance, kainic acid or

135 113 pilocarpine-induced SE in adult rats have been shown to result in two types of pyramidal cell death: early necrosis ( 1 day) (Araújo et al., 2008; Wang et al., 2008) and delayed cell death with apoptotic features ( 3 days) (Narkilahti et al., 2003a; Weise et al., 2005; Wang et al., 2008). This finding is consistent with the early and delayed times of pyramidal cell death we observed within the dorsal and ventral CA1, and may apply to the other neuronal populations in regions (LaDL, LaVM, BLP and BMP areas of the amygdala, VPM and VPL areas of the thalamus) exhibiting similar patterns of neuronal death. Further studies are required to determine if different cell death mechanisms between regions and within the same neuronal populations underlie different patterns of neuronal death, and if so, what proportion of degenerating neurons at a specific time point are necrotic versus apoptotic Conclusion The present study demonstrates that the RLDP procedure for the induction of SE results in widespread neuronal death, and that different regions, even within the same structure, may exhibit differences in both the temporal profiles and the severity of neuronal loss following SE. Neuronal loss was detected as early as 1 hr after SE in some brain regions, with significant brain damage in all regions (except CA2 and dentate gyrus) present by 24 hrs. Although neuronal death occurred between 1 and 14 days in regions within the hippocampus and amygdala, and extended beyond 14 days in the thalamic somatosensory nuclei, it only contributed minimally to the total damage in these brain structures caused by SE. Furthermore, with the possible exception of the thalamic somatosensory nuclei, the development of SRSs did not appear to contribute significantly to the neurodegeneration observed in epileptic rats.

136 114 Figure 4.14: The number of damaged brain regions as a function of increasing recovery time after 60-min of SE. Grey bars represent the number of brain regions with initial neuronal loss significantly different from corresponding shams. Black bars represent the number of brain regions exhibiting maximal neuronal loss. With the exception of CA2, all brain regions sustained early damage within 24 hours after SE. There is a delay, however, between initial neuronal loss and maximal neurodegeneration for most brain regions. Brain regions exhibiting initial neuronal damage and maximal neuronal damage at specific recovery times are listed in tables 4.2 and 4.3.

137 115

138 116 Table 4.2 Temporal progression of brain regions exhibiting initial neuronal loss significantly different from corresponding shams. Brain structure 1 hr Recovery 3 hr recovery 6 hrs Recovery 12 hrs recovery 24 hrs recovery Hippocampus Dorsal CA1 Dorsal CA1 Ventral CA3 Hilus Dorsal CA1 Ventral CA3 Hilus Dorsal CA4 Dorsal CA1 Ventral CA3 Hilus Dorsal CA4 Dorsal CA3 Dorsal CA1 Ventral CA3 Hilus Dorsal CA4 Dorsal CA3 Ventral CA1 Thalamus LDDM LDDM Rt LDVL Po LDDM Rt LDVL Po VPM VPL LDDM Rt LDVL Po VPM VPL LDDM Rt LDVL Po VPM VPL Amygdala and piriform cortex LaDL LaDL PMCo BMP LaDL PMCo BMP LaVM PPC LaDL PMCo BMP LaVM PPC BLP LaDL PMCo BMP LaVM PPC BLP All of the following brain regions showed statistically significant neuronal loss compared to shams. Hippocampus: Dorsal (dca1) and ventral (vcal) CA1, CA3, CA4, and hilus. Thalamus: laterodorsal thalamic nucleus, dorsomedial part (LDDM), laterodorsal thalamic nucleus, ventrolateral part (LDVL), posterior thalamic nuclei (Po), ventral posteromedial thalamic nucleus (VPM), ventral posterolateral thalamic nucleus (VPL), reticular thalamus nucleus (Rt). Amygdala: lateral amygdaloid nucleus, dorsolateral part (LaDL), lateral amygdaloid nucleus, ventrolateral part (LaVM), basolateral amygdaloid nucleus, posterior part (BLP), basomedial amygdaloid nucleus, posterior part (BMP), posteromedial cortical amygdaloid nucleus (PMCo). Posterior piriform cortex (PPC).

139 117 Table 4.3 Temporal progression of brain regions exhibiting maximal neuronal death following SE Brain structure 3 hr recovery 6 hrs recovery 12 hrs recovery 24 hrs recovery 3 days recovery 7 days recovery 14 days recovery 3 months recovery Hippocampus Hilus Ventral CA3 Hilus Ventral CA3 CA4 Hilus Ventral CA3 CA4 Dorsal CA3 Hilus Ventral CA3 CA4 Dorsal CA3 Hilus Ventral CA3 CA4 Dorsal CA3 Dorsal CA1 Hilus Ventral CA3 CA4 Dorsal CA3 Dorsal CA1 Ventral CA1 Hilus Ventral CA3 CA4 Dorsal CA3 Dorsal CA1 Ventral CA1 Hilus Ventral CA3 CA4 Dorsal CA3 Dorsal CA1 Ventral CA1 Thalamus Rt Rt Po Rt Po Rt Po LDDM LDVL Rt Po LDDM LDVL Rt Po LDDM LDVL Rt Po LDDM LDVL Rt Po LDDM LDVL VPM VPL Amygdala and piriform cortex PMCo PMCo PMCo PPC PMCo PPC PMCo PPC PMCo PPC BLP BMP PMCo PPC BLP BMP LaDL LaVM PMCo PPC BLP BMP LaDL LaVM All of the following brain regions showed statistically significant (and maximal) neuronal loss compared to shams. Hippocampus: Dorsal (dca1) and ventral (vcal) CA1, CA2, CA3, CA4, and hilus. Thalamus: laterodorsal thalamic nucleus, dorsomedial part (LDDM), laterodorsal thalamic nucleus, ventrolateral part (LDVL), posterior thalamic nuclei (Po), ventral posteromedial thalamic nucleus (VPM), ventral posterolateral thalamic nucleus (VPL), reticular thalamus nucleus (Rt). Amygdala: lateral amygdaloid nucleus, dorsolateral part (LaDL), lateral amygdaloid nucleus, ventrolateral part (LaVM), basolateral amygdaloid nucleus, posterior part (BLP), basomedial amygdaloid nucleus, posterior part (BMP), posteromedial cortical amygdaloid nucleus (PMCo). Posterior piriform cortex (PPC).

140 118 Chapter 5 Neuroprotection following status epilepticus by targeting protein interactions with PSD Introduction Status epilepticus (SE), defined as 30 min or greater of continuous seizure activity, is a neurological emergency resulting in high mortality and morbidity (DeLorenzo et al., 1996; Fountain, 2000). Sustained activation of N-methyl-D-aspartate receptors (NMDARs), which mediate excitatory neurotransmission in the central nervous system, is necessary for the induction of SE (Rice et al., 1998; Deshpande et al., 2008). Overactivation of NMDARs mediates excitotoxicity, causing widespread neurodegeneration in humans (Fujikawa et al., 2000a) and in animal models of epilepsy, including kindling (Cavazos and Sutula, 1990), and the administration of chemoconvulsants such as pilocarpine ( Turski et al., 1983b; Nevander et al., 1985; Cavalheiro et al., 1987) and kainic acid (Ben-Ari, 1985; Fujikawa et al., 2000b). Consistent with a critical role for NMDARs in SE-induced neuropathology, the systemic administration of NMDAR antagonists provides substantial neuroprotection in rodent models of epilepsy, even when given after the onset of SE (Fariello et al., 1989; Clifford et al., 1990; Fujikawa et al., 1994; Fujikawa, 1995). The clinical efficacy of NMDAR antagonists, however, is limited because of their associated side effects, including the induction of psychosis in humans ( Krystal et al., 1994; Lahti et al., 1995; Malhotra et al., 1996; Rowland et al., 2005) and neurotoxicity in rats (Olney et al., 1989; Olney et al., 1991; Fix et al., 1993). A proposed alternate approach to preventing NMDAR-mediated neurotoxicity is to prevent excitotoxic signaling from the NMDAR, such as the production of nitric oxide, by blocking interaction of the receptor with downstream signaling molecules (Aarts et al., 2002). PSD-95 is a critical scaffolding protein that links the NMDAR to signaling enzymes within the postsynaptic density, and suppression of the expression of PSD-95 has selectively attenuated excitotoxicity triggered via NMDARs (Sattler et al., 1999). Tat-NR2B9c is a synthetic peptide consisting of the C- terminal 9 amino acids of the NR2B subunit of NMDARs fused to the membrane transduction domain of the HIV-1-Tat protein, that was designed to disrupt neurotoxic signaling from the NMDAR by interfering with protein interactions involving the PDZ1 and PDZ2 domains of PSD-95 (Aarts et al., 2002; Cui et al., 2007). Tat-NR2B9c was reported to provide significant

141 119 neuroprotection and to preserve cognitive function following transient stroke in rats (Aarts et al., 2002; Sun et al., 2008). Because activation of NMDA receptors is critically involved in the neuropathological outcomes of SE (Rice et al., 1998; Deshpande et al., 2008), we investigated the ability of Tat-NR2B9c to provide neuroprotection following SE induced by lithium/pilocarpine. The results demonstrate that Tat-NR2B9c, when administered 3 hrs following the termination of SE, significantly reduces neuronal cell death in the hippocampus of adult rats. 5.2 Methods Induction of status epilepticus All procedures were approved by the University of Toronto Animal Care Committee and were in accordance with the guidelines established by the Canadian Council on Animal Care. Male Wistar rats (Charles River Laboratories, Sherbrooke, Quebec, Canada) weighing between 300 and 350 g were individually housed with free access to food and water for at least 7 days in 12 h light/dark cycles before experimental use. For the induction of SE, rats were pretreated with lithium chloride (3mEq/kg, i.p.) 24 hours before the injection of pilocarpine, and received methylatropine nitrate (10 mg/kg, i.p.) 30 min prior to pilocarpine. Pilocarpine (10 mg/kg, i.p.) was administered every 30 min as described by Glien et al., (2001) until the rat experienced a generalized, class 4/5 seizure, since rats generally developed SE shortly thereafter. Animals that did not develop SE within 30 min of the first class 4/5 seizure received additional pilocarpine injections at 30-min intervals up to a maximum of 6 injections. Animals that did not develop SE after the sixth injection of pilocarpine were not used in this study. Duration of seizure activity was controlled by the administration of diazepam (4 mg/kg, i.p.) 1, 3 and 5 hours following the onset of SE. Sham animals underwent identical procedures but received saline in place of pilocarpine, and did not exhibit any seizures. Pilocarpine, lithium chloride and methylatropine nitrate were purchased from Sigma (St Louis, Missouri, USA) and dissolved in 0.9% saline prior to administration. Diazepam was purchased from CDMV (Saint-Hyacinthe, Quebec, Canada) and used as the commercial solution (5mg/ml). The behavioural progression of pilocarpine-induced seizures was assessed using a modified Racine scale (Racine, 1972; Cammisuli et al., 1997), as follows: class 1, facial clonus; class 2,

142 120 head nodding; class 3, forelimb clonus; class 4, forelimb clonus and rearing; class 5, forelimb clonus, rearing and one fall (loss of postural control); class 6, forelimb clonus, rearing and multiple falls; and class 7, running and jumping. Animals were continuously monitored following the first injection of pilocarpine. The average maximum seizure activity was determined by averaging the highest class of seizure that occurred in each 5 minute interval following the initiation of SE. Following the termination of SE, animals were kept in a quiet room for 3 days. Immediately following the first injection of diazepam, animals received 5 ml 0.9% saline (3ml i.p. and 2ml s.c.), and this was repeated in the morning and evening of the following day (5ml i.p.) as described by Glien et al., (2001). Starting on the second day after SE, animals were tube-fed softened rat chow mixed with applesauce for 3 days on average. Softened rat chow was also provided in dishes until the rats commenced to eat hard pellets Administration of peptides Tat-NR2B9c, a synthetic peptide consisting of the C-terminal 9 amino acids of the NR2B subunit of the NMDA receptor (Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val) fused to the cell membrane protein transduction domain of the HIV-1-Tat protein (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg- Arg-Arg) disrupts protein interactions involving the PDZ1 and PDZ2 domains of PSD-95 (Cui et al., 2007; Kornau et al., 1995) and was a generous gift of nono Inc. (Toronto, ON, Canada). Control peptide (Tat-NR2BAA) consisted of Tat-NR2B9c in which the COOH-terminal serine and valine of the NMDAR peptide were changed to alanine residues (Lys-Leu-Ser-Ser-Ile-Glu- Ala-Asp-Ala). The altered PDZ-binding motif is not expected to bind PDZ domains (Kornau et al., 1995; Aarts et al., 2002; Cui et al., 2007) and was devoid of neuroprotective properties in a model of ischemia (Aarts et al., 2002). Peptides were dissolved in saline at a concentration of 3mM and stored at -80 o C until use. For the administration of peptides, animals underwent femoral vein cannulation and were allowed at least 4 days recovery prior to SE induction. Briefly, animals were anaesthetized with a continuous supply of 3-5% isoflurane (CDMV, Saint-Hyacinthe, Quebec, Canada) and oxygen. A 2 cm ventral skin incision was made along the crease formed by the abdomen and right thigh. The right femoral vein was cannulated with PE10 tubing prefilled with saline and secured in place with suture. A 0.5 cm dorsal, midline skin incision was made between the

143 121 scapulae. The PE10 tubing was fed underneath the connective tissue to the dorsal incision using a sterile stainless metal tube, immobilized, sutured with a surgical button, and occluded with a pin. Rats received temgesic (0.1 mg/kg, i.p.; CDMV, Saint-Hyacinthe, Quebec, Canada) for postoperative pain relief. Peptides were administered (3 or 9 nmols/gm) via the cannula over 4 to 5 minutes, either 10 minutes following the onset of SE, or 3 hrs following the termination of SE. Saline control animals were treated identically except that they received saline (0.3 ml) in place of peptide. In all cases the experimenter was blinded to the nature of the injection NeuN Immunohistochemistry Two weeks following the termination of SE, animals were anaesthetized with a mixture of xyaline (26 mg/kg i.p., Rompun, CDMV, Saint-Hyacinthe, Quebec, Canada) and ketamine (174 mg/kg i.p., Ketalar, CDMV, Saint-Hyacinthe, Quebec, Canada), and perfused transcardially with 90 ml of 0.1 M phosphate-buffered saline, ph 7.4 (PBS) followed by ml of 4% (w/v) paraformaldehyde in PBS. Brains were removed and left overnight at 4 C in the paraformaldehyde solution. The following day, brains were soaked for cryoprotection in 30% (w/v) sucrose in PBS until they sank at room temperature. Next, brains were frozen in 2- methylbutane at -35 C and stored at -80 C. Forty µm coronal sections were prepared with a freezing microtome. Sections were placed in 24-well culture plates containing antifreeze solution (0.05 M sodium phosphate buffer, ph 7.4, 30% (w/v) ethylene glycol, 15% (w/v) glucose), and stored at -20 C. For each animal, 3 coronal brain sections containing the dorsal hippocampus were selected between Bregma -3.2 mm through mm (Paxinos and Watsons rat brain atlas, 5 th edition), with the first section presenting all hippocampal subfields, exhibiting only the dorsal portion of the lateral ventricle, and containing the most ventral portions of the capsular division (CeC) and lateral division (CeL) of the central amygdala. The subsequent sections were selected ventrally at 240 µm intervals, and exhibited the lateral ventricle extending to the ventral portion of the brain, replacing the structures CeC and CeL detected in the initial section. Section were rinsed in PBS (3 x 5 min washes), reacted overnight at 4 C with NeuN antibody (1:1000; Chemicon, Billerica, MA, USA) in 0.2% (v/v) goat serum, 0.3% (v/v) Triton X-100 in PBS followed by 2 hours at room temperature with Cy3 conjugated secondary antibody (1:200; Chemicon, Billerica, MA, USA) in 0.2% (v/v) goat serum, 0.3% (v/v) Triton X-100 in PBS.

144 122 Neurons were imaged using a Zeiss LSM510 Laser Scanning Confocal microscope equipped with a 40x/1.3 oil-immersion objective lens, and neuronal density determined using the unbiased optical dissector technique as described by West and Gundersen (1990). Briefly, three counting frames (120 X 60 µm) were positioned in the CA1, CA3 and posterior piriform cortex (PPC), two counting frames were positioned in the CA4 region, and 1 counting frame was placed in CA2 of the left brain hemisphere as shown in Figures 5.1 A and C. Right brain structures were visually inspected and found to be comparable to the left side. Neurons were identified as NeuN-positive cells that contained a relatively large (>8µm) soma (Shi et al., 2004). Chromophilic somas contained within each counting frame, or touching the inclusion borders of the frame (upper and right borders) were counted (West and Gunderson, 1990). The dissector height equivalent to the known tissue height prior to staining (40 µm) was used in all calculations (Hatton and Von Bartheld, 1999), and upper or lower exclusion borders in the z plane were excluded as previous studies demonstrated that no significant variation in neuronal density occur whether such borders were used or not (Harding et al., 1994; Azcoitia et al., 2005; Gardella et al., 2003). Unless otherwise specified, cell densities for individual animals represent the average densities of all counting frames in a particular region for 3 brain sections. All results are expressed as neurons per mm Statistical Analysis Statistical analysis was performed using Statistica 6.0 software. Significant differences were determined using one-way analysis of variance (ANOVA). The Newman-Keuls post-hoc test was used to determine difference between treatment groups. Chi-square analysis was performed to analyze mortality rates. Significance was set at a p-value of 0.05 or less. 5.3 Results Induction of status epilepticus SE was induced by the repeated administration of low doses of pilocarpine (10 mg/kg) to animals that had been pre-treated with lithium chloride and methyl atropine nitrate as described by Glien et al., (2001). Of 63 animals that received pilocarpine, 16 (25%) did not develop SE after 6 injections of pilocarpine, and were not used for the present study. The other animals developed SE after one (n=2), two (n=23), three (n=16) or four (n=6) pilocarpine injections, with

145 123 SE developing within minutes (mean 68 +/- 22 min) following the initial pilocarpine injection. The overall fatality rate for animals that entered SE was 30% and did not differ among experimental groups (Table 5.1). All animals which entered SE displayed stage 4/5 convulsive seizures within the first 10 minutes. During the 60 minutes of SE, animals experienced seizures varying between 2 and 4/5 on the Racine scale (1972) and did not regain consciousness. There was no significant difference between groups with respect to the average maximum seizure activity recorded during SE, which approximated to 3.0 for all groups (Table 5.1). All groups experienced similar weight gain during the recovery period (Table 5.1) so that by 14 days there was no difference in mean body weight between groups (Table 5.1)

146 124 Table 5.1: Comparison of the effect of treatment on mortality, seizure severity and weight gain following SE. Treatment Total Survived 1 Died 1 SE Severity 2 SE + Saline (administered following SE) SE + Tat-NR2B9c (3 nmol/gm, administered following SE) SE + Tat-NR2B-AA (3 nmol/gm, administered following SE) SE + Tat-NR2B9c (3 nmol/gm, administered during SE) SE + Tat-NR2B9c (9 nmol/gm, administered during SE) (27.3%) (28.6%) (27.3%) (42.9%) (25.0%) Initial Weight 3 (gm) Final Weight 3 (gm) 3.06 ± ± ± 18 (16.6%) 2.98 ± ± ± 22 (9.2%) 2.90 ± ± ± 12 (14.2%) 3.13 ± ± ± 15 (12.3%) 3.09 ± ± ± 27 (11.2%) Sham (no SE) No SE 379 ± ± 11 (14.5%) 1. The number of animals that survived or died within 14 days following SE. Numbers in ( ) represent the % of animals that died. 2. Average maximum seizure activity for all animals determined as in Methods (mean ± SD). 3. Initial and final group weights evaluated immediately before, and at 2-weeks following, SE induction (mean ± SD). Values in () indicate % weight gain. No significant differences in mortality rates (chi-square analysis), seizure severity (one-way ANOVA) or % weight gain (one-way ANOVA) were detected between treatment groups.

147 SE induced by repeated low doses of pilocarpine results in neurodegeneration in the hippocampus and piriform cortex The hippocampus and piriform cortex play critical roles in the development and maintenance of limbic seizures, and are the most susceptible brain regions to SE-induced damage (Turski et al., 1983b; Druga et al., 2003; Andre et al., 2007; Chen et al., 2007;). We therefore focused our analyses on these regions. Preliminary experiments demonstrated that neurodegeneration in both the dorsal hippocampus and the piriform cortex occurred rapidly within the first few days following the termination of SE, and was complete within 2 weeks (see chapter 4). The effect of SE on neuronal cell death was therefore assessed 14 days following the termination of seizures. Because the effect of SE induced by the repeated administration of low doses of pilocarpine on neurodegeneration has not previously been described, we initially determined the effect of SE on neuronal cell density in animals that experienced SE but that did not receive any additional treatment. Preliminary comparisons of pyramidal cell counts in each of the 3 coronal brain sections showed that the extent of SE-induced cell death did not vary between sections. For example, the average cell densities in the CA1 region before and after SE were 110,796 ± 23,841 and 33,654 ± 24,097, 106,271 ±24,551 and 33,234 ± 24,207, 104,692 ± 18,128 and 35,879 ± 21,056 for the 3 sections from the most proximal to the most distal from Bregma. Similar results were obtained for the other hippocampal subfields. Cell densities were therefore determined by averaging the densities for all counting frames in all 3 sections for a particular region. The results presented in Figure 5.1 show that SE resulted in a marked loss of pyramidal neurons in the CA1, CA3, and CA4 regions of the dorsal hippocampus, and in layer ІІ of the posterior piriform cortex (PPC). Neuronal densities (cells/mm 3 ± SD) prior to and following SE were 104,993 ± 18,982 and 18,982 ± 22,093 (70% cell loss) in the CA1 region, 64,705 ± 3,575 and 37,616 ± 12,412 (34% cell loss) in the CA3 region, 64,704 ± 9,697 and 31,084 ± 20,344 (58% cell loss) in the CA4 region, and 48,417 ± 12,123 and 5,851 ± 5,103 (88% cell loss) in the PPC. SE did not result in a significant change in cell density in the CA2 region (81,018 ± 10,195 cells/mm 3 in naive animals, 68,297 ± 12,678 cells/mm 3 in SE rats), and for this reason the effect of Tat-NR2B9c on the CA2 region was not assessed. Overall, pyramidal cell loss with the current procedure for SE induction was similar to neuronal loss reported in previous studies using other variations of the pilocarpine model (Turski et al., 1983a; Clifford et al., 1987; Covolan and Mello, 2000; Fujikawa, 1996).

148 126 We next compared the effect of SE on cell density in individual counting frames to determine if SE-induced pyramidal cell loss was uniform within discrete structures. The data in Table 5.2 show that the extent of cell loss in individual counting frames was similar regardless of position within the specific hippocampal subfield or in the PPC, indicating that the effect of SE on neurodegeneration was independent of cell location within the region. As also illustrated in Table 5.2, the boundary between hippocampal subfields with respect to neurodegeneration was quite sharp. For example, in counting frame 3c, on the CA3 side of the border between CA3 and CA4, 29.9 ± 22.4% of the pyramidal cells were lost following SE, whereas in counting frame 4a, on the CA4 side of the border, the decrease was 64.3 ± 36.4% (p<0.05). Table 5.2: Comparison of SE-induced pyramidal cell loss in individual counting frames Brain Region Counting frame a Counting frame b Counting frame c CA ± ± ± 12.2 CA ± ± ± 22.4 CA ± ± PPC 85.3 ± ± ± 20.2 Individual counting frames were positioned in hippocampal subfields and the PPC as depicted in Figure 5.1 A and C. Numbers represent % decrease of pyramidal cells ± SD (n=5). Pyramidal cell loss within individual regions was similar regardless of the placement of the counting frame (one-way ANOVA).

149 127 Figure 5.1 Neurodegeneration depicted in NeuN-stained coronal sections of the rat dorsal hippocampus and posterior piriform cortex (PPC) 14 days following status epilepticus (SE). A, B: Representative images of the left dorsal hippocampus from a sham rat (A) or from a rat 14 days after SE (B). Open arrowheads define the borders of CA1, whereas the dotted line indicates the border between CA3 and CA4. Pyr depicts the pyramidal cell layer. C, D: Representative images of the PPC from a sham (C) or from a rat 14 days after SE (D). Boxes represent placement of counting frames in the CA1 (1a-c), CA2 (2a), CA3 (3a-c) and CA4 (4a,b) regions of the hippocampus, and in the PPC (a-c). Scale bar = 500 µm. Low-powered images captured at 25X magnification. E: Cell densities in sham (grey bars, n=5) and SE (black bars, n=5) animals at 2 weeks recovery (mean density ± SD). Significant pyramidal cell loss occurred in the CA1, CA3, CA4 and PPC, but not in the CA2. * depicts significant difference from naive animals (p<0.05).

150 128 E * * * *

151 Tat-NR2B9c reduces SE-induced neurodegeneration in the hippocampus We next determined the effect of Tat-NR2B9c on SE-induced neuronal loss. For these experiments, animals received Tat-NR2B9c, its inactive homologue Tat-NR2B9AA, or saline 3 hrs following the termination of SE, and cell densities were determined 2 weeks later. Results for individual animals are plotted in Figure 5.2. SE-induced cell loss in animals that received saline was similar to that reported above (compare Fig 5.2 and Fig 5.1). The administration of Tat- NR2B9c resulted in a statistically significant overall reduction in pyramidal cell loss in hippocampal subfields CA1 and CA4 relative to saline treated animals, although there was considerable animal to animal variation in the efficacy of the peptide. In CA1, the decrease in pyramidal cell density in animals that received Tat-NR2B9c was 38 ± 35% (mean± SD) as compared to a decrease of 66 ± 18% in the saline treated animals (p<0.05). Similarly in CA4, administration of Tat-NR2B9c reduced SE-induced cell loss from 60 ± 28% in saline controls, to 26 ± 39% in animals that received the peptide (p<0.05). In neither case did Tat-NR2B9AA, the inactive homologue of Tat-NR2B9c, exhibit any neuroprotective effect, consistent with the action of the active peptide being mediated via its interaction with PDZ domains. In contrast to the results for CA1 and CA4, Tat -NR2B9c failed to reduce overall cell loss in either CA3 or the PPC when compared to saline treated animals (Fig 2).

152 130 FIGURE 5.2 Tat-NR2B9c reduces pyramidal cell loss in the dorsal hippocampus when administered 3 hours after SE. Animal received saline, Tat-NR2B9c or Tat-NR2B9AA 3 hr following the termination of SE, and were sacrificed 14 days later. Coronal sections were stained with NeuN and cell densities determined as in Methods. A: Representative NeuN stained images of the pyramidal cell layer of indicated regions of the hippocampus and of layer II of the posterior piriform cortex (PPC). The scale bar = 20 µm. High-powered images captured at 400X magnification. B. Administration of Tat-NR2B9c reduced SE-induced cell loss in the CA1 and CA4, but not in the CA3 or PPC. Cell densities for individual animals were plotted as open circles. The dash represents the mean cell density for the group. Mean cell densities for each brain region were as follows. CA1: shams ( ± 4339 cells/mm 3, mean ± S.E.M., n=11), Saline (36699 ± 7002 cells/mm 3, n=8), Tat-NR2B9c (66049 ± cells/mm 3, n=10), Tat-NR2B9AA (30767 ± 7606 cells/mm 3, n=8). CA3: shams (62812 ± 1354 cells/mm 3 ), Saline (44618 ± 5702 cells/mm 3 ), Tat-NR2B9c (52916 ± 4124 cells/mm 3 ), Tat-NR2B9AA (41354 ± 6401 cells/mm 3 ). CA4: shams (67847 ± 2923 cells/mm 3 ), Saline (27391 ± 7657 cells/mm 3 ), Tat-NR2B9c ( ± 8238 cells/mm 3 ), Tat-NR2B9AA (32118 ± 7012 cells/mm 3 ). PPC: shams (49961 ± 3033 cells/mm 3 ), Saline (8611 ± 3991 cells/mm 3 ), Tat-NR2B9c (9104 ± 3691 cells/mm 3 ), Tat-NR2B9AA (12935 ± 3437 cells/mm 3 ). * denotes a significant difference from sham group (p<0.05), # denotes a significant difference from saline and Tat-NR2B9AA groups (p<0.05), ^ indicates a significant difference from saline group (p<0.05), and approaching significance with Tat-NR2B9AA group (p=0.08).

153 131

154 Preferential neuroprotection of Tat-NR2B9c is found within specific regions of the CA1 and CA3 The above results demonstrate that the administration of Tat-NR2B9c 3 hrs following SE resulted in reduced neurodegeneration in the CA1 and CA4 hippocampal subfields, but not in CA3 or the PPC. These findings were obtained by averaging the cell densities of all counting frames placed in the different brain regions. To determine if the neuroprotective effects of Tat- NR2B9c were uniform within individual regions, we compared the effect of Tat-NR2B9c on neuronal cell densities in individual counting frames. Consistent with the results presented in Table 5.2, the extent of SE-induced neurodegeneration was similar in each of the counting frames placed within CA1, CA3, and CA4 and in the PPC in animals that received either saline or the inactive peptide, Tat-NR2B9AA (Figure 5.3, grey and oblique stripped bars). However, in contrast to the uniform effect of SE on cell death, comparison of cell densities within individual counting frames identified differences in the neuroprotective actions of Tat-NR2B9c within CA1 and CA3. The neuroprotective effect of Tat-NR2B9c varied in the medial to lateral direction across CA1, increasing with proximity to the subicular border (Figure 5.3A, black bars). Thus, in the presence of the peptide, 81 ± 13% (86,226 ± 14,027 cells/mm 3 in animals that received Tat-NR2B9c vs 106,691 ± 5051 in control animals, p<0.05) of pyramidal neurons remained in counting frame 1a, adjacent to the subicular border, following SE, whereas only 44 ± 9% (47,685 ± 9,707 cells/mm 3 in animals that received Tat-NR2B9c vs 109,638 ± 4542 in control animals) remained in counting frame 1c, adjacent to CA2 (Figure 5.3A, black bars). Similarly, although Tat-NR2B9c had no overall protective effect in CA3 (Figure 5.2), comparison of individual counting frames showed that whereas there was no protective effect in the areas represented by counting frames 3a and 3b, there was nearly complete protection in the area represented by counting frame 3c, the area proximal to the transitional border with CA4 (Figure 5.3B, black bars) either CA4 or PPC. Differential neuroprotective effects of Tat-NR2B9c were not observed in

155 133 FIGURE 5.3 Tat-NR2B9c exhibits differential neuroprotection within different regions of the CA1 and CA3 subfields of the hippocampus. Neuronal densities in individual counting frames in hippocampal subfields were compared between treatment groups. Results are expressed as mean ± SEM. Open bars: shams (n = 11); grey bars: saline (n=8); black bars: Tat- NR2B9c (n=10); oblique stripped bars: Tat-NR2B9AA (n=8). * denotes significant difference from sham group, # denotes significant difference from saline and Tat-NR2B9AA groups, ^ indicates a significant difference from saline group (p<0.05), and approaching significance relative to Tat-NR2B9AA group (p=0.067). ** indicates significant difference between counting frames a and c.

156 134 A. CA cells/mm 3 x # * * * * * * ** * * * cells/mm 3 x 10-3 B. CA * ** * * * ** # * * * cells/mm 3 x a 1b 1c 3a 3b 3c C. CA4 D. PPC # * * * 4a *^ * * 4b cells/mm 3 x * ** * * * a b c * * *

157 Tat-NR2B9c did not provide neuroprotection in CA1 when administered during SE The above findings demonstrate that administration of Tat-NR2B9c 3 hrs following the termination of SE results in significant protection of pyramidal neurons in the hippocampus. We also determined if Tat-NR2B9c would provide protection if it was administered during SE. For these experiments, animals received Tat-NR2B9c after 10 minutes of continuous seizure activity, and SE was allowed to continue for an additional 50 minutes. Administration of the peptide at this time had no affect on seizure severity or survival rates (Table 5.1). The results presented in Figure 5.4 show that under these conditions Tat-NR2B9c offered no neuroprotection in CA1. Increasing the dosage of peptide from3 nmols/g to 9 Mols/g did not increase the neuroprotective properties of the peptide under these conditions.

158 136 FIGURE 5.4 Tat-NR2B9c is not neuroprotective when administered 10 minutes following the onset of SE. SE was induced as in Methods section and animals received saline or Tat-NR2B9c (3 or 9 nmol/gm) following 10 minutes of continuous behavioural seizures. Seizures were allowed to continue and SE was terminated after 60 minutes and animals sacrificed 14 days later. A: Representative images of NeuN stained pyramidal cells in the CA1 region of the dorsal hippocampus. The scale bar = 20 µm. High-powered images captured at 400X magnification. B: NeuN stained neurons in the CA1 were quantified as in Methods section and cell densities plotted. Cell densities for individual animals were plotted as open circles for sham (n=10) and saline (n=8) groups. Solid shapes denote cell densities for individual animals that received Tat-NR2B9c at doses of 3 nmols/gm (circles, n=4), or 9 nmols/gm (triangles, n=3). The dash represents the mean cell density for the group. Mean cell densities were: shams ( ± 4339 cells/mm 3, mean ± S.E.M.), Saline (36996 ± 7002 cells/mm 3 ), Tat-NR2B9c at 3nMol/gm (26427 ± 5847 cells/mm 3 ), Tat-NR2B9c at 9nMol/gm (28340 ± cells/mm 3 ). * denotes significant difference when compared to sham group (p<0.05).

159 137

160 Discussion In the current study we determined the ability of Tat-NR2B9c, a synthetic peptide designed to selectively disrupt the NMDAR signaling complex (Aarts et al., 2002), to reduce SE-induced neuropathology. Previously, it was reported that Tat-NR2B9c markedly reduced brain damage following an ischemic challenge (Aarts et al., 2002; Sun et al., 2008), while maintaining function of the NMDAR ion channel (Sattler et al., 1999; Aarts et al., 2002). Cui et al (Cui et al., 2007), analyzed the interaction of Tat-NR2B9c with all known potential human binding partners and found that it was highly specific for the PDZ2 domain of PSD-95, suggesting that its neuroprotective action is due to the disruption of protein interactions involving PSD-95. Although NMDAR antagonists are highly neuroprotective in rodent models of epilepsy, even when administered after the onset of SE (Fariello et al., 1989; Clifford et al., 1990; Fujikawa et al., 1994; Fujikawa, 1995), clinical trials with these compounds have failed because of poor tolerance and efficacy (Dyker et al., 1999; Davis et al., 2000; Albers et al., 2001; Ikonomidou and Turski, 2002; Muir, 2006). Tat-NR2B9c offers an alternative approach to disrupting NMDAR-mediated pro-death signaling, while sparing signaling pathways linked to survival and plasticity (Aarts et al., 2002; Arundine and Tymianski, 2003; Aarts and Tymianski, 2004) Tat-NR2B9c provided significant neuroprotection in the hippocampus The primary finding of the present study is that Tat-NR2B9c significantly attenuated hippocampal damage induced by an episode of SE. Importantly, substitution of the C-terminal ser and val of Tat-NR2B9c by alanine residues to prevent interaction with the PDZ domains of PSD-95 (Kornau et al., 1995; Aarts et al., 2002; Cui et al., 2007), completely eliminated the neuroprotective effect of the peptide, supporting the hypothesis that neuroprotection is due to disruption of protein interactions involving the PDZ domains of PSD-95. We initially reported that Tat-NR2B9c preferentially reduced the co-immunoprecipitation of NR2B and PSD-95 from rat brain extracts (Aarts et al., 2002). More detailed binding analysis subsequently demonstrated that Tat-NR2B9c has high affinity for the PDZ2 domain of PSD-95, and inhibits the binding of NR2 subunits, as well as nnos, to this domain with IC50 values in the low um range (Cui et al., 2007). Neuronal nitric oxide synthase (nnos) is a component of the NMDAR complex and is activated by the influx of Ca 2+ ions via the NMDAR ion channel (Forder and Tymianski, 2009). NO production is enhanced following SE (Gupta and Dettbarn, 2003), and inhibition of nnos is

161 139 neuroprotective (Montecot et al., 1998; Murashima et al., 2000) and antiepileptogenic (Rajasekaran et al., 2003; Sardo and Ferraro, 2007). Taken together, these findings support a model in which the neuroprotective actions of Tat-NR2B9c result from disruption of the NMDAR signaling complex, and in particular dissociation of nnos from PSD-95, by competing for PDZ domain binding sites on PSD-95, and are consistent with known mechanisms of SE-induced cell death (Arundine and Tymianski, 2003; McNamara et al., 2006) Regional specificity of neuroprotection by Tat-NR2B9c within CA1 and CA3 An unexpected observation was the regional specificity of neuroprotection by Tat-NR2B9c within CA1 and CA3, the greatest protection of pyramidal neurons within these regions occurring proximal to the CA1/subicular border and CA3/CA4 border, respectively. molecular basis for the differential neuroprotective effects of Tat-NR2B9c remains unclear but the finding suggests the presence of previously unidentified differences in the properties of these neurons. The severity of SE-induced neurodegeneration was similar across all counting frames within CA1 or CA3, indicating that the differential effects of Tat-NR2B9c cannot be explained by variable sensitivities to the initial insult. The projection patterns of pyramidal cells change gradually as one moves across CA3 from the border with CA2 to that with CA4 (Lorente De Nó, 1934; Li et al., 1994) and it is tempting to speculate that the increase in neuroprotection from CA3a (essentially no protection) to CA3c (nearly complete protection), may be related to the varying pattern of connectivity of CA3 pyramidal neurons. Alternatively, subunit composition is an important determinant of NMDAR function (Nakanishi et al., 1994; Cull-Candy et al., 2001) and may affect association of the receptor with members of the PSD-95 family of membrane associated guanylate kinases (Cousins et al., 2008; Cousins et al., 2009) as well as with downstream signaling molecules, such as nnos (Lynch and Guttmann, 2002; Al-Hallaq et al., 2007). The results therefore suggest the occurrence of differences between protected and nonprotected neurons in subunit composition of the NMDAR and/or in protein interactions involving PSD-95 within the NMDAR signaling complex. The Although differences in the subunit composition of NMDARs between hippocampal subfields and between dorsal and ventral CA1 have been reported (Coultrap et al., 2005; Pandis et al., 2006; Papatheodoropoulos, 2007), we are not aware of any studies examining either NMDAR subunit composition or the NMDAR

162 140 signaling complex within different regions of the same hippocampal subfield, and this remains an important subject for future investigation Neuroprotective effect of tat-nr2b9c is dependent on time of administration Although the administration of Tat-NR2B9c significantly reduced neuronal loss when the peptide was given after the termination of SE, it was ineffective if given during SE. Previous studies have shown that forty to sixty minutes of continuous seizure activity is required before neuron loss occurs (Nevander et al., 1985; Fujikawa, 1996), suggesting that molecular mechanisms that occur in the earlier stages of SE are distinct from the pro-death signaling that occurs following prolonged SE. In general accord with this suggestion, SE results in numerous changes in the composition and properties of the postsynaptic density, including the recruitment and activation of several signaling molecules (Niimura et al., 2005; Wyneken et al., 2001), enhanced phosphorylation of the NMDA receptor and other proteins (Niimura et al., 2005; Huo et al., 2006), and changes in protein interactions or molecular organization (Moussa et al., 2001). These seizure-induced changes to the composition and structure of the postsynaptic signaling apparatus may alter the way in which Tat-NR2B9c interacts with its binding partners and render the neuron more susceptible to the neuroprotective actions of the peptide. Alternatively, although peptides fused to the Tat protein transduction domain readily penetrate the blood brain barrier (Schwarze et al., 1999; van Vliet et al., 2007; Fabene et al., 2008) and enter neurons (Aarts et al., 2002), enhanced efficacy of Tat-NR2B9c at later times may also result from increased accessibility to neurons as a result of seizure-related damage to the blood brain barrier (Fabene et al., 2008). In contrast to the present findings, NMDA receptor antagonists were reported to provide protection to several brain regions, including the hippocampus and piriform cortex, when administered only 15 min into SE induced with pilocarpine (Fujikawa, 2004), or when administered 90 min into SE induced by kainic acid (Ebert et al., 2002; Brandt et al., 2003b). The difference between these results and those described here with Tat-NR2B9c presumably relate to the different mechanisms of action of the drugs, the antagonists preventing entry of Ca 2+ ions via the NMDA receptor ion channel and Tat-NR2B9c affecting signalling downstream of the receptor.

163 Conclusion In summary, the present study demonstrates that the use of Tat-NR2B9c for the targeted disruption of the NMDAR signaling complex represents a viable approach for limiting the neuropathological consequences of SE. Because preservation of neurons within the hippocampus reduces the behavioural consequences of SE (Brandt et al., 2006), we subsequently investigated whether Tat-NR2B9c also offered long-term protection against SE-induced neurological deficits (see chapters 6 and 7).

164 142 Chapter 6 Long-lasting behavioural and anxiolytic changes in rats following status epilepticus 6.1 Introduction Depression and anxiety disorders, followed by psychoses, are the most common interictal behavioural disturbances observed in patients with epilepsy (Boro and Haut, 2003; Devinsky, 2004a; Gaitatzis et al., 2004; Garcia-Morales et al., 2008). Moreover, several studies have found that these comorbid psychiatric disorders are an independent risk factor for a poor quality of life in epileptic patients (Cramer, 2002; Gilliam, 2002; Cramer et al., 2003; Boylan et al., 2004; Johnson et al., 2004). The prevalence rates of psychiatric comorbidities are significantly higher in temporal lobe epilepsy (TLE) than in other types of epilepsies (Perini et al., 1996; Matsuura et al., 2003; Garcia-Morales et al., 2008), with up to 50% of patients with TLE experiencing profound interictal disturbances in emotional behaviour (D. Blumer, G. Montouris and B. Hermann, 1995). The control of seizure activity with pharmacologic treatment does not always alleviate these disorders (Engel et al., 1991) and the causal relationship between TLE and psychiatric disorders is poorly understood (Devinsky, 2003; Swinkels et al., 2005). The lithium/pilocarpine model of SE in rats recapitulates most clinical and neuropathological features of human TLE (Curia et al., 2008). In adult rats, the injection of lithium and pilocarpine leads to status epilepticus (SE) and subsequent development of spontaneous recurrent seizures (SRSs) within 2 to 6 weeks. Recent literature suggests that the seizure-free period following SE, also referred to as epileptogenesis, is when neuropathological changes occur to develop an epileptic brain (McNamara et al., 2006; Curia et al., 2008; Pitkänen and Lukasiuk, 2009). During epileptogenesis, neuronal loss, gliosis, and synaptic reorganization participate in the constitution of a hyperexcitable circuit that underlies the occurrence of SRSs (Fujikawa, 2005; Acharya et al., 2008). The affective symptoms associated with epilepsy are believed to be secondary to seizures, and are also derived from these neuropathological changes (Sayin et al., 2004). Previous studies have shown that SE in rats causes extensive neuronal loss in the hippocampus, cortex, amygdala and thalamus (Honchar et al., 1983; Turski et al., 1983b; Covolan and Mello, 2006). These structures are known to be functionally important in anxiety and exploration (Davis, 1992; Degroot and Treit, 2002; Moreira et al., 2007; Carvalho et al.,

165 ), and damage to them following SE is likely to produce behavioural impairment. This idea is supported by findings that neuroprotection within these regions attenuates SE-induce anxiolytic changes in rats when tested in the elevated plus-maze and open field (dos Santos et al., 2005; Brandt et al., 2006). While administration of epileptogenic agents is known to produce long-term behavioural disruption (Rice et al., 1998; Narkilahti et al., 2003b; dos Santos et al., 2005; Detour et al., 2005; Szyndler et al., 2005; Brandt et al., 2006; de Oliveira et al., 2008; Cardoso et al., 2009), the relationship between behavioural disruption and the development of status epilepticus remains unclear. In chapter 4, we demonstrated that the majority of SE-induced neuronal death occurs within 3 days after the termination of SE. Because neurodegeneration is associated with cognitive and behavioural morbidity (Milgram et al., 1988; Mikati et al., 2001; Wu et al., 2001; dos Santos et al., 2005; Brandt et al., 2006), our first objective was to examine the time course of behavioural changes following SE. We assessed short-term behavioural disturbances at 1 and 2 weeks following SE, and long-term behavioural disturbances at 12 weeks to determine if these changes were persistent or were affected by the development of SRSs. Rats treated with the repeated low-dose pilocarpine (RLDP) protocol exhibited less severe status and a lower mortality rate compared to rats treated with the LDP protocol (see Results sections and 3.3.3). Although neuropathology in rats treated with the two seizure-inducing protocols was similar (see Results section 3.3.5), it is possible that differences in other factors not assessed (i.e., changes in synaptic plasticity, neurogenesis, synaptic reorganization) may be present and as a result, cause differences in behavioural responses in rats treated with the two procedures. Therefore, our second objective was to compare the subsequent behavioural effects of the two seizure-inducing protocols. Because we demonstrated protection within the CA1, CA3 and CA4 subfields of the dorsal hippocampus following administration of tat-nr2b9c (see Results 5.3.3), our third objective was to determine the effect of tat-nr2b9c on behaviour. Our behavioural assessment protocol consisted of two common tests for anxiety-like behaviour, the open-field (Prut and Belzung, 2003) and the elevated plus maze (File, 1993), and four hyperexcitability tests (Rice et al., 1998).

166 Methods Animals All procedures were approved by the University of Toronto Scarborough Animal Care Committee and were in accordance with the guidelines established by the Canadian Council on Animal Care. Male Wistar rats (Charles River Laboratories, Sherbrooke, Quebec, Canada) weighing between 300 and 350 g were individually housed with free access to food and water for at least 7 days in 12 h light/dark cycles before experimental use Induction of status epilepticus and administration of peptides Two separate studies were performed. In the first, the effects of the low-dose pilocarpine (LDP) and the repeated low-dose pilocarpine (RLDP) seizure-inducing protocols on behaviour were compared. SE was induced in adult Wistar rats ( g) using either the LDP or RLDP methods exactly as described in Methods section SE was terminated by the administration of diazepam (4 mg/kg, i.p.) at 1, 3 and 5 hours following the onset of SE. Ten animals developed SE following the LDP procedure and were used for the subsequent behavioural studies (Table 6.1). For the RLDP method, rats were pretreated with lithium chloride (3mEq/kg, i.p.) approximately 24 hours before and methylatropine nitrate (10 mg/kg, i.p) 30 min before the initial injection of pilocarpine. Pilocarpine (10 mg/kg, i.p.) was administered every 30 min as described by Glien et al., (2001) until the rat experienced a generalized, class 4/5 seizure, since rats generally developed SE shortly thereafter. Animals that did not develop SE within 30 min of the first class 4/5 seizure, received additional pilocarpine injections at 30-min intervals up to a maximum of 6 injections. Diazepam (4 mg/kg, i.p.) was administered at 1, 3 and 5 hours following onset of SE to terminate seizure activity. Twenty-seven animals developed SE using the RLDP procedure (Table 6.1) The second study assessed the effect of the peptide tat-nr2b9c on behavioural outcomes in rats following SE. SE was induced with the RLDP procedure and peptides, tat-nr2b9c and tat- NR2BAA, were administered as described in Methods section Two control groups were used: (1) Sham rats (n=19) received a mock femoral vein cannulation and were treated identically to SE animals except that they received saline in place of pilocarpine; they did not

167 145 develop SE. In a pilot study, sham rats were found to behave identically in the behavioural tests (open field, elevated-plus maze, and hyperexcitability tests) to naïve rats not subjected to the mock surgery, indicating that femoral vein cannulation had no effect on the behavioural outcome. (2) Pilo no-se rats (n=22) were treated with the RLDP protocol but did not develop SE. Figure 6.1 summarizes the SE-inducing protocols and experimental animal groups used in the behavioural studies (chapters 6 and 7). Animals were continuously monitored following the first injection of pilocarpine, and seizure activity was recorded as described in Methods section All rats received post-seizure care as described in Methods section

168 146 Figure 6.1 Schematic of SE-induction protocols and treatments in SE and non-se groups (see Methods section 6.2.2).

169 147

170 Behavioural tests Pre-observational period to behavioural testing All animals were observed for 1 hr prior to testing. If the rat was seizure-free within the observation period, the animal was tested as described below. If a rat exhibited recurrent seizures within the observation period, than the test was postponed until the following day. Any trial in which a rat exhibited SRSs was excluded from the final analyses of the data. The open field test and hyperexcitability tests were performed 1, 2 and 12 weeks following SE. The elevated plus maze was performed 12 weeks after SE. The elevated plus maze test was performed on separate days from the open field and hyperexcitability tests. In the behavioural studies, all SRSs observed during handling, during the testing or observation periods, or by direct observation of the rats in their home cages were recorded. A rat was considered epileptic after exhibiting one or more SRSs Open-field test The open field test measures anxiety-like behaviour and locomotor activity (Prut and Belzung, 2003). Rats were placed in the center of a square open field (diameter 100 cm) enclosed with walls 36 cm high, and created from clear Plexiglas. The apparatus was divided into 16 equally spaced squares, with the 4 centre squares defined as the centre arena. The open field was placed inside a light- and sound-attenuated room. Before each trial, the field was cleaned with 70% alcohol solution. Behaviour was recorded with a video camera for 10 min, and later analyzed independently by 2 experimenters blinded to animal treatment. Experimenters were not present in the room during the test. For each rat, the total number of squares crossed (with four paws) in the peripheral and center regions of the open field, the number of entries (with four paws) into the centre arena, and the frequency and duration of rearing activity was assessed Elevated plus maze test The elevated-plus maze is used to assess anxiety in rodents (Hogg, 1996). The procedure consists of allowing rats to freely explore two elevated open and two elevated closed arms. In such a situation, rats spontaneously prefer the darker closed arms and avoid the anxietyprovoking open arms. The apparatus consisted of painted black wood in the form of two open

171 149 arms (50 X 10 cm), and two enclosed arms (50 X 10 X 40 cm), annexed to a central platform (10 X 10 cm) to form a plus sign. Grip was facilitated by lining the enclosed and open arms with black textured rubber. The apparatus was elevated 50 cm above the floor level. The test was conducted inside a light- and sound-attenuated room, without the presence of an experimenter. Before each trial, the maze was cleaned thoroughly with 70% alcohol. At the beginning of the trial, rats were placed on the central platform always facing the same closed arm. Behaviour was recorded with a video camera for 5 min, and later analyzed independently by 2 experimenters. For each rat, the time spent and number of entries (with four paws) in different sections of the maze (open and closed arms), frequency of risk assessments (with two paws in the open arm, head extended over the ledge), and rearing frequency was assessed. Occasionally, SE groups fell from the ledge of the outer arm to the floor of the elevated plus maze. In these cases, the SE groups were retested the following day; if the animal fell during the second trial, the data was eliminated from final analyses Hyperexcitability tests Rice et al., (1998) described four behavioural tests that potentially discriminate hyperexcitability differences between SE groups and non-se rats. These tests were taken from the functional observational battery described by Moser et al., (1988). The four tests are described below: 1. Approach-response test: A pen held vertically is moved slowly toward the face of the animal. Responses were scored as 1, no reaction; 2, the rat sniffs at the object, 3, the rat moves away from the object; 4, the rat freezes; 5, the rat jumps away from the object; and 6, the rat jumps at or attacks the object. 2. Touch-response test: The animal is gently prodded in the rump with the blunt end of a pen. Responses were scored as 1, no reaction; 2, the rat turns toward the object; 3, the rat moves away from the object; 4, the rat freezes; 5, the rat jerks around toward the touch; 6, the rat turns away from the touch; and 7, the rat jumps with or without vocalizations. 3. Finger-snap test: A finger snap several inches above the head of the animal is performed. Responses were scored as 1, no reaction; 2, the rat jumps slightly or flicks the ear (normal reaction); and 3, the rat jumps dramatically. 4. Pick-up test: The animal is picked up by grasping around the body. Responses were scored as 1, very easy; 2, easy with vocalizations; 3, some difficulty, the rat rears and

172 150 faces the hand; 4, the rat freezes (with or without vocalizations); 5, difficult, the rat avoids the hand by moving away; and 6, very difficult, the rat behaves defensively, and may attack the hand. A minimum of three observers independently recorded the behaviour for each rat, and the means of their scores were calculated for each animal in each test. The tests were accomplished in the home cage, and were conducted at least 30 min apart Statistical Analysis Analysis of variance (ANOVA) and chi-square analysis were performed using Statistica 9.0 software. Non-parametric testing was performed using GraphPad Prism 5 software. Significance was set at a p-value of 0.05 or less.

173 Results SE induction SE induction rates and mortality rates between groups were assessed using chi-square analysis. The number of pilocarpine injections administered in the RLDP protocol and weight gain between groups was assessed by one-way ANOVA. As demonstrated in Table 6.1, there was no significant difference between groups with respect to seizure susceptibility, as assessed by the number of repeated low-dose pilocarpine injections (10 mg/kg) administered in the RLDP protocol, and by the percentage of rats entering SE. By 3 months after SE, however, rats treated with the LDP protocol exhibited a higher mortality rate compared to rats treated with the RLDP protocol. All experimental groups experienced similar weight gain at 3 months following SE. By 3 months, all rats that developed SE were observed to exhibit at least 1 recurrent seizure (stage 4/5), and therefore were considered to be epileptic. Survived 4 Died 4 Weight 5 Table 6.1: Comparison of experimental groups in seizure susceptibility and mortality at 3 months following SE Treatment Total # # of Latency to SE SE of rats pilocarpine onset for LDP induction 3 (g) per group injections for RLDP protocol 1 protocol 2 (min) Saline RLDP ± (67%) 27 9 (25%)* 550 ± 43 (Saline SE) Tat-NR2B9c ± (70%) 17 6 (26%)** 548 ± 29 SE Tat ± (66%) 23 7 (23%)* 545 ± 41 NR2BAA SE Wistar LDP ± (68%) 10 7 (41%) 552 ± 60 Sham ± 49 Pilo non-se ± Number of low dose pilocarpine injections (10 mg/kg) administered in the RLDP protocol (average ± SD). 2. Latency to SE onset in the LDP protocol (average ± SD). 3. Number of animals that developed SE for 60 min. Numbers in () represent % of animals that entered SE. 4. Number of animals that surivived or died within 3 months following SE. Numbers in () represent the % of animals that died. * Lower mortality rate detected in rats treated with the RLDP protocol compared with the LDP protocol p<0.005, **p< Weight in grams (average ± SD) at 3.5 months after SE. Data assessed using one-way ANOVA or chi-square analysis.

174 Open field test The open field data was first assessed by repeated measures ANOVA followed by the Newman- Keuls post-hoc test to determine group differences. No significant effect in locomotor activity as assessed by the total number of squares crossed between SE and non-se groups, week at which the test was conducted, and no significant interaction between test week and group was detected. These results indicate no presence of motor impairment between groups or between testing sessions. However, there were group differences in rearing. SE groups exhibited a lower frequency and a lower duration of rearing activity when compared to non-se groups within the first week after SE, and this difference in behaviour remained unchanged in the second and twelfth week (Figure 6.2, A,B). No difference was detected between SE groups and non-se groups in the number of entries into the centre, or the number of centre squares crossed (Figure 6.2, C,D). Instead, all groups showed substantially higher locomotor activity in the peripheral region of the open field (Figure 6.2, E). Rats treated with either the LDP or the RLDP seizureinducing protocol exhibited similar performance in the open field test (Figure 6.3).

175 153 Figure 6.2: The effect of SE on behaviour in the open field. SE was induced with either the RLDP (n=27) or the LDP (n=10) procedure and terminated after 60 min as in Methods section Non-SE control groups included pilo no-se rats (n=22) treated with pilocarpine and did not develop SE, and sham rats (n=19) treated with saline instead of pilocarpine. Behaviour in the open field assessed 1 week (black bars), 2 weeks (grey bars) and 12 weeks (oblique-striped bars) after SE (see Methods section ). (A) Illustrates the frequency of rearing during the 10-min trial. (B) Illustrates the total duration of rearing activity (sec). (C) Illustrates the frequency of entries (with four paws) into the centre square. (D) Illustrates number of centre squares crossed (with four paws). (E) Illustrates number of peripheral squares crossed (with four paws). (F) Illustrates total number of squares crossed. * Significant difference from non-se control groups across all three times assessed (p<0.05). Bars represent group average ± SEM. Data analyzed by one-way ANOVA followed by Newman-Keuls post-hoc test.

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177 Hyperexcitability tests Because behaviour in the hyperexcitability tests was assessed using a 3- to 6- grade ordinal scale, data from each week assessed was first analyzed using the non-parametric Kruskall-Wallis ANOVA by ranks, followed by the Dunn s test to determine individual group differences. Among the four hyperexcitability tests described by Rice et al., (1998), SE groups and non-se groups exhibited similar responses on the approach-response test and the finger-snap test (Figure 6.3, A, C). By contrast, a clear difference was observed in the touch-response test in that the SE groups were significantly more touch-sensitive than the non-se groups (Figure 6.3, B). In addition, the SE groups reacted more aggressively than the non-se groups in the pick-up test (Figure 6.3, D). The difference in responses between SE and non-se groups was detected within the first week after SE, and persisted in the second and twelfth week. The nonparametric Friedman s test was used to measure the performance stability for each group all along the 3 testing sessions. This analysis showed that for each group, animal responses in the first week did not change in the second and twelfth week in any of the four hyperexcitability tests. The behaviour in the hyperexcitability tests of the RLDP SE group was indistinguishable from the LDP SE group.

178 156 Figure 6.3 The effect of SE on behaviour in the four hyperexcitability tests. SE was induced with either the RLDP (n=27) or the LDP (n=10) procedure and terminated after 60 min as in Methods section Non-SE control groups included pilo no-se rats (n=22) treated with pilocarpine and did not develop SE, and sham rats (n=19) treated with saline instead of pilocarpine. Behaviour in the hyperexcitability tests assessed 1 week (black bars), 2 weeks (grey bars) and 12 weeks (oblique-striped bars) after SE. See Methods section for behavioural responses assessed in the following tests: (A) approach-response test; (B) touch-response test; (C) finger-snap test; (D) pick-up test. * Significant difference from non-se control groups across all three times assessed (p<0.05). Bars represent average ± SEM. Data analyzed by Kruskal-Wallis (ANOVA for non-parametric data) test followed by Dunn s test for individual differences.

179 157

180 Elevated-plus maze: Behaviour in the elevated-plus maze was determined 12 weeks following SE and data was first analyzed using one-way ANOVA. The Newman-Keuls post-hoc test detected differences between SE groups and non-se groups. Similar to the findings in the open field test, SE groups showed attenuated rearing activity when compared to non-se groups in the elevated plus maze (Figure 6.4, A). The frequency of risk assessments was also decreased in the SE groups, indicating decreased exploratory behaviour (Figure 6.4, B). Non-SE groups spent most of their time within the protected closed arms of the elevated-plus maze (Figure 6.4, C-F). However, unlike controls, the SE groups spent a greater amount of time in the open unprotected arms, and had fewer entries into the closed arms of the maze (Figure 6.4, C-F), indicating decreased anxiety-like behaviour. Rats treated with either the LDP or the RLDP protocol had similar behavioural responses in the elevated-plus maze. Some SE groups failed to remain on the elevated-plus maze, and fell off the ledge of the open arm in two separate 5-min trials. The results from these incomplete trials were eliminated from the final analyses. Chi-square analysis revealed no statistically significant difference between SE groups in terms of the number of rats excluded: 3 from the LDP SE experimental group, 6 from the RLDP SE (or saline SE ) group, 2 from the tat-nr2b9c SE group, and 3 from the tat-nr2baa SE group. All rats in the non-se groups remained on the elevated-plus maze during the duration of testing.

181 159 Figure 6.4 The effect of SE on behaviour in the elevated-plus maze. SE was induced with either the RLDP (grey bar, n=21) or the LDP (checker-patterned bar, n=7) procedure and terminated after 60 min as in Methods section Non-SE control groups included pilo no- SE rats (white bar, n=22) treated with pilocarpine and did not develop SE, and sham rats (black bar, n=19) treated with saline instead of pilocarpine. Behaviour in the elevated-plus maze assessed 12 weeks after SE (see Methods section ). (A) illustrates the frequency of rearing during the 5-min trial. (B) illustrates the frequency of lookouts (with two paws in the open arm, head extended over the ledge). (C) illustrates the frequency of entries (with four paws) into the open arm. (D) illustrates the frequency of entries (with four paws) into the closed arm. (E) illustrates time spent in the open arm (sec). (F) illustrates time spent in the closed arm (sec). * Significant difference from non-se control groups (p<0.05). ** Significant difference from sham group. Bars represent average ± SEM. Data assessed by one-way ANOVA followed by Newman-Keuls multiple comparison post-hoc test.

182 160

183 The effect of Tat-NR2B9c on behaviour following SE In chapter 5, we demonstrated that tat-nr2b9c is neuroprotective within the CA1, CA3 and CA4 pyramidal cell layers of the dorsal hippocampus. Because the dorsal hippocampus is involved in behaviours such as anxiety and exploration (Davis, 1992; Moreira et al., 2007; Carvalho et al., 2008; Degroot and Treit, 2002), we determined whether the neuroprotection provided by tat- NR2B9c would mitigate SE-induced behavioural morbidity. The results showed that administration of tat-nr2b9c did not alter the behavioural outcomes in the open field test (figure 6.5), the hyperexcitability tests (figure 6.6) or the elevated-plus maze test (figure 6.7). Data for each test was analyzed as described above.

184 162 Figure 6.5 Tat-NR2B9c had no effect on behaviour in the open field. In drug-treated animals, tat-nr2b9c (n=16), tat-nr2baa (n=19) or saline (n=27) was administered 3 hr following the termination of SE induced by the RLDP procedure (see Methods section 6.2.2). Shams (n=19) received saline instead of pilocarpine. Behaviour in the open field assessed 1 week (black bars), 2 weeks (grey bars) and 12 weeks (oblique-striped bars) after SE (see section ). (A) illustrates the frequency of rearing during the 10-min trial. (B) illustrates the total duration of rearing activity (sec). (C) illustrates the frequency of entries (with four paws) into the centre square. (D) illustrates number of centre squares crossed (with four paws). (E) illustrates number of peripheral squares crossed (with four paws). (F) illustrates total number of squares crossed. * Significant difference from shams across all three times assessed (p<0.05). Bars represent average ± SEM. Data analyzed by one-way ANOVA followed by Newman-Keuls post-hoc test.

185 163

186 164 Figure 6.6 Tat-NR2B9c had no effect on behaviour in the four hyperexcitability tests. In drug-treated animals, tat-nr2b9c (n=16), tat-nr2baa (n=19) or saline (n=27) was administered 3 hr following the termination of SE induced by the RLDP procedure (see Methods section 6.2.2). Shams (n=19) received saline instead of pilocarpine. Behaviour in the hyperexcitability tests assessed 1 week (black bars), 2 weeks (grey bars) and 12 weeks (obliquestriped bars) after SE. See section for behavioural responses assessed in the following tests: (A) approach-response test; (B) touch-response test; (C) finger-snap test; (D) pick-up test. * Significant difference from shams across all three times assessed (p<0.05). Bars represent average ± SEM. Data analyzed by Kruskal-Wallis (ANOVA for non-parametric data) test followed by Dunn s test for individual differences.

187 165

188 166 Figure 6.7 Tat-NR2B9c had no effect on behaviour in the elevated-plus maze. In drugtreated animals, tat-nr2b9c (black bars, n=14), tat-nr2baa (oblique-striped bars, n=16) or saline (grey bars, n=21) was administered 3 hr following the termination of SE induced by the RLDP procedure (see Methods section 6.2.2). Shams (white bar, n=19) received saline instead of pilocarpine. Behaviour in the elevated-plus maze assessed 12 weeks after SE (see section ). (A) illustrates the frequency of rearing during the 5-min trial. (B) illustrates the frequency of lookouts (with two paws in the open arm, head extended over the ledge). (C) illustrates the frequency of entries (with four paws) into the open arm. (D) illustrates the frequency of entries (with four paws) into the closed arm. (E) illustrates time spent in the open arm (sec). (F) illustrates time spent in the closed arm (sec). * Significant difference from shams (p<0.05). Bars represent average ± SEM. Data assessed by one-way ANOVA followed by Newman-Keuls multiple comparison post-hoc test.

189 167

190 Discussion In the present study, the behavioural responses of rats were assessed at 1 week, 2 weeks and 12 weeks following SE in the open field test and in four hyperexcitability tests, and at 12 weeks in the elevated plus maze test (described in Methods section 6.2.3). The study showed (1) that SE in rats causes anxiolytic changes in behaviour when assessed in the open field and elevated-plus maze, and increased hyperexcitability in the touch-response test and in the pick-up test; (2) these behavioural changes appear within the first week after SE and persist with the occurrence of SRSs and; (3) treatment with tat-nr2b9c did not modify the post-se behavioural response. These findings are discussed below SE causes anxiolytic changes in behaviour and increased hyperexcitability In the present study, we demonstrated that rats following SE showed anxiolytic changes in behavior and increased hyperexcitability. Although previous findings have reported behavioural changes in rats following SE (Detour et al., 2005; dos Santos et al., 2005; Szyndler et al., 2005; Brandt et al., 2006; Cardoso et al., 2009; Groticke et al., 2008), there are some differences between our observations and previously published results. In the open field and elevated-plus maze, we observed decreased rearing activity following SE. This observation is similar to the findings reported in Dos Santos et al., (2006) with Wistar rats using high-dose pilocarpine (320 mg/kg), and in Groticke et al., (2008) with mice using intrahipppocampal injection of kainate, and has been interpreted as indicating that rats following SE display hyporeactivity to a stressful environment (dos Santos et al., 2005; Groticke et al., 2008). On the contrary, elevated rearing activity in Sprague Dawley rats was reported after SE induced by the low-dose pilocarpine procedure (Detour et al., 2005), and after self-sustaining status epilepticus (SSSE) induced by electrical stimulation of the basolateral amygdaloid nucleus (Brandt et al., 2006); in these studies, increased rearing activity was suggested to reflect disinhibited hyperactive behaviour (dos Santos et al., 2005). Although our results and previous findings demonstrated anxiolytic behavioural changes in the elevated-plus maze, with SE groups spending a greater amount of time in the unprotected open arm (dos Santos et al., 2005; Detour et al., 2005; Brandt et al., 2006; de Oliveira et al., 2008; Cardoso et al., 2009), Groticke et al., (2008) failed to find behavioural changes with this task in SE mice. Additional behavioural responses not observed in the current study but previously reported include increased locomotor activity (Milgram et al.,

191 ; Santos et al., 2000; Kubova et al., 2004; Sayin et al., 2004; Detour et al., 2005; dos Santos et al., 2005; Szyndler et al., 2005; Brandt et al., 2006; Müller et al., 2009; Cardoso et al., 2009; Sun et al., 2009), and elevated activity in the central square of the open field (Detour et al., 2005; Szyndler et al., 2005; Brandt et al., 2006). For the hyperexcitability tests, our results are similar to Rice et al., (1998) with Sprague-Dawley rats using high-dose pilocarpine (320 mg/kg), demonstrating that SE groups scored significantly higher on the touch-response and pick-up tests. In contrast, Brandt et al.,(2006) only detected a significant difference in the touch-response test in Sprague-Dawley rats after SSSE induced by electrical stimulation of the basolateral amygdaloid nucleus. Because rat strain or use of different seizure protocols have been shown to result in differences of behavioural outcomes (Cardoso et al., 2009; Hort et al., 2000), these factors may account for the differences between our findings and previously published results. An alternative possibility is that our results differ from others because the rats were retested multiple times in the open field and in the hyperexcitability tests. Previous studies have shown that repeated testing in the open field can result in decreased open field activity directed to searching the environment, and is considered an adaptive response (Stafstrom et al., 1993; Erdogan et al., 2005). However, we detected no behavioural differences between test times in SE and non-se animal groups, and consider that the interval between each test session was long enough not to cause habituation in the open field or in the hyperexcitability tests. In chapter 3, we demonstrated that rats treated with the RLDP protocol exhibited a lower mortality rate and less severe seizure activity compared to rats treated with the LDP protocol. Despite these differences, we found that the RLDP SE groups and the LDP SE groups exhibited similar behavioural responses in the elevated-plus maze, open field and hyperexcitability tests. The significance of these findings is discussed in greater detail in section 7.4.1, but in general they support the conclusion that the RLDP protocol is a better method for generating seizures. Although the open field and elevated-plus maze both measure behavioural responses to anxiety and stress in rodents, we found that the elevated-plus maze was the more sensitive test in distinguishing SE groups from controls. As discussed above, we found SE groups to be different from non-se groups in several behavioural measures assessed with the elevated-plus maze. Briefly, these differences consisted of SE groups exhibiting a decrease in rearing activity, a lower frequency of outlooks, less entries and time spent in the closed arm, and increased entries

192 170 and time spent in the open arm. Although rearing activity in SE groups was attenuated in the open field, other parameters assessed (i.e., entries and activity in the center of the maze) were found to be similar to non-se groups. In general, we found that with the lithium/pilocarpine model, the elevated-plus maze was more sensitive in detecting behavioural alterations in rats caused by SE than the open field Behavioural changes in rats following SE are long-lasting SE-induced behavioural alterations in the hyperexcitability tests and in the open field test appeared within the first week after SE and were still apparent 12 weeks later, indicating that these changes were unaltered by development of SRSs. Our results are in agreement with Rice et al., (1998) who detected behavioural changes in the touch-response and pick-up tests as early as 2 days after SE. Severe and persistent behavioural changes are also reported in other seizure models. For instance, kainic acid-treated rats showed increased hyperreactivity in response to handling and increased activity in the open field 24-hours after SE, with behavioural changes present 2 months later (Milgram et al., 1988). Many authors have suggested that early behavioural changes are caused by neuronal injury resulting from continuous seizure activity persisting longer than 30 minutes (Milgram et al., 1988; dos Santos et al., 2005; Detour et al., 2005; Szyndler et al., 2005; Brandt et al., 2006; Groticke et al., 2008). The hippocampus and amygdala strongly mediate the unconditioned fear response resulting from exposure to a threatening situation (Pitkanen et al., 2000; Kjelstrup et al., 2002). The ventral hippocampus projects to the prefrontal cortex and is closely connected to the bed nucleus of the stria terminalis and the amygdala, as well as other subcortical structures which are associated with the hypothalamic-pituitary-adrenal axis (Pitkanen et al., 2000; Bannerman et al., 2004). Most of the amygdaloid nuclei have some reciprocal projections with the hippocampal formation, although this is most pronounced for the basal and lateral nuclei (Pitkanen et al., 2000; Bannerman et al., 2004). Detour et al., (2005) suggested that disruption of these networks which are involved in fear expression causes misevaluation of threatening situations, which could in turn reduce anxiety (as we observed in the open field and elevated-plus maze), and enhance impulsive maladapted behaviour (as we observed in the hyperexcitability tests).

193 171 Because we conducted an intensive time-course evaluation of SE-induced neurodegeneration (refer to chapter 4), we were able to compare the progression of neuron loss with the occurrence of behavioural changes in rats following SE. We demonstrated that extensive neuronal loss occurs within the hippocampus, amygdala, thalamus and piriform cortex as early as 3 hrs after SE, with the majority of damage present by 3 days (see chapter 4, section 4.3.2). The acute loss of neurons we observed in rats after SE is consistent with the early development of behavioural deficits described by Rice et al., (1998), by Milgram et al., (1988), and with our results in the present study (see above). Although we are unable to link neuropathology with behavioural disruption, our data suggest that the occurrence of neuronal loss caused by SE is accompanied by early behavioural disturbances, and that these behavioural changes are not altered by the occurrence of SRSs Treatment with tat-nr2b9c did not have neuroprotective effects as assessed behaviourally. In Chapter 5, we reported that administration of tat-nr2b9c resulted in decreased loss of pyramidal cells in the CA1, CA3, and CA4 subfields of the dorsal hippocampus. In the present study, we investigated whether this protection was associated with reversal of SE-induced behavioural changes. Although traditionally associated with learning and memory, recent studies show that the dorsal hippocampus also exhibits a modulatory role in anxiety-related behaviours. For instance, pharmacological stimulation of the GABAergic, serotonergic or cholinergic receptor systems in the dorsal hippocampus produces robust anxiolytic effects in several animal models of anxiety (Engin and Treit, 2007). Similarly, lesion studies by Bannerman et al., (1999) in either dorsal or ventral hippocampus produces anxiolytic effects in the elevated-plus maze (Bannerman et al., 1999). More specifically, Menard et al., (2001) demonstrated that the pyramidal cells within the dorsal hippocampus sends glutamatergic efferents to the septal nucleus, and that these regions act in concert to regulate some (open arm avoidance in elevated-plus maze test) but not all (e.g. shock-probe burying test) anxiety-related behaviours (Menard and Treit, 2001). Despite the evidence that the dorsal hippocampus is involved in anxiety-related behaviours, we found that the degree of neuroprotection provided by tat-nr2b9c within this region had no effect on any of the behavioural tests analyzed. While some studies support a role for neuroprotection in the improvement of behavioural functioning, other studies have failed to establish this connection. Congruent with our findings,

194 172 Narkilahti et al., (2003b) demonstrated that although inhibition of caspase-3 after SE provided partial protection to CA1 and CA3c, it did not modify behavioural deficits as assessed in the open field and elevated-plus maze. In contrast, Brandt et al., (2006) demonstrated that nearly complete neuroprotection within the hippocampus in valporate-treated rats reduced anxiolytic effects in the open field, in the elevated-plus maze and in the touch-response test. Dos Santos et al., (2005) also demonstrated that treatment with cycloheximide four hours after SE prevented tissue shrinkage of the dorsal hippocampus, and spared behavioural functioning in the open field and in the elevated-plus maze. A possible explanation for the difference in findings between these studies may be related to variations in the amount of protection under the different conditions. While Brandt et al., (2006) and dos Santos et al., (2005) observed nearly complete preservation of the dorsal hippocampus, only partial protection was described by Narkilahti et al., (2003b) and by us (see chapter 5). It is also possible that the various treatments resulted in differential protection of other brain regions relevant to the behaviours analyzed, and/or affected other neurpathological factors that may contribute to altered behaviour (i.e., neurogenesis and synaptic plasticity). The administration of Tat-NR2B9c did not prevent the development of SRSs, in spite of its neuroprotective effect. All rats that developed SE were determined to be epileptic within 3 months regardless of whether or not they received the peptide. This result is in line with several other studies in which neuroprotection did not prevent the development of epilepsy (Andre et al., 2001; Brandt et al., 2003b; Brandt et al., 2006), and indicates that other factors such as axonal sprouting, neurogenesis and/or synaptic reorganization may play a more important role in the development of SRSs (Curia et al., 2008). The effect of tat-nr2b9c on these processes has yet to be assessed Conclusion In the present study, we found that treatment of rats with lithium and pilocarpine produced longlasting behavioural modifications that appeared within the first week after SE, and remain unchanged by development of SRSs at 3 months. These modifications consisted of decreased exploratory behaviour as assessed in the open field test, and increased hyperreactivity as assessed in the pick-up test and in the touch-response test. SE also produced anxiolytic effects as measured in rats by the elevated-plus maze at 3 months following treatment. The behavioural

195 173 effects were accompanied by histopathological changes in the brain structures (i.e., hippocampus, thalamus, amygdala, and piriform cortex) involved in the control of exploration and anxiety. However, further research is necessary to establish the link between early SEinduced histopathological changes and behavioural alterations. In sum, the obtained results indicate that lithium/pilocarpine-induced SRSs are also accompanied by profound behavioural disturbances, and thus could model anxiety-related disorders observed in humans with TLE.

196 174 Chapter 7 The effect of SE on performance in the Morris water maze and use of exploratory strategies 7.1 Introduction Impaired learning and memory is common in patients with epilepsy (Motamedi and Meador, 2003; Vingerhoets, 2006). The type of neuropathology associated with epilepsy may affect the type of cognitive dysfunction. For instance, temporal lobe epilepsy (TLE) with hippocampal sclerosis is often associated with memory impairment (Motamedi and Meador, 2003). The most reliable observations in TLE patients are deficits in declarative memory (ability to acquire facts and events related to one s personal past, which is often compared with visual-spatial learning in rats; (Guerreiro et al., 2001) and in the performance of visuospatial tasks (Hermann et al., 1997; Gleissner et al., 1998; Abrahams et al., 1999)). When administered systemically pilocarpine results in a condition similar to human TLE (Honchar et al., 1983; Turski et al., 1989; Persinger et al., 1993; Curia et al., 2008). Rats receiving pilocarpine undergo convulsive status epilepticus (SE), which is followed by development of spontaneous recurrent seizures (SRSs), as well as memory and learning impairment (Persinger et al., 1993; Rice et al., 1998; Curia et al., 2008). The Morris water maze (MWM) has frequently been used to investigate spatial learning and memory (Morris, 1984; Vorhees and Williams, 2006). During testing, a rat is placed into a pool of water and swims to find a hidden (submerged) platform. It is now recognized that acquisition of the water maze task has two main components: (1) procedural strategy learning and (2) spatial learning (Morris and Frey, 1997; Inglish and Morris, 2004). The first component involves learning behavioural strategies that allow the rat to move around in its spatial environment and to learn the most effective strategies for finding and reaching its target. To accomplish this, the rat must learn to search away from the pool wall (where the escape platform is never placed), realize that the escape platform offers the only refuge in the pool, and suppress attempts to climb out of the pool by clawing at the wall (Whishaw, 1989). During spatial learning the rat builds a spatial cognitive map correlating context information (extramaze cues) with platform location (Morris, 1984; DiMattia and Kesner, 1988; Fenton and Bures, 1993). Once this map is created, the rat is able to swim directly to the escape platform from any point of the circumference of the tank. Success in the spatial learning component requires prior learning

197 175 of the procedural-strategy component (Morris, 1989;; Whishaw, 1989; Saucier and Cain, 1995; Bannerman et al., 1995; Baldi et al., 2003). Although previous studies have showed impaired spatial learning during MWM testing in rats following SE (Persinger et al., 1993; Rice et al., 1998; Hort et al., 1999; Hort et al., 2000; Wu et al., 2001; McKay and Persinger, 2004; dos Santos et al., 2005; Frisch et al., 2007; Zhou et al., 2007; Cunha et al., 2009;), little attention has been given to the effect of SE on search strategy use. In the present study, we conducted a more refined analysis that included assessment of the acquisition and application of search strategies in SE rats for several reasons. First, there appear to be separate neural systems required for spatial learning and procedural-strategy learning (McDonald and White, 1994; McDonald and White, 1993; Miranda et al., 2006), and both systems are involved in acquisition of the MWM task. Evidence strongly supports the role of the hippocampus and posterior parietal cortex in spatial mapping and memory (Cain et al., 2006). Conversely, the prefrontal cortex, caudate nucleus, cerebellum and medial thalamus appear to be involved in acquisition of procedural-search strategies (Packard and McGaugh, 1996; Leggio et al., 1999; Cain et al., 2006). Because all of these brain regions are damaged in rats following SE (Turski et al., 1983b; Clifford et al., 1987; Fujikawa, 1996;), the impaired spatial learning of these animals is most likely attributed not only to poor spatial memory, but also to impaired use of search strategies. Second, Brody et al., (2006) demonstrated that poor search strategy use largely contributed to impaired MWM performance in transgenic mice following traumatic brain injury (TBI). Since TBI results in diffuse brain damage comparable to the pattern of injury we reported in rats following SE (see chapter 4), use of less efficient search strategies may similarly underlie impaired MWM performance in SE animals. Finally, the clinical relevance of this analysis is supported by impaired cognitive strategy use detected in humans following traumatic brain injury (Kennedy et al., 2003; Burke et al., 2004; Salmond et al., 2005) and in patients with TLE (Kennedy et al., 2003; Burke et al., 2004; Salmond et al., 2005; Butman et al., 2007; Labudda et al., 2009). Our first objective was therefore to investigate the effect of SE on performance in the MWM, with particular emphasis on search strategy use. We analyzed the predominate search strategy used in each trial during hidden platform testing according to the schema proposed by Graziano et al., (2003). In chapter 3, we demonstrated that rats treated with the RLDP protocol exhibited less severe seizures and had a lower mortality rate compared to rats treated with the LDP protocol. To

198 176 determine whether the advantage in using the RLDP protocol to reduce mortality results in similar cognitive and behavioural changes as compared to the LDP protocol, our second objective was to compare the MWM performance of rats following SE induced by the two procedures. Finally, studies have reported that neuroprotection within the hippocampus following SE improves performance in visual-spatial learning (Rice et al., 1998; Yang et al., 2007; Cunha et al., 2009; Jun et al., 2009). Because we demonstrated neuroprotection within the CA1, CA3 and CA4 subfields of the dorsal hippocampus following administration of tat- NR2B9c (see chapter 5), brain regions implicated in spatial learning (Broadbent et al., 2004; Gaskin et al., 2009), our third objective was to determine whether protection within these regions would improve MWM performance in SE rats. 7.2 Methods Animals All procedures were approved by the University of Toronto Scarborough Animal Care Committee and were in accordance with the guidelines established by the Canadian Council on Animal Care. Male Wistar rats (Charles River Laboratories, Sherbrooke, Quebec, Canada) weighing between 300 and 350 g were individually housed with free access to food and water for at least 7 days in 12 h light/dark cycles before experimental use. All experiments were conducted during daylight hours between 7 a.m. and 7 p.m Induction of status epilepticus and administration of peptides SE was induced using both the low-dose pilocarpine (LDP) and the repeated low-dose pilocarpine (RLDP) procedures, and peptides (tat-nr2b9c or tat-nr2baa) were administered as described in chapter 6 (Methods section 6.2.2). Saline control rats were treated identically except that they received saline (0.3 ml) following SE in place of the peptide. The non-se control groups consisted of the following: Pilo no-se rats (n=22) were treated with the RLDP protocol and did not develop SE. Shams (n=19) received saline instead of pilocarpine. SEinducing protocols and experimental animal groups are shown in Figure 6.1. SE and non-se groups used in the present study are the same animals used in chapter 6, and are illustrated in Figure 6.1. Animals were tested in behavioural tasks (i.e., elevated plus maze, open field, hyperexcitability tests) described in chapter 6 on separate days and prior to commencement of MWM testing.

199 Morris water maze testing Pre-observational period to behavioural testing MWM testing began 3 months after SE induction. All rats were observed for recurrent seizures at least 1 hr prior to testing. If the rat was seizure-free within the observation period, the animal was tested as described below. If a rat exhibited recurrent seizures within the observation period, than the test was postponed for at least 3 hours, at which time the rat was again observed for recurrent seizures. On the rare occasion that a rat exhibited a seizure during testing, the trials for that day were removed from the final analysis. No difference in the number of trials removed from each experimental group was detected: in RLDP (or saline-treated) SE rats, a total of 24 trials (or 1% of trials) were removed; in LDP SE rats, 18 trials (or 2.1% of trials); in tat- NR2B9c-treated SE rats, 24 trials (or 1.8% of trials); in tat-nr2baa-treated SE rats, 18 trials (or 1.2% of trials) Apparatus The water maze was a blue circular pool (2 m diameter) located in the centre of a room, with distal visual cues (geometric shapes, posters) on the walls and features (doors, cabinets, shelving, etc.) that served as visual cues. The pool was filled with water (19-22 C) and made opaque by adding non-toxic white paint. Four regions surrounding the pool were designated as north (N), east (E), south (S) and west (W), dividing the pool into four quadrants: NE, SE, NW and EW (not true compass headings). A clear Plexiglas escape platform (16.5 cm height, 5.5 cm diameter) placed in the centre of a quadrant was used to allow the rat to escape from the water Visible platform (cued) testing This portion of the test controls for differences between groups in motivation to escape from the water, swimming ability, and visual acuity (Vorhees and Williams, 2006). On the first day of testing, a 60-s free swim with no platform was performed to familiarize the animals to the pool. During visible platform learning, the escape platform was elevated 2.5 cm above the water and cued with a multi-coloured flag that was extended by 12 cm above the water. The platform was rotated between the NE, NW and SE quadrants of the pool during subsequent trials in a pseudorandom order, as determined by the Research Randomizer Form V4.0 computer program. Each rat was tested with 6 trials per day, for up to 3 consecutive days. At the beginning of each trial, the rat was gently grasped around the midsection and lowered into the pool facing the SW

200 178 wall. The experimenter subsequently left the room and was not present during testing. During the 60-s inter-trial intervals and following testing, rats were towel dried and placed next to a space heater. Each trial lasted a maximum of 90 s, and at the end of each trial, the rat was guided to the platform and/or allowed to stay on the platform for 15 s. The criteria to pass visual acuity consisted of the following: 1) the rat located the platform three consecutive times in less than 30 seconds, or 2) the rat located the platform in less than 30 seconds in 4 out of the 6 trials, and located the platform in the remaining 2 trials. Once the rat passed visual acuity, it was not required to repeat the task on the following day. Animals that failed to pass this test were removed from the study. Both the latency to platform (i.e. the amount of time necessary for the rat to reach the platform) and swimpaths were recorded as described in Methods Hidden platform testing During spatial acquisition, the rats must learn to use distal cues to navigate a direct path to the hidden platform (Vorhees and Williams, 2006). For these tests, the platform was submerged 2.5 cm beneath the surface of water, and remained in a constant position. The platform was placed in the SW quadrant of the pool for 56% of the rats, and in the NE quadrant for the remaining animals. A total of 6 trials per day for each animal were conducted over 14 consecutive days. Rats were placed twice into each of the 3 quadrants that did not contain the platform in a pseudorandom order. Both the path taken and latency to platform were recorded (see Methods section ). Each trial lasted a maximum of 60 s, and at the end of each trial, the rat was guided to the platform and/or allowed to stay on the platform for 15 s. The score for each animal was calculated as the average time of the 6 trials. Spatial reversal evaluates the ability of the rat to extinguish its initial learning of the platform path (spatial acquisition), and acquire a direct path to the new goal position (Vorhees and Williams, 2006). The protocol was the same as described above for spatial acquisition, except that the platform was relocated to the opposite quadrant, and testing was conducted over 5, rather than 14, consecutive days. Spatial reversal was conducted the day following the probe test for spatial acquisition (see Methods )

201 Probe trials Probe trials assess reference memory and are determined by preference for the platform area when the platform is absent (Vorhees and Williams, 2006). The probe trials were administered 24 hr following the last day of the spatial acquisition test and the spatial reversal test. Rats were tested for only a single 60 s trial, and were lowered into the opposite quadrant from where the platform was located during spatial learning. The number of crossings in the area where the platform was previously located in each trial was recorded Search strategy analysis Explorative strategies were classified by two independent investigators, blinded to treatment of the animals, into seven prototypical behavioural categories as defined by Graziano et al., (2003) (Figure 7.1). The data analyzed in the present study were based on the swim paths categorized by the principle investigator, and had a 96% inter-observer agreement on strategy classification. In reanalysis of a subset of trials by the principle investigator 1 month later, the intra-observer agreement on strategy classification was 98%. Consequently, we were confident that the swim paths could be reliably sorted into one of the seven search strategies (Graziano et al., 2003). As illustrated in Figure 7.1, the seven behavioural categories were divided into 3 broader categories that included spatial strategies, systematic non-spatial strategies and repetitive looping-based strategies (Brody and Holtzman, 2006). Spatial strategies include the following categories: 1) spatial direct, which consists of swimming directly to the platform, 2) approaching, characterized by adjusting trajectory while approaching the platform, and 3) self-orienting, characterized by approaching and missing the platform by several degrees, followed by returning to the start point and making another attempt to reach the platform, or briefly re-orienting elsewhere in the pool and then reaching the platform. Spatial strategies rely on distal cues to efficiently reach the platform. Non-spatial, systematic strategies do not use distal cues to locate the platform, but instead involve procedural strategies (i.e. swimming away from the pool wall) that increase the likelihood of locating the platform. The following categories are defined as non-spatial, systematic strategies: 1) scanning, whereby the searching pattern is focused primarily on the center of the pool, and the subject swims back to the centre of the pool immediately if it touches

202 180 the edge and 2) random searching, in which the rat moves in jagged paths through the entire pool, with sudden changes in direction. Strategies involving repetitive looping do not involve the use of distal cues and are the least efficient in locating the platform, They consisted of the following two categories: 1) circling, in which the rat moves away from the wall forming circular trajectories, but often remains too close to the periphery and often fails to locate the platform, and 2) thigmotaxis, characterized by swimming almost exclusively at the periphery and repeatedly searching for contact with the tank wall. Swim paths in which the rat formed tight circles with some net directional movement were also categorized as thigmotaxis.

203 181 Figure 7.1 Behavioural categories. Each row illustrates a few prototypical tracks for each search strategy.

204 182

205 Data Acquisition A digital video camera mounted to the ceiling directly above the center of the pool was relayed to a tracking system (SMART, San Diego Instruments) in a separate room. During Morris water maze testing, escape latency (s) and swim speed (cm/s) were recorded. Swim paths were traced with a mouse on the monitor during testing in a separate room from where the water maze tank was located, and later classified as described in section Statistical Analysis Statistical analysis was performed using Statistica 6.0 software. For Morris water maze performance data, repeated measures ANOVAs were used. For probe trial data, assessment was performed by one-way ANOVAs. The Newman-keuls post-hoc test was used to determine difference between treatment groups. For comparison of strategy use between groups, chi-square analysis was performed. Significance was set at a p-value of 0.05 or less. 7.3 Results SE induction See Results section and Table Visible platform testing As illustrated in Figure 7.2, SE rats required a greater number of trials to pass the visible platform test when compared to rats in both the pilo no-se and sham control groups. Eight SE animals failed to locate the platform by the end of 3 days and were eliminated from the study. There were no differences between SE groups in terms of the number of rats excluded: 0 from the LDP SE experimental group, 4 from the RLDP SE (or saline SE ), 1 from the tat- NR2B9c SE, and 3 from the tat-nr2baa SE (p=0.47). Of the disqualified SE rats, some consistently clung to the wall of the pool (classified as thigmotaxis), which may suggest sensory or motor impairment in these animals, and/or exhibited frequent recurrent seizures during testing. In contrast, thigmotaxis and circling in SE rats during hidden platform testing did not appear to reflect a visual or motor deficit or a lack of motivation, because they performed successfully during visible platform testing, during which they displayed incentive to escape from the pool, and exhibited similar swim speed to corresponding non-seizure controls (Figure 7.4 C).

206 184 Figure 7.2 Number of trials performed to reach criterion during visible platform testing. Criterion to pass visible acuity was assessed as described in Methods section SE was induced with either the RLDP (n=23) or the LDP (n=10) procedure and terminated after 60 min. In drug-treated animals, tat-nr2b9c (n=16) or tat-nr2baa (n=19) was administered 3 hr following the termination of SE induced by the RLDP procedure (see Methods section 6.2.2). Pilo no-se rats (n=22) were treated with the pilocarpine and did not develop SE. Shams (n=19) received saline instead of pilocarpine. * different from shams and pilo no-se control groups (p<0.05). Data expressed as average ± SEM and analyzed by one-way ANOVA followed by Newman-Keuls post-hoc.

207 185

208 The effect of SE on spatial acquisition The position of the platform (i.e., SW or NE quadrant) during testing had no impact on the final performance within any of the experimental groups. As illustrated in Figure 7.3, both SE groups showed significantly greater latencies than the non-se control groups over the 14-day testing period. The sham and pilo no-se control groups performed equally, with average escape latencies of 21.0±2.2 s and 20.2±2.0 s in the initial 4 days of testing, reduced to 9.2±1.0 s and 9.9±1.2 s in the final 4 days, respectively. No significant difference in performance was detected between the LDP and the RLDP SE rats (p=0.17), with the average escape latencies of 42.7± 2.8 s and 41.1± 3.6 s in the initial 4 days of testing, improving to 32.9± 4.0 s and 34.3± 4.7 s in the final 4 days, respectively (Figure 7.3) The effect of SE on spatial reversal During spatial reversal, the hidden platform was placed in the opposite quadrant from the quadrant in which it was located during spatial acquisition (Figure 7.3). In SE and non SE groups, an increase in latencies was observed on the first day of spatial reversal when compared to the last day of spatial acquisition (Figure 7.3, compare day A14 with day R1); however, reversal latencies remained lower than those for day 1 of spatial acquisition (Figure 7.3, compare day R1 with day A1). Whereas both non-se control groups exhibited reduced latencies by the second day of reversal training, no improvement in performance was detected in either of the SE experimental groups.

209 187 Figure 7.3 The effect of SE on hidden platform testing in the Morris water maze. SE was induced with either the RLDP (red circles, n=23) or the LDP (blue circles, n=10) procedure and terminated after 60 min as in Methods. Pilo no-se rats (black diamonds, n=22) were treated with the pilocarpine and did not develop SE. Shams (green triangles, n=19) received saline instead of pilocarpine. Each data point represents the average of 6 trials per day (± SEM). Each group was tested for 14 days of spatial acquisition (A1 A14) and for 5 days of spatial reversal (R1 R5). # Performance in SE groups different from non-se control groups (p<0.05). * During acquisition training, latencies in final 4 days (A11-A14) were reduced compared to initial 4 days (A1-A4) in SE groups and in non-se control groups (p<0.05). ** Latencies reduced in day R1 compared to day A1 in the RLDP SE group and in both non-se control groups (p<0.05). *** Latencies increased in day R1 compared to day A14 in SE and non-se control groups (p<0.05). ^ During reversal training, latencies reduced in day R5 compared to day R1 in non-se control groups (p<0.05). Data was analyzed by repeated-measure ANOVA. Dashed lines represent the predicted performance of corresponding groups based solely on search strategy use (see appendix III, section 3.1).

210 188

211 Acquisition and reversal probe tests SE rats exhibited impaired spatial memory in the acquisition and reversal probe tests. As illustrated in Figure 7.4 (A, B), rats following SE exhibited a lower number of platform crossings compared to both non-se control groups.

212 190 Figure 7.4 Number of platform crossings in spatial acquisition (A) and spatial reversal (B) probe trials and swim speed (C). SE was induced with either the RLDP (n=23) or the LDP (n=10) procedure and terminated after 60 min as in Methods section In drug-treated animals, tat-nr2b9c (n=16) or tat-nr2baa (n=19) was administered 3 hr following the termination of SE induced by the RLDP procedure (see Methods section 7.2.2). Pilo no-se rats (n=22) were treated with the pilocarpine and did not develop SE. Shams (n=19) received saline instead of pilocarpine. A, B: SE groups exhibit poor retention in location of the hidden platform: * different from shams and pilo no-se control groups (p<0.05). C: Swim speed was similar across all animal groups during the probe tests. Black bars depict swim speed during the spatial acquisition probe trial. Grey bars depict swim speed during the spatial reversal probe trial. Data expressed as average ± SEM. Data analyzed by one-way ANOVA followed by Newman-Keuls post-hoc.

213 191

214 Effect of SE on search strategy use during spatial acquisition Because differences in escape latencies may reflect the type of search strategy used (Brody and Holtzman, 2006), we next analysed the use of different search strategies by the various groups. The distribution of each search strategy used in SE and non-se groups is illustrated in Figure 7.5, and a comparison in search strategy use between groups is demonstrated in Figure 7.6. In control groups, both the sham and the pilo no-se rats employed similar strategies at all times assessed: on day 1 of spatial acquisition, these animals used a high proportion of spatial strategies (sham: 37%, pilo non-se: 34%) and non-spatial systematic strategies (sham: 52%, non-se: 59%, Figure 7.6A, B), and a low fraction of repetitive looping-based strategies (Sham: 11%, Non-SE: 8%, Figure 7.6C). As their performance improved, control rats used spatial strategies nearly exclusively (Figure 7.6, A). In contrast to non-se rats, rats following SE employed a lower proportion of spatial strategies and a greater proportion of non-spatial, systematic and/or repetitive looping-based strategies on all days tested (p<0.05, Figure 7.6). Over the 14-days of testing, RLDP SE and LDP SE rats increased their use of spatial strategies and decreased their use of non-spatial, systematic strategies and repetitive-looping based strategies (Figure 7.6) Effect of SE on search strategy use during spatial reversal The above results show that SE rats exhibit impaired use of search strategies when compared to non-se groups, but still increased the use of more efficient strategies over 14-days of spatial acquisition. We next assessed the effect of the spatial reversal task on search strategy use by the various groups. Following relocation of the submerged platform to the opposite quadrant, both SE and non-se groups exhibited a decrease in the use of spatial strategies, and an increase in the use of non-spatial, systematic strategies (Figure 7.6, compare days A14 and R1). During spatial reversal, non-se rats used more efficient search strategies (i.e., a greater proportion of spatial strategies and a lower proportion of non-spatial, systematic strategies and repetitive looping-based strategies) compared to SE rats across all days assessed (Figure 7.6, p<0.05). Both the sham and the pilo no-se rats showed similar improvement in strategy use over 5-days of testing, with spatial strategies being used nearly exclusively by day 5 (Figure 7.6A). Although RLDP SE rats exhibited an increase use of spatial strategies over this period (Figure 7.6A), more than half of the strategies selected still consisted of non-spatial, systematic

215 193 and repetitive-based looping strategies on day 5 (Figure 7.6B, C). In contrast to RLDP SE rats, LDP SE rats showed no change in search strategy use (Figure 7.6A).

216 194 Figure 7.5 The effect of SE on the distribution of search strategies used during spatial acquisition and spatial reversal testing. SE was induced with either the RLDP (n=23) or the LDP (n=10) procedure and terminated after 60 min as in Methods section Pilo no-se rats (n=22) were treated with the pilocarpine and did not develop SE. Shams (n=19) received saline instead of pilocarpine. Animals tested in the MWM and search strategies assessed as in Methods section 7.2.3on day 1 (A1), day 7 (A7) and day 14 (A14) of spatial acquisition, and on day 1 (R1) and day 5 (R5) of spatial reversal. Results are presented as the percentage of trials and as the total number of trials that all animals in each group used a given strategy.

217 195

218 196 Figure 7.6 Summary of search strategy use between groups during Morris water maze testing. SE was induced with either the RLDP (n=23) or the LDP (n=10) procedure and terminated after 60 min as in Methods section Pilo no-se rats (n=22) were treated with the pilocarpine and did not develop SE. Shams (n=19) received saline instead of pilocarpine. Animals tested in the MWM and search strategies assessed. Results presented as the percentage of trials that all animals in each group used a given strategy: (A) spatial strategies, (B) nonspatial, systematic strategies, and (C) repetitive looping-based strategies. * Search strategy use different from day A1 (p<0.05). ** Search strategy use on day R5 different from day R1 (p<0.05). ^ Search strategy use on day R1 different from day A14 (p<0.05). # Search strategy use in SE rats different from non-se control groups on corresponding day (p<0.05). Chi-square analysis was used for each comparison

219 197

220 Quantitative assessment of the contribution of search strategy to overall performance The above results demonstrating relatively poor performance of the SE rats in the probe test (a measure of spatial memory) suggested that the reduction in escape latencies during acquisition testing in the MWM might primarily reflect improved use of more efficient search strategies by these animals. We therefore determined the extent to which the shift in search strategies contributed to improved performance. An alternative possibility was that the rats became better at using one or more of the individual strategies over the 14-days of testing and that this contributed to improved performance. We first calculated the average escape latency for each individual strategy on the first and last day of acquisition learning. As shown in Table 7.1, both of the non-se control groups exhibited improved performance in the use of both spatial strategies and non-spatial, systematic strategies. In contrast, improved performance in the RLDP SE rats occurred only with the scanning strategy, and no significant improvement in any of the strategies was detected in the LDP SE rats.

221 199 Table 7.1: Performance as a function of strategy use in hidden platform Morris water maze testing Strategy RLDP SE day 1 RLDP SE day 7 RLDP SE day 14 LDP SE day 1 LDP SE day 7 LDP SE day 14 Repetitive Looping 58.5 ± 2.5 n = 36 Thigmotaxis 59.2 ± 0.3 n = 26 Circling 56.7 ± 0.7 n = 10 Non-spatial, systematic 51.2 ± 3.9 n = 73 Random Searching 57.4 ± 1.7 n = 42 Scanning 42.9 ± 2.7 n= 31 Spatial 13.2 ± 1.5 n = 23 Orienting 18.4 ± 1.0 n = 13 Approaching 7.3 ± 3.2 n = 8 Direct Find 2.7 ± 0.7 n= ± 2.3 n = ± 3.4 n = ± 2.9 n = ± 2.6 n = 47* 56.9 ± 2.8 n = ± 2.8 n = 27* 8.3 ± 0.8 n = ± 1.5 n = ± 0.6 n = n= ± 4.1 n = ± 5.4 n = ± 5.9 n = ± 2.5 n = 53* 51.2 ± 4.1 n = ± 2.7 n = 31* 9.2 ± 0.9 n = ± 1.6 n = ± 0.8 n = ± 0.2 n = ± 1.9 n = ± 1.7 n = n = ± 3.9 n = n = ± 4.7 n = ± 1.2 n = n = ± 1.2 n = 4 1 n = ± 3.4 n = ±18.7 n = ±16.8 n = ± 2.8 n = ± 2.4 n = ± 3.0 n = ± 3.6 n = ± 1.9 n = ± 6.5 n = 8 N/A 56.4 ± 6.0 n = ± 5.3 n = ±20.3 n = ± 3.4 n = ± 2.0 n = ± 3.4 n = ± 3.1 n = ± 3.8 n = ± 5.3 n = 9 Latency in seconds (mean ± standard error). n: number of individual trials during hidden platform testing where a given strategy was used. N/A: not applicable since no example of this search strategy was found. Shaded rows: classes of strategies, values reflect the pooled results from the individual strategies listed below each shaded row. * Performance significantly different from day 1 (p<0.05). Data assessed using one-way ANOVA. N/A

222 200 Table 7.1: continued Strategy Sham day 1 Repetitive Looping 42.6 ± 5.2 n = 12 Thigmotaxis 42 ± 5.2 n = 12 Sham day 7 Sham day 14 Pilo no- SE day 1 N/A N/A 54.5 ± 3.5 n = 10 N/A N/A 54.5 ± 3.5 n = 10 Pilo no- SE day 7 Circling N/A N/A N/A N/A N/A N/A Non-spatial, systematic 42.5 ± 2.3 n = 59 Random Searching 58.3 ± 1.7 n = 20 Scanning 34.4 ± 2.4 n = 39 Spatial 11.4 ± 0.1 n = 42 Orienting 15.9 ± 1.7 n = 13 Approaching 9.6 ± 1.0 n = 28 Direct Find 3.4 n = ± 4.0 N = 9 * 24.9 ± 3.6 n=16 * 47.6 ± 1.9 n = 72 N/A N/A 59.9 ± 0.1 n = ± 4.0 n = 9 * 5.8 ± 0.6 n = 104* 11.5 ± 2.2 n = ± 0.6 n = 69* 2.8 ± 0.02 n = ± 3.6 n = 16 * 7.0 ± 0.6 n = 97* 13.8 ± 1.5 n = ± 0.5 n = 62* 2.3 ± 0.2 n = ± 2.5 n = ± 1.0 n = ± 1.4 n = ± 0.8 n = n = 1 N/A N/A 24.4 ± 3.3 n = 27 * 60 n = ± 2.7 n = 25 * 6.3 ± 0.5 n = 98* 11.3 ± 1.2 n = 20* 5.4 ± 0.6 n = 67* 6.3 ± 0.5 n = 98 Pilo no- SE day 14 N/A N/A 19.5 ± 3.8 n = 12 * N/A 19.5 ± 3.8 n = 12 * 5.3 ± 0.3 n = 114* 8.4 ± 0.5 n = 21* 5.0 ± 3.4 n = 74* 2.7 ± 0.2 n= 19 Latency in seconds (mean ± standard error). n: number of individual trials during hidden platform testing where a given strategy was used. N/A: not applicable since no example of this search strategy was found. Shaded rows: classes of strategies, values reflect the pooled results from the individual strategies listed below each shaded row. * Performance significantly different from day 1 (p<0.05). Data assessed using one-way ANOVA.

223 201 We next compared the importance of the shift in strategies relative to the improved efficiency when using each strategy. Quantitative analyses were based on calculations described by Brody et al., (2006). As demonstrated in section 3.1 of Appendix III, convolution analysis was used to determine the predicted performance of the RLDP SE rats based on shifts in strategy use for all days of spatial acquisition, while removing the changes in the performance of each strategy from the assessment. The predicted performance based on this analysis closely matched the actual data collected in the RLDP SE and the LDP SE groups (Figure 7.2, dashed lines). In RLDP SE rats, the convolution analysis predicted that performance would be improved by 33% from day 1 to day 14, a value that is close to the 40% improvement observed. A similar analysis conducted in LDP SE rats predicted a 34% improvement in performance from day 1 to day 14, and is close to the 32% improvement observed. The converse analysis was also performed. To obtain the predicted performance, the change in performance of each strategy was used in the assessment, while the effect of shift in strategy use was removed. Calculations are illustrated in section 3.2 of Appendix III. This analysis predicted an improved performance of only 11% from day 1 to day 14 in the RLDP SE rats, and an improved performance of only 0.1% in the LDP SE rats. These values markedly underestimated the actual improvement in performance of 40% and 32% in the RLDP SE and the LDP SE rats, respectively. Based on this analysis, we concluded that the improved performance s between days 1 and 14 was mainly accounted for by a shift in strategy use. A similar analysis was conducted for the control groups. For shams, the convolution analysis based on strategy shift predicted a 68% improvement over the 14 days of testing, similar to the observed improvement of 74%. When the analysis was based on improved performance within each strategy, the predicated improvement was 48%. Predicted values were similar in pilo no- SE rats, with a 73% improvement attributable to a shift in strategy use, and a 53% improvement based on increased efficacy in performance within each strategy. The predicted value based on shift in strategy use was closest to the observed improvement of 72% in pilo no-se rats. Accordingly, in non-se rats, shifts in strategy use predominately contributed to improved performance during spatial acquisition. However, improvements in the use of each individual strategy also played an important role in improved performance.

224 SE results in differential impairment in Morris water maze performance and search strategy use A closer examination in the performance of individual RLDP SE rats during spatial acquisition showed that SE differentially affects MWM performance, despite each rat receiving similar treatment for SE induction. In this analysis, individual rats were partitioned into groups based on escape latencies in the initial 4 days compared to the final 4 days of spatial acquisition testing. Here, performances of individual rats clearly fell into three discrete groups. As illustrated in Figure 7.7, 8 of the RLDP SE rats (group 1) showed no improvement in performance over 14- days of testing, and were significantly different from 10 of the RLDP SE rats (group 2) that showed significant reduction in latencies within this period. These 2 groups also exhibited differences in search strategy use (Figure 7.8). The 8 RLDP SE rats that showed no improvement in MWM performance also had no shift in strategy use during testing. In contrast, the 10 RLDP SE rats that exhibited improved performance also showed an increase in use of spatial strategies (Figure 7.8A), and a decrease in use of non-spatial, systematic strategies (Figure 7.8B). All of these animals showed impaired performance and use of less efficient strategies when compared to shams. Five of the RLDP SE rats (group 3) exhibited no impairment during MWM testing, with performance (Figure 7.7) and selection of search strategies (Figure 7.8) similar to shams. As shown in Table 7.2, there was no significant difference between groups with respect to seizure susceptibility and seizure severity. All groups exhibited similar swim velocities when compared to shams, indicating no presence of motor impairment. Reference memory, as assessed by the number of platform crossings in the spatial acquisition and spatial probe tests, was different between the three RLDP SE groups. Although group 1 had a lower number of platform crossings when compared to group 2, both groups performed poorly when compared to shams. On the other hand, the number of platform crossings in group 3 RLDP SE rats was similar to shams in the two probe tests.

225 203 Table 7.2: Comparison of RLDP SE rats that exhibit differences in Morris water maze performance Treatment /group # 1. RLDP SE (n=8) 2. RLDP SE (n=10) 3. RLDP SE (n=5) Shams (n=19) # of pilocarpine injections for RLDP protocol 1 Seizure Severity 2 Swim velocity during spatial acquisition probe test 3 # of platforms crossed during spatial acquisition probe test 4 Swim velocity during spatial acquisition probe test 3 # of platforms crossed during spatial acquisition probe test ± ± ± ± 0.5* ^ 27.0 ± ± 0.7* ^ 3.9 ± ± ± ± 0.8* 24.6 ± ± 1.3* 3.8 ± ± ± ± ± ± ± ± ± ± Number of repeated low-dose pilocarpine injections (10 mg/kg) administered in the RLDP protocol (average ± SD). 2. Average maximum seizure activity for all animals determined as in Methods (average ± SD). 3. Average swim velocity (cm/s) (average ± SD). 4. # of platforms crossed in probe trial (average ± SD). * different from shams (p<0.05). ^ different from group 2 RLDP SE rats (p<0.05). Data analyzed by one-way ANOVA followed by Newman-Keuls post-hoc.

226 204 Figure 7.7 SE results in differential impairment in Morris water maze performance. SE was induced with the RLDP (n=23) procedure and terminated after 60 min as in Methods section RLDP SE animals were subdivided into 3 groups based on performance (see section 7.3.9). Shams (black circles, n=19) received saline instead of pilocarpine. Each data point represents the average of 6 trials per day (± SEM). * During acquisition training, latencies in final 4 days (A11-A14) reduced compared to initial 4 days (A1-A4) in RLDP SE groups 2 and 3 and in shams (p<0.05). # Performance in RLDP SE groups 1 and 2 are different from RLDP SE group 3 and shams (p<0.05). Data analyzed by repeated-measure ANOVA.

227 205

228 206 Figure 7.8 SE results in differential use of search strategies during Morris water maze testing. SE was induced with the RLDP (n=23) and terminated after 60 min as in Methods section SE animals were subdivided into 3 groups based on performance (see section 7.3.9). Shams (n=19) received saline instead of pilocarpine. Animals tested in the MWM and search strategies assessed as in Methods section Results presented as the percentage of trials that all animals in each group used a given strategy: (A) spatial strategies, (B) non-spatial, systematic strategies, and (C) repetitive looping-based strategies. * Search strategy use different from day A1 (p<0.05), ** p=0.1. # Search strategy use different from shams on corresponding day (p<0.05). Chi-square analysis was used for each comparison.

229 207

230 The effect of Tat-NR2B9c on visual-spatial learning and use of search strategies following SE In chapter 5, we demonstrated that tat-nr2b9c is neuroprotective within the CA1, CA3 and CA4 pyramidal cell layers of the dorsal hippocampus. Because the dorsal hippocampus is involved in visual-spatial learning, we determined whether the neuroprotection provided by tat-nr2b9c would improve spatial learning and search strategy use following SE. Rats treated with tat- NR2B9c following SE had similar swim velocities when compared to both non-se control groups, indicating no presence of motor impairment (Figure 7.4C). Figure 7.9 demonstrates that administration of tat-nr2b9c did not improve escape latencies during either spatial acquisition or spatial reversal testing, and did not improve reference memory as assessed in the probe tests (Figure 7.4A, B). Furthermore, search strategy use as a function of time was not altered by the drug treatment during spatial acquisition (Figure 7.10), with all SE experimental groups using less efficient search strategies when compared to shams.

231 209 Figure 7.9 Tat-NR2B9c did not improve performance in SE rats during hidden platform learning. Escape latencies (mean ± SEM) during spatial acquisition (A1 A14) and spatial reversal testing (R1 R5) in tat-nr2b9c (n = 16; purple squares), tat-nr2baa (n=17, orange triangles) and saline-treated (n = 24; red circles) rats 3 months following 60-minutes SE and in shams (n=19, black diamonds) were determined as in Methods. Each data point represents the average of 6 trials per day: # Performance in SE rats different from shams (p<0.05). Data analyzed by repeated-measure ANOVA followed by Newman-Keuls post-hoc.

232 210

233 211 Figure 7.10 Tat-NR2B9c has no effect on the distribution of search strategies used during spatial acquisition in rats following SE. SE was induced with the RLDP procedure and terminated after 60 min as in Methods. Tat-NR2B9C (n=16), Tat-NR2BAA (n=19) or saline (n=23) was administered 3 hr following the termination of SE. Shams (n=19) received saline instead of pilocarpine. Search strategies were assessed as in Methods. Results are presented as the percentage of trials for which all rats in a group used a given strategy. Similar shift in search strategy use was detected in rats treated with saline, tat-nr2baa and tat-nr2b9c (p=0.51). * Search strategy use different from day A1. # Search strategy use different from shams. Chisquare analysis was used for each comparison.

234 212

235 Discussion Although many studies have examined the impact of SE on spatial learning during MWM testing (Persinger et al., 1993; Rice et al., 1998; Hort et al., 1999; Hort et al., 2000; Wu et al., 2001; McKay and Persinger, 2004; dos Santos et al., 2005; Frisch et al., 2007; Zhou et al., 2007), little attention has been given to the effect of SE on the use of search strategies. In the present study, we analyzed the effect of SE on strategy use during spatial acquisition and spatial reversal testing. The main findings include: (1) SE groups treated with either the RLDP or LDP seizureinducing protocol exhibit impaired performance in the MWM, and show some improvement in performance over prolonged training. (2) SE groups use less efficient search strategies than non- SE groups. (3) Increased use of more efficient search strategies, rather than improvement in performance of individual strategies, most closely predicted the improved performance in both the SE and non-se groups. (4) Within the same group of animals with SE, variability in behaviour of individual rats during MWM testing occurred. While the majority of rats show impaired performance and use of poor search strategies, but differed in the ability to show improvement over prolonged training, some rats exhibited no behavioural impairment when compared to non-se rats. (5) Finally, neuroprotection within the dorsal hippocampus provided by tat-nr2b9c did not modify MWM performance in rats following SE. These results are discussed below SE rats exhibit impaired performance in the MWM and improve during prolonged training In agreement with previous studies, we found that MWM performance in rats following SE was markedly impaired (Rice et al., 1998; Hort et al., 2000; Wu et al., 2001; McKay and Persinger, 2004; Frisch et al., 2007; Zhou et al., 2007; dos Santos et al., 2005; Cunha et al., 2009). However, we found that with prolonged training, SE rats were capable of improving their performance. This finding is generally similar to that of dos Santos et al., (2006) but differs from that of several other groups that found no improvement in performance (Rice et al., 1998; Hort et al., 2000; Wu et al., 2001; McKay and Persinger, 2004; Zhou et al., 2007; Cunha et al., 2009); in these latter studies, MWM testing was conducted for up to a maximum of 5 days. Since we did not observe a significant change in performance within the first 5 days of testing, this duration may not have been long enough for improved performance to occur in SE rats described in the other studies (Rice et al., 1998; Hort et al., 2000; Wu et al., 2001; McKay and

236 214 Persinger, 2004; Zhou et al., 2007; Cunha et al., 2009). In general, our results suggest that rats following SE retain the ability to improve performance in MWM testing with extended training. We previously demonstrated that rats treated with the RLDP protocol exhibited a lower mortality rate and less severe status compared to rats treated with the LDP protocol (see chapter 3). Despite these differences, we found that the MWM performance was similar between the RLDP SE and the LDP SE groups. These results are congruent with our previous findings that the two seizure-inducing protocols also cause similar behavioural impairments in rats when assessed in the open field, elevated plus maze, and hyperexcitability tests (see chapter 6). These results are not unexpected, since we previously found that neuronal loss in the hippocampus and piriform cortex was similar in RLDP SE and LDP SE rats, brain regions associated with cognitive (see section 7.1) and behavioural performance (see section 6.4.2). Our general finding is that the RLDP protocol has the advantage to reduce mortality in Wistar rats following SE, without affecting SE-induced morbidity and neuropathology, and therefore serves as a more beneficial method in generating seizures for this rat strain (see section 7.4.6) SE rats use less efficient strategies in the MWM One of our main findings was that rats following SE showed impaired search strategy use in the spatial acquisition and spatial reversal tasks. SE groups displayed a greater proportion of less efficient search strategies (repetitive-looping based strategies and non-spatial, systematic strategies) while using fewer spatial strategies compared to non-se groups. Nonetheless, with increased training times, epileptic rats learned how to use more efficient strategies and the increased use of these strategies was able to account for the improved performance. The observation that SE rats showed poor spatial memory compared to non-se rats (as assessed by the probe tests) further supports the idea that the reduced latencies during spatial acquisition resulted from improvements in the procedural-strategy component of the MWM task as oppose to the spatial component. The prolonged training necessary for improved performance in SE groups is also congruent with the fact that procedural learning is a form of habit-like learning that can take weeks rather than days to acquire, and does not involve cognitive factors (e.g., memory and expectancy) that occur during place learning (Kirsch et al., 2004; Bayley et al., 2005). In the present study, once the more efficient strategies were learned by SE rats, they were retained, as indicated by the observation that performance and strategy selection was better on

237 215 initial day of spatial reversal than at the beginning of spatial acquisition. These findings indicate that in spite of the initial poor performance on the MWM, the rats following SE were able to form and retain a procedural memory of newly acquired search strategies. During the spatial reversal task, no improvement in overall performance was observed in rats treated with either induction protocol. Although there was no change in strategy use by the LDP SE rats, RLDP SE rats increased their use of more efficient strategies over the 5 days of reversal training. It is not clear why the change in strategy use in RLDP SE rats did not coincide with improved performance during reversal training, or why strategy use was different in rats treated with the LDP protocol compared to the RLDP protocol. Because spatial reversal assesses different factors (i.e., cognitive flexibility) than those assessed during spatial acquisition (Vorhees and Williams, 2006), the differences in the procedures may account for the differences in behavioural responses of SE rats detected during spatial acquisition when compared to spatial reversal. Another possibility is that testing in the spatial reversal task was only 5-days, and that this duration was not long enough to detect improvement in performance in SE rats, as was detected over 14-days of testing in the spatial acquisition task Improvement in search strategy selection contributed to improved performance epileptic rats In the present study, we found that the RLDP SE group and the non-se groups improved their performance in using individual search strategies. To further assess the importance of a change in strategy use relative to improved efficiency in use of individual strategies in contributing to improved performance, we conducted the convolution analyses as described by Brody et al., (2006). The general finding was that changes in strategy use more closely predicted and therefore contributed mostly to the change in performance observed in the SE groups. A shift in strategy use was also found to predominately contribute to improved performance in non-se groups. Brody et al., (2006) found similar results in transgenic mice following traumatic brain injury. This emphasizes that the use of less efficient search strategies in rats following SE, and perhaps in brain trauma more generally (Brody et al., 2006), along with poor spatial memory, are important factors underlying the impaired performance in these animals.

238 The pathological effects of SE may interfere with the selection of more efficient search strategies Our results indicate that search strategy use does not operate based on a simple hierarchical hypothesis as was suggested by Sutherland et al (1982) (Sutherland et al., 1982). In this hypothesis, Sutherland et al., (1982) proposed that mice capable of using spatial information will proceed to exclusively use spatial strategies, whereas those incapable of using spatial information will predominately use nonspatial, systematic strategies. We found, however, that SE rats showing little evidence of true spatial memory in the probe tests showed improvement in use of spatial strategies during spatial acquisition. This finding is similar to that previously reported for transgenic and wild type mice subjected to traumatic brain injury (Brody and Holtzman, 2006). In addition, when the platform was changed to the opposite quadrant, SE rats exhibited a decrease in performance that correlated with the use of fewer spatial strategies, and the use of more non-spatial, systematic strategies (in Figures 7.2 and 7.4, compare day 16 with day 14). These results suggest that SE rats are capable of acquiring spatial memory, but nevertheless continue to predominately use less efficient search strategies and to perform poorly in the spatial probe tests. In this respect, they behave similarly to caudate-putamen lesioned rats, which although capable of using spatial strategies, exhibit a preference for alterative, possibly simpler strategies (Whishaw et al., 1987). Wishaw et al., (1987a) hypothesized that damage to the caudate-putamen, which has previously been reported in rats following SE (Fujikawa, 1996; Clifford et al., 1987), may disrupt the ability of the animal to select the most appropriate behavioural strategy. Although the physiological basis for determining strategy choice during spatial navigation tasks is not fully understood, studies have indicated that: 1) the cholinergic system is involved (Sutherland et al., 1982; Whishaw and Petrie, 1988; Whishaw, 1989; Chang and Gold, 2003; McIntyre et al., 2003), and that 2) different neural systems control whether spatial or non-spatial strategies are employed (McDonald and White, 1993; McDonald and White, 1994; Miranda et al., 2006). Muscarinic blockade with atropine does not disrupt performance in a non-spatial version (visible platform) of the Morris water maze, but impairs strategies in searching for the platform in a subsequent spatial version (hidden platform) (Whishaw and Petrie, 1988; Whishaw, 1989). In addition, the level of acetylcholine in the striatum and in the hippocampus determines whether tasks are solved using either response (egocentric) or place (allocentric) mechanisms,

239 217 respectively (Chang and Gold, 2003; McIntyre et al., 2003). These results suggest that when spatial information is required for the solution of a task the hippocampus is more active, whereas when stimulus-response learning is required the striatum is more active (Miranda et al., 2006). Several authors have suggested that the striatal and hippocampal neural systems interact through competitive mechanisms, so that in determining which strategy is best suited to solve a task, the more efficient system is used (McDonald and White, 1993; McDonald and White, 1994; Epp et al., 2008). Previous studies have demonstrated that widespread neurodegeneration (Honchar et al., 1983; Turski et al., 1983b; Peredery et al., 2000) and changes in the cholinergic system (Jope and Gu, 1991; Mingo et al., 1997; Baran et al., 2004; Mendes de Freitas et al., 2005) occur in rats following SE. Consequently, these and other pleiotropic effects of SE on the nervous system most likely contribute to the impaired selection of search strategies by SE rats Rats following SE exhibited variability in behaviour during MWM testing One of the main findings was that variability in behaviour during MWM testing occurred within the same group of rats with SE. Although the majority of rats following SE showed impaired performances during MWM testing, there were differences in their ability to improve performances over prolonged training. Within the RLDP SE group, 44% of the rats learned how to use more efficient strategies, and the increased use of these strategies was able to account for the improved performance in these animals. On the other hand, 34% of the SE rats exhibited no improvement in escape latencies or changes in strategy use with extended training. Since these rats simply swam around the pool and rarely located the platform, this behaviour can account for why these animals had a worst performance in the probe tests when compared to SE rats that showed improved performance, and utilized strategies that increased their likelihood of encountering the platform. Five out of 23 rats exhibited no behavioural impairment, with performances in the MWM and probe tests indistinguishable from controls. We previously demonstrated that rats following SE sustain major neuronal loss in brain regions associated with MWM performance (i.e., hippocampus and thalamus, see chapters 4 and 5). However, variability in the severity of brain damage between individual SE rats was also observed. Therefore, it is possible that differences in the pattern of brain damage involved in learning of the MWM task may contribute to the variability in performance between rats following SE. In support of this idea, McKay and Persinger (2004) found that ketamine-treated

240 218 SE rats performed equivalently to nonseized controls in spatial and non-spatial water maze tasks, whereas acepromazine-treated SE rats performed poorly. Despite cognitive sparing in ketaminetreated SE rats, extensive brain damage was still present. However, the pattern of brain damage (i.e., amygdala, hippocampus and thalamus) was different between the two groups, and may have contributed to the differences in MWM performance between the ketamine-treated SE rats and the acepromazine-treated SE rats (McKay and Persinger, 2004). Further research is necessary to determine whether the variability in MWM performance we observed in rats following SE is correlated with differences in neuropathology Neuroprotection of the dorsal hippocampus by tat-nr2b9c did not modify performance in the MWM Administration of tat-nr2b9c resulted in neuroprotection within the CA1, CA3 and CA4 subfields of the dorsal hippocampus (see Chapter 5). In spite of its protective action, the administration of Tat-NR2B9c did not result in improved performance in the MWM, or in the use of more efficient search strategies during MWM testing. In contrast to our results, several studies have reported a connection between neuroprotection and improved cognitive functioning. Cunha et al., (2009) found that repeated daily treatments of diazepam, carbamazipine and phenytoin immediately following SE provided substantial neuroprotection, resulting in less than 30% neuronal loss in the CA1, CA3 and dentate gyrus; this protective effect correlated with improved spatial learning during MWM testing. Similarly, Yang et al., (2007) found that treatment of rats with erythropoietin prior to SE induction resulted in neuroprotection of the CA1 and CA3 (although the extent was not specified), as well as improved cognitive functioning and use of spatial strategies in the MWM (Jun et al., 2009). Rice et al., (1998) also showed that pretreatment of rats with MK-801 resulted in almost complete protection of the CA1 and greatly improved spatial learning. A possible explanation for the difference in findings from these studies compared to our results may be the amount of protection that was afforded in the hippocampus following the different treatments. For instance, Broadbent et al., (2004) demonstrated that spatial learning was impaired by bilateral dorsal hippocampal lesions that encompassed 30 to 50 percent of the total hippocampal volume (Broadbent et al., 2004). Consequently, the partial neuroprotection of the hippocampus by tat-nr2b9c may not have been enough to result in cognitive sparing.

241 219 An alternative possibility is that other damaged brain regions not affected by tat-nr2b9c may contribute to cognitive impairment during MWM testing. As previously discussed, McKay and Persinger (2004) demonstrated that different patterns of brain damage (i.e., amygdala, hippocampus, thalamus) following SE can differentially affect performance in the MWM (see section 7.4.5). Although our results suggest that neuroprotection within the dorsal hippocampus does not modify SE-induced alterations in spatial learning and behavioural strategies, it is also possible that there was not enough protection within the dorsal hippocampus in the present study to have a noticeable behavioural effect Conclusion The present study demonstrates that suboptimal search strategy use, along with poor spatial learning and memory, contributes to the impaired performance of epileptic rats during spatial learning tasks in the MWM. With extended training, the majority of rats following SE were able to improve their performance by learning how to use more efficient search strategies during spatial acquisition. Partial neuroprotection within the dorsal CA1 provided by tat-nr2b9c was insufficient to improve spatial learning, suggesting either that the amount of neuroprotection in the hippocampus was insufficient to rescue behaviour or that other damaged brain regions not affected by Tat NR2B9c may contribute to the decrease in learning ability. In general, our results indicate that systematic assessment of behavioural strategies and their contribution to overall performance is an important factor to consider when assessing morbidity in animal models of epilepsy. Patients with TLE may have analogous poor use of cognitive strategies.

242 220 Chapter 8 General Discussion 8.1 The lithium/pilocarpine model of mesial temporal lobe epilepsy The lithium/pilocarpine rodent model recapitulates the main clinical and neuropathological features of mesial temporal lobe epilepsy with hippocampal slerosis (MTLE-HS), the most common form of human epilepsy (see section 1.1). Both in the rodent pilocarpine model and in the human condition the seizure disorder is initiated by an initial precipitating injury (IPI) followed by a latent period, whereby the subjects appear neurologically normal with subsequent development of unremitting epileptic seizures (Wieser, 2004; Curia et al., 2008). In both conditions, widespread neuronal loss is observed in many brain regions including the hippocampus, neocortex, piriform and entorhinal cortices, septum, amygdaloid and thalamic structures (Moran et al., 2001; Jutila et al., 2002; Bernasconi et al., 2003; Bonilha et al., 2010a; Bonilha et al., 2010b). A high prevalence of cognitive impairment and behavioural disturbances (e.g., aggression, anxiety) is also reported in human MTLE-HS ( Boro and Haut, 2003; Gaitatzis et al., 2004; Devinsky, 2004a; Swinkels et al., 2005; Cornaggia et al., 2006; Marcangelo and Ovsiew, 2007; Garcia-Morales et al., 2008) and is observed after SE in rodents (Rice et al., 1998; Hort et al., 1999; Hort et al., 2000). Nonetheless, the causal relationship between neuronal death, epileptogenesis, and cognitive and behavioural morbidity following SE remains unclear (see sections 1.5 and 1.6). Our general hypothesis is that genesis of SRSs, cognitive impairment and behavioural alterations are caused by SE-induced neuronal death. This thesis focused on five specific hypotheses that further characterized neuronal death and behavioural/cognitive morbidity in rats following SE induced by the lithium/pilocarpine model (see chapter 2). The causal relationship between neuronal death, epileptogenesis and cognitive and behavioural morbidity was investigated by: (1) comparing the temporal onset between neuronal death and behavioural alterations following SE (section 8.6), and (2) assessing the effect of partial neuroprotection within the hippocampus on the incidence of SRSs, behavioural alterations and cognitive deficits after SE in rats (section 8.7). In the subsequent sections, we summarize our main findings and discuss the significance of these results to clinical human data.

243 Comparison of the low-dose (LDP) and repeated low dose lithium/pilocarpine (RLDP) procedures High mortality in rodents is a major drawback in using the lithium/pilocarpine model (reviewed in section 1.3.4). Glien et al., (2001) showed that when compared to a single dose of pilocarpine (30 mg/kg, LDP protocol), repeated administration with reduced doses of pilocarpine (10 mg/kg, RLDP protocol) at 30-min intervals until SE onset significantly reduced mortality in Wistar rats. Still, it is difficult to determine a best-of protocol since a comparison of the effect of the LDP and the RLDP procedures on neurodegeneration, behavioural alterations and cognitive impairment has not been completed. This type of analysis becomes complex since most studies have used multiple outbred rat strains such as Sprague-Dawley, Wistar, and Long-Evans hooded (LEH) in epilepsy research. Interstrain differences have been detected in post-status epilepticus models, including kainic acid (Sanberg and Fibiger, 1979; Golden et al., 1995; Xu et al., 2004), pentylenetetrazol (Becker et al., 1997a) or pilocarpine (Xu et al., 2004; Hort et al., 2000). Nonetheless, the potential interstrain differences in the LDP and RLDP lithium/pilocarpine procedures have not been determined. In chapter three, we compared the effect of the LDP and RLDP procedures on the rates of SE induction, intensity of behavioural seizures and mortality rates in Wistar rats and in LEH rats. Our findings showed that the RLDP protocol reduced mortality and severity of SE in the Wistar rats when compared to the LDP protocol, but had no significant effect in LEH rats. Consequently, Wistar rats were used in all other experiments presented in this thesis to minimize the number of experimental animals used. Next, we showed that in spite of the differences in mortality rates and in intensity of behavioural seizures between Wistar rats treated with either the LDP or the RLDP protocols, severity of pyramidal cell death in the hippocampus and in the piriform cortex was similar at 3 months of recovery (reviewed in section 3.4). Likewise, we demonstrated animals that developed SE, whether from the LDP or the RLDP protocol, did not differ in anxiolytic behavioural assessments (reviewed in section 6.4.1) or in Morris water maze (MWM) performance (reviewed in section 7.4.1). Although our results suggest that the RLDP procedure is the most effective in reducing mortality in Wistar rats, it is not necessarily the best option to use in other strains as it had no effect in LEH rats. We were able to validate that the RLDP protocol produced reliable neuronal loss and behavioural and cognitive deficits in Wistar rats that were similar to the LDP protocol; these results are discussed in the subsequent sections.

244 The severity and pattern of neuronal death in the lithium/pilocarpine model We provided the first analysis of neurodegeneration with the RLDP lithium/pilocarpine protocol for the induction of SE, and found that the extent of neuronal death in regions of the hippocampus, amygdala, thalamus and piriform cortex was comparable to that previously reported in the LDP lithium/pilocarpine protocol, and in the high-dose pilocarpine model (Honchar et al., 1983; Turski et al., 1983a; Turski et al., 1983b; Clifford et al., 1987; Fujikawa, 1996; Motte et al., 1998; Covolan and Mello, 2000; Peredery et al., 2000; Poirier et al., 2000) (see section 4.4.1) Pattern of neuronal death in the hippocampus The pattern of hippocampal pyramidal cell death we report in rats following SE closely replicates classical hippocampal sclerosis in humans, with more severe neuronal death occurring in the CA1 compared to the CA3, CA4 and hilus (Mathern et al., 1996; Wieser, 2004; Löscher and Brandt, 2010). In addition, we detected no neuronal death in the CA2 or the granule cell layer of the dentate gyrus. The CA2 and dentate gyrus are often referred to as the resistant sectors since these areas are also relatively spared in classical human hippocampal sclerosis (Mathern et al., 1996; Wieser, 2004). Although the lithium/pilocarpine model closely replicates classic hippocampal sclerosis in humans, this pattern of neuronal loss is not comparable across all poststatus epilepticus models (see section 1.2.2). For instance, when compared to the lithium/pilocarpine and pilocarpine models, SE elicited by kainic acid results in preferential pyramidal cell death to the CA3 compared with CA1 (Schwob et al., 1980; Golden et al., 1995; Tokuhara et al., 2007), and probably reflects the higher distribution of hippocampal kainate receptors in CA3 (Dudek et al., 2006). Recent findings have suggested that hippocampi from patients with MTLE can be assigned to several distinct groups based on the severity and distribution of neuronal loss (Mathern et al., 2002; de Lanerolle et al., 2003; Blümcke et al., 2007; Mueller et al., 2009; Thom et al., 2010). In these studies, a classic pattern of hippocampal sclerosis with severe pyramidal cell loss in CA1 and moderate neuronal loss in all other subfields excluding CA2 ranged between 19 to 70 percent of cases evaluated (Mathern et al., 2002; de Lanerolle et al., 2003; Thom et al., 2010; Blümcke et al., 2007; Mueller et al., 2009). Other frequently observed sub-categories included

245 223 severe to varying degrees of pyramidal cell loss in all hippocampal subfields, neuronal loss restricted to the CA1 area, and neuronal loss restricted to the hilar region (de Lanerolle et al., 2003; Thom et al., 2005; Blümcke et al., 2007; Mueller et al., 2009; Thom et al., 2010). Up to 19 percent of cases evaluated were reported to have an absence of hippocampal sclerosis (and therefore, were referred to as paradoxical TLE) (de Lanerolle et al., 2003; Thom et al., 2005; Blümcke et al., 2007; Mueller et al., 2009; Thom et al., 2010). The different patterns of hippocampal neuronal loss have been suggested to reflect different pathoetiologies and clinical subtypes (de Lanerolle et al., 2003; Thom et al., 2005; Blümcke et al., 2007; Mueller et al., 2009; Thom et al., 2010). For instance, clinicopathological correlations have been demonstrated between different patterns of HS and the age of an IPI (Van Paesschen et al., 1997; Blümcke et al., 2007; Thom et al., 2010). Furthermore, different subcategories of HS have been found to be predictive of postsurgical outcome; patients exhibiting a classic HS, or varying damage within all hippocampal subfields, had the best outcome of remaining seizure-free up to 2 years following surgery when compared with patients exhibiting damage restricted to the hilar region, the CA1 region, or with no HS (de Lanerolle et al., 2003; Thom et al., 2005; Blümcke et al., 2007; Mueller et al., 2009; Thom et al., 2010). Further research is necessary to assess the significance of different patterns of HS in determining the causes and potential treatment of what may be different subcategories of MTLE. Although the lithium/pilocarpine model closely replicates classic HS in humans, it remains unknown how effective this or other post-status epilepticus models are in representing other potential patterns of HS in human MTLE Pattern of neuronal death in extrahippocampal structures Although the quintessential pathology of human MTLE has been HS, a combination of neuronal loss, synaptic reorganization and gliosis (see section ), these pathological alterations have also been reported in extra-hippocampal structures (Hudson et al., 1993; Du et al., 1993; Wolf et al., 1997; Juhász et al., 1999; Yilmazer-Hanke et al., 2000; Bernasconi et al., 2003; Natsume et al., 2003; Bernasconi et al., 2005; Dawodu and Thom, 2005; Thivard et al., 2005). We comparably showed a broad distribution of neuronal loss within the hippocampus, thalamus, amygdala and piriform cortex in rats after SE. Previous studies have additionally reported other pathological changes including synaptic reorganization, gliosis and cell genesis in regions of the hippocampus, thalamus, amygdala, and piriform and entorhinal cortices (Bertram and Scott, 2000; Roch et al., 2002; Andre et al., 2007; Jung et al., 2009; Ben-Ari and Dudek, 2010). These

246 224 pathological changes are purported to cause the increased seizure propensity of these regions in human MTLE ( Babb, 1986; Swanson, 1995; Masukawa et al., 1996; Pitkanen et al., 1998; Aliashkevich et al., 2003; Bartolomei et al., 2005; Koch et al., 2005; Majores et al., 2007; Williamson and Patrylo, 2007; Wittner et al., 2009) and in rats after SE (Bertram et al., 1998; Mangan et al., 2000; Bertram et al., 2001; Aroniadou-Anderjaska et al., 2008). Based on the broad distribution of the brain pathology, Bertram et al., (2009) suggested that seizure onset is not just focused in the hippocampus, but involves widespread neuronal circuitry of independent limbic structures. Additional data from animal studies further support the concept that seizures arise within a distributed limbic system. First, electrophysiological recording of multiple limbic sites in chronically epileptic rats showed that the epileptogenic zone is broad, and indicated that the substrate for seizure generation is distributed over several brain structures (e.g., hippocampus, amygdala, and piriform cortex) (Bertram, 1997). Second, kindling studies have demonstrated that the same type of behavioural seizure may be induced by stimulation of a number of different limbic sites (Goddard et al., 1969; Bertram, 2007). However, the ease with which the seizure is induced and the rapidity with which the seizure spread depends on which site is stimulated. For instance, although kindling of limbic seizures occurred with stimulation of the medial dorsal thalamic nucleus, the threshold for electric current to induce the seizure was significantly higher than the threshold in the amygdala and hippocampus (Bertram et al., 2008). Furthermore, the seizures generalized more rapidly from the medial dorsal thalamic nucleus compared to the limbic structures (Bertram et al., 2008). Further studies showed that inactivating the medial dorsal thalamic nucleus pharmacologically or manipulating the excitability of this region had a profound effect on seizure activity in the hippocampus (Bertram et al., 2001; Bertram et al., 2008). In general, while the hippocampus, amygdala and entorhinal and piriform cortices are proposed to act as multiple independent generators of seizures, the thalamus displays characteristics that suggest this structure may act as a physiological synchronizer (Bertram et al., 1998; Avoli et al., 2002; Bertram, 2009). Other sites (although unknown) are proposed to act as neuromodulatory input to the epileptogenic network that affects the foci propensity to seize (Bertram, 2009). Similar findings supporting the involvement of distinct epileptogenic structures in human MTLE have been reported: (1) in pathological studies that showed a broad distribution of

247 225 neuropathological changes (e.g., neuronal loss, synaptic reorganization, neurogenesis) in hippocampal and extrahippocampal structures (discussed above), (2) in electrophysiological studies that have demonstrated multifocal or synchronized regional seizure onset (Spencer and Spencer, 1994; Blumenfeld et al., 2004; Kobayashi et al., 2009; Bartolomei et al., 2010; Wendling et al., 2010), or the presence of multiple epileptogenic zones (Maillard et al., 2004; Bartolomei et al., 2008a; Bartolomei et al., 2008b; Chassoux et al., 2008; Wendling et al., 2010), (3) in functional imaging studies (PET scans) that have shown broad regional hypometabolism (indicative of an epileptogenic zone) in the mesial temporal structures (Henry et al., 1990; Sperling et al., 1990; Ryvlin et al., 1991; Duncan, 1997; Benedek et al., 2004), and (4) in studies assessing surgical outcomes of seizure control in patients with MTLE-HS. For instance, although removal of limbic areas of the medial temporal lobe has led to a high rate of seizure control (varying 50 to 93% between studies), there exist a significant number of patients for whom seizure control is incomplete, indicating the presence of other seizure focii (Guénot, 2004; Téllez-Zenteno et al., 2005; Schramm, 2008; Bertram, 2009). This idea is further supported by studies which demonstrated that the greater extent of resection of mesial temporal structures correlated with a better prognosis of remaining seizure-free (Nayel et al., 1991; Bonilha et al., 2004; Vinton et al., 2007); however, others have failed to establish this relationship ( McKhann et al., 2000; Schramm, 2008). Finally, the presence of extra-temporal epileptogenic zones (e.g., thalamus and neocortex) has been found to decrease the effectiveness of surgical intervention in patients with refractory complex partial seizures (Newberg et al., 2000; Choi et al., 2003; Téllez- Zenteno et al., 2005; Treiman, 2010). Altogether, these data emphasize the importance of assessing neuropathological changes not only in the hippocampus, but also in extrahippocampal structures, with the ultimate goal of improving surgical outcomes and developing other nonsurgical therapies to improve seizure control Differences in the pattern of neuronal loss between human MTLE and rats after SE The symmetry of neuronal death between the cerebral hemispheres is different in human MTLE- HS compared to rats following SE. In patients with MTLE-HS, atrophy of the hippocampus is often bilateral but asymmetrical, with the side ipsilateral to the seizure focus exhibiting more severe sclerosis (Mintzer et al., 2004; Wieser, 2004). The same pattern of neuronal loss also occurs in extrahippocampal structures, with greater atrophy detected on the side ipsilateral to the

248 226 seizure focus (Bonilha et al., 2010a; Pail et al., 2010). In contrast, our findings and others noted bilateral and symmetrical neurodegeneration of hippocampal and extrahippocampal structures in rats following SE (Ben-Ari et al., 1980; Turski et al., 1983b; Clifford et al., 1987; Covolan and Mello, 2000). The difference in pattern of neuronal loss may be contributed to species differences in neural connectivity between the left and right cerebral hemispheres (Engel et al., 2008). For instance, a major difference exists in the progression of seizure activity in rodents compared to primates. In the rat, focally evoked seizures appear bilaterally symmetrical from time of onset, as evident by both behavioural and cortical manifestations (Ben-Ari et al., 1979; Peterson et al., 1992); seizure activity can spread rapidly mostly via commissural pathways such as those in the anterior commissure and hippocampus (Engel et al., 2008). In contrast, in primates, as in humans, behavioural and electrophysiological seizure manifestations remain lateralized longer, and typically stay asymmetrical even as the seizure progress to engage brainstem circuitry (Loddenkemper and Kotagal, 2005; Engel et al., 2008; Götz-Trabert et al., 2008; Jan et al., 2010; Napolitano and Orriols, 2010); this occurs since the path of least resistance involves seizure activity spreading down the neuraxis rather than across the forebrain commissures (e.g., corpus collosum, hippocampal commissure, and anterior commissure) (Lieb et al., 1987; Lieb et al., 1991; Engel et al., 2008). 8.4 The effect of increasing survival time on SE-induced neuronal death Majority of neuronal death occurs early in rats following SE Because previous studies have been limited by the semi-quantitative assessment of tissue damage and by the limited number of recovery times assessed (Fujikawa, 1996; Motte et al., 1998; Covolan and Mello, 2000; Peredery et al., 2000; Poirier et al., 2000) (see sections and 4.4.1), our knowledge remains restricted on how SE-induced neuronal death in different brain regions evolves over time. In chapter 4, we extended these findings by providing a detailed, quantitative time-course comparison of SE-induced neuronal death in 19 brain regions within the hippocampus, amygdala, thalamus and piriform cortex. Stereological estimates of NeuN positive neurons were assessed at ten different time intervals after SE (from 1 hour to 3 months). Our results demonstrated the the majority of neuronal death evolved rapidly after SE in rats, with the majority of neuronal loss in all brain structures present by 24 hours. We were the first to demonstrate that maximal neuronal loss occurred as early as 3 hours after SE in the hilus, ventral

249 227 CA3, reticular thalamic nucleus (Rt) and posteromedial cortical amygdaloid nucleus (PMCo), by 6 hours in the dorsal CA4 and posterior thalamic nuclei, and by 12 hours in the dorsal CA3 and posterior piriform cortex (PPC) (see Table 4.3). The time course of this degeneration was different for individual regions of the hippocampus, amygdala, thalamus and piriform cortex. These findings are extensively discussed in section 4.4. Clinical studies have provided evidence that the acute neuronal loss we observe in rats following lithium/pilocarpine-induced SE may closely resemble the evolution of brain damage observed in humans following an IPI. In studies using magnetic resonance imaging, evidence of acute neuronal injury was detected in patients when assessed within 1 to 5 days after a prolonged febriles seizure (Scott et al., 2002; Sokol et al., 2003; Scott et al., 2003). An elevation of serum neuron-specific enolase, a marker for acute neuronal injury, was also elevated in adult patients when assessed within 24 hours following SE (DeGiorgio et al., 1996; DeGiorgio et al., 1999) Majority of neuronal death is the consequence of SE and not of spontaneous recurrent seizures (SRSs) Because it is difficult to determine whether delayed neuronal loss is caused by SE, the occurrence of SRSs, or both, the literature remains unclear as to the effect of SRSs on neuronal loss (Dudek et al., 2002) (see section ). Although we did not determine the latency to onset of SRSs in rats after SE, our findings did demonstrate that by 3 months of recovery, all rats were considered epileptic, defined as having exhibited at least 1 SRSs. Glien et al., (2001) demonstrated that following 60 minutes of SE induced by the RLDP protocol in Wistar rats, the average latency to the first SRS was 40 days (range days). Thus, it is reasonable to assume that any neuronal death preceding 40 days after SE is the consequence of SE and not of SRSs. This suggestion is supported by our findings that: (1) with the exception of the thalamic somatosensory nuclei (VPM and VPL), all other brain regions assessed showed no further progression of neuronal loss 2 weeks after SE, a period in which SRSs were observed to occur in rats analyzed at 3 months of recovery, and (2) Fluoro-Jade B staining, a marker for degenerating neurons, was not detected in epileptic rats analyzed at 3 months after SE. In support of the suggestion that SE primarily contributes to neurodegeneration, Liu et al., (1994) used the highdose pilocarpine procedure and showed neuronal death at 3 weeks recovery in the dorsal CA1 and CA3. The authors reasoned that since SRSs appeared approximately 2 to 2 ½ weeks after SE in these animals, but did not contribute to any significant additional neuronal loss 6 to 12

250 228 weeks later, that neuronal death primarily resulted from the pilocarpine-induced SE (Liu et al., 1994). In our studies, the thalamic somatosensory nuclei were the only regions to show additional neuronal death between 2 weeks and 3 months after SE, and therefore, may have resulted from SE, SRSs, or both. Overall, our results indicate that the vast majority of neuronal death is the result of SE and that little, if any additional death, occurs subsequent to the start of SRSs. These results are further discussed in section Our findings suggest that SE as the IPI, and not the subsequent development of SRSs, primarily contributes to neuronal death in rats. This appears to parallel findings in human data that support the existence of brain damage prior to development of human MTLE-HS (Mathern et al., 1996; Wieser et al., 2004), and the lack of evidence of substantial neuropathology evolving during the chronic stage of SRSs (Cendes et al., 1993; Bower et al., 2000; Moran et al., 2001). Regarding the causes of brain damage, previous studies have suggested a high incidence of an IPI, which includes febrile convulsions, SE, encephalitis, stroke or traumatic brain injury, as a predisposing factor in human MTLE-HS (Mathern et al., 1996; Mathern et al., 2002). The most common type of IPI recognized is prolonged febrile seizures reported in 30 to 80% of MTLE-HS patients (French et al., 1993; Lewis, 1999; Cendes and Andermann, 2002; Mathern et al., 2002). Clinical data determining whether MTLE-HS is a progressive disease remains ambiguous. There is evidence from some (Briellmann et al., 2001; Briellmann et al., 2002; Fuerst et al., 2003), but not all (Liu et al., 2002; Holtkamp et al., 2004), longitudinal MRI studies that chronic epileptic seizures result in progressive hippocampal changes suggesting cumulative damage. Furthermore, while some studies found increased hippocampal neuronal loss in patients with a longer duration of temporal lobe epilepsy (Mathern et al., 1996; Mathern et al., 2002; Fuerst et al., 2003), or those with a greater total seizure number (Kalvianinen and Salmenpera, 2002), others have failed to establish these relationships (Cendes et al., 1993; Moran et al., 2001; Thom et al., 2005). Still, there appears to be a general consensus that in patients with MTLE-HS, the majority of neuronal loss occurs prior to the onset of habitual seizures, and that less substantial neuronal loss may occur with an increased duration of the epileptic syndrome (Mathern et al., 1996; Mathern et al., 2002; Wieser, 2004).

251 Cognitive and behavioural alterations following lithium/pilocarpine-induced SE MTLE-HS in humans is often associated with co-morbid interictal disorders, including anxiety, depression and psychosis (Boro and Haut, 2003; Gaitatzis et al., 2004; Devinsky, 2004a, Garcia- Morales et al., 2008), as well as learning and memory impairments (Motamedi and Meador, 2003; Vingerhoets, 2006) (see section 1.6). Studies have shown that co-morbid psychiatric disorders are an independent risk factor for a poor quality of life in human epilepsies, and can be equal or more detrimental to the individual s overall function than the seizures themselves (Cramer, 2002; Gilliam, 2002; Cramer et al., 2003; Boylan et al., 2004; Johnson et al., 2004). Consequently, it is important that animal models not only recapitulate the neuropathology and genesis of SRSs as observed in human MTLE-HS, but also allow researchers to characterize and to identify treatments that can potentially mitigate disease-related cognitive and behavioural morbidity (Stafstrom et al., 2006). In chapter 6, we demonstrated that SE in rats reduced exploratory behaviour as assessed in the open field (defined in section ), and increased hyperreactivity (or aggression) as assessed in the pick-up test and in the touch-response test (defined in section ). These behavioural alterations appeared within the first week after SE and remained unchanged by development of SRSs at 3 months (see section 8.6). SE also produced anxiolytic effects as measured in rats by the elevated-plus maze (defined in section ) at 3 months of recovery. In chapter 7, we demonstrated that rats 3 months after SE exhibited impaired performance in the MWM (i.e., poor spatial learning and use of behaviouralsearch strategies) (defined in section ). These results are extensively discussed in sections 6.4 and 7.4, respectively. While we are the first to report cognitive changes (e.g., spatial learning and memory) in rats treated by the RLDP procedure, Detour et al., (2005) is the only other group to have characterized behavioural changes with this protocol (discussed in section 6.4.1). In general, our data show that lithium/pilocarpine-induced SE and/or SRSs are accompanied by profound behavioural and cognitive alterations, and thus could model affective and cognitive disorders observed in human MTLE-HS. the sections that follow The effect of SE on spatial learning and memory Specific aspects of these alterations are discussed in The lithium/pilocarpine model allows us to not only investigate how seizures alter behaviour, but also which aspect(s) of behaviour are most at risk for impairment (Stafstrom et al., 2006).

252 230 Acquisition of the hidden platform version of the Morris water maze (MWM) task has two main components, behavioural strategies learning and spatial learning (see section ). Although previous studies have reported impaired spatial learning and memory in rats after SE, little attention has been given to determine the effect of SE on behavioural strategies (see section ). In chapter 7, we extended previous findings by completing a systematic assessment of behavioural search strategies, and quantifying their contribution to overall cognitive performance in rats after SE. We first showed that SE in rats exhibited impaired spatial learning and memory. This finding is supported by the observations that rats after SE (1) still reached criterion in the visual acuity test and exhibited swim speeds comparable to controls, indicating an absence in gross motor or visual impairment, (2) performed poorly on the spatial probe tests, and (3) predominately used repetitive-looping based search strategies and non-spatial, systematic search strategies; these strategies do not rely on use of spatial information. Our results are consistent with previous studies that have demonstrated poor spatial learning and memory in rats after SE (see section ). Comparable deficits in visual-spatial learning and memory are reported in human MTLE-HS (Hermann et al., 1997; Gleissner et al., 1998; Abrahams et al., 1999; Glikmann-Johnston et al., 2008). For example, spatial and non-spatial, working and reference memory in MTLE-HS patients were evaluated using a Nine-Box Maze (Abrahams et al., 1997; Abrahams et al., 1999), a task analogous to the radial-arm maze used in rodents (reviewed in: Lanke et al., 1993; Hodges, 1996). Results showed that MTLE-HS patients exhibited specific deficits in the working and reference spatial memory components of the task (Abrahams et al., 1997; Abrahams et al., 1999). Furthermore, spatial memory errors significantly correlated with volumetric measures (e.g., degree of atrophy) of mesial temporal lobe structures, indicating a role of the hippocampal region in spatial memory (Abrahams et al., 1999). In another study, Glikmann-Johnston et al., (2008) assessed three measures of spatial learning in patients with MTLE-HS: navigation, object location and plan drawing. The authors demonstrated that MTLE-HS patients exhibited compromised behavior in all 3 measures. These results indicate that the poor spatial learning and memory we observed in rats following SE are comparable to cognitive deficits reported in human MTLE-HS.

253 The effect of SE on use of behavioural search strategies Impaired selection of behavioural search strategies We next showed that SE in rats dramatically impaired search strategy use in the spatial acquisition and spatial reversal MWM tasks. Rats following SE displayed a greater proportion of less efficient search strategies (repetitive-looping based strategies and non-spatial, systematic strategies) while using fewer spatial strategies compared to controls (see section 7.4.2). Several specific findings indicate that the application of poor search strategies in the SE group occurred as a result of behavioural inflexibility (e.g., the inability to select appropriate behavioural strategies): (1) Although the SE group showed impaired MWM performance over the entire 14 days of acquisition training compared to controls, the majority of these animals were able to reduce escape latencies to finding the platform within this period (discussed in section 7.4.1). By conducting the convolution analyses (described in section 7.3.8), we showed that changes in search strategy use primarily contributed to improved MWM performance in these animals. This finding is further discussed in section 7.4.3, and suggests that the higher escape latencies in rats after SE may not be solely attributed to an inability to acquire behavioural strategies. Although rats after SE required more trials to pass the visible platform test, indicating impairment in behavioural strategies, these animals were still capable of acquiring the necessary skills of swimming away from the wall of the pool and climbing onto the escape platform. (2) We noted that although the SE group was capable of acquiring spatial memory, these animals continued to predominately select less efficient search strategies and to perform poorly in the spatial probe tests. This observation was supported by the fact that the SE group showed improvement in use of spatial strategies during spatial acquisition. Moreover, when the platform was changed to the opposite quadrant, these animals exhibited an increase in escape latencies that correlated with the use of fewer spatial strategies, and the use of more non-spatial, systematic strategies. These findings are further discussed in section Altogether, our results suggests that rats after SE are capable of acquiring behavioural strategies, and are even capable of using spatial search strategies. However, SE elicited in rats may cause deficits in the ability to inhibit non-efficient escape strategies (i.e., behavioural inflexibility).

254 232 These results are comparable to previously reported observations in rats treated with anticholinergic drugs (Whishaw, 1989; Whishaw and Tomie, 1987; Day and Schallert, 1996), in rats and mice following traumatic brain injury (Brody and Holtzman, 2006; Thompson et al., 2006), and in rats with lesions to specific brain structures (i.e., hippocampus, caudate-putamen) (Whishaw and Petrie, 1988; Day et al., 1999). These studies used different testing paradigms in the MWM task (see chapter 9) to dissociate spatial learning, behavioural-strategy learning and the ability to switch between behavioural strategies. Although the experimental rats in these studies were capable of behavioural strategy learning and spatial learning, in adaptive testing paradigms (i.e., where animals were required to switch use of behavioural strategies), they perseverated in using the least efficient strategy (see section 9.2) Impaired acquisition of behavioural search strategies One of our main findings was that although the majority of rats with SE exhibited impaired MWM performance there were between animal differences in the ability to improve performance over prolonged training, and we were able to subdivide rats into three categories based on performance during spatial acquisition. These results are discussed in section Of the animals that showed impaired MWM performance compared to controls, 44% learned how to use more efficient search strategies, and the increased use of these strategies accounted for their improved performance, suggesting that they were capable of acquiring new behavioural strategies. In contrast, 34% of the rats showed no improvement in escape latencies or changes in search strategy use with extended training. As noted above, acquisition of the MWM has two main components: behavioural strategy learning and spatial learning. The animal must first acquire behavioural strategies before success in spatial learning can be achieved. Acquisition of behavioural strategies involves the rat learning how to navigate in its spatial environment and using the most effective strategies for searching and reaching its target (see section ). Failure to swim away from the wall to search the inner region of the pool can inflate escape latencies, and reduce the usefulness of escape latencies as a measure of spatial learning (Schenk and Morris, 1985; Whishaw and Tomie, 1987; Cain, 1997). Although elevated escape latencies are most often interpreted as a spatial learning impairment, others have reported dissociations between this measure and more specific and accurate measures of spatial learning (Schenk and Morris, 1985; Cain, 1997).

255 233 Consequently, escape latencies should be interpreted cautiously, preferably in conjunction with behavioural strategy analyses, and other more specific and accurate measures of spatial memory (e.g., probe test analyses). Several authors have suggested that under conditions of marked thigmotaxic swimming, elevated escape latencies are best interpreted as impairment in behavioural strategy learning (Schenk and Morris, 1985; Morris, 1989; Whishaw, 1989; Cain, 1997). The subset of rats in our study that exhibited no improvement during spatial acquisition had parallel impairments in both escape latencies and thigmotaxic swimming, indicating that they were also impaired in the acquisition of behavioural strategies. In sum, we showed that both impairment in search strategy use, and poor spatial learning and memory, contribute to the impaired performance of epileptic rats during spatial learning tasks in the MWM. The ability to both learn behavioural strategies and to select the most appropriate behavioural strategy appears to operate on overlapping and separable neural systems. For instance, the ability to select between allocentric (e.g., based on distal cues) and egocentric (e.g., based on placement in the environment) search strategies relies on a competitive interaction between the hippocampal and the striatal neural systems, respectively (see section 7.4.4). The amygdala has also been shown to influence search strategy selection (Packard, 2009; Hawley et al., 2011). In contrast, behavioural strategy learning has been shown to require multiple neural systems, including the prefrontal cortex, striatum, cerebellum, medial thalamus, and hippocampus (see section ). Although in the present study it appears that SE in rats impaired the ability to select the most appropriate search strategy, a subset of these rats may instead exhibit deficits in behavioural strategy learning. Future studies using different testing paradigms are necessary to more directly delineate the effect of SE on spatial learning, behavioural strategy learning, and behavioural flexibility (see chapter 9) Impaired use of behavioural strategies in human MTLE-HS Patients with MTLE-HS may have analogous poor use of behavioural strategies. Recent studies indicate that MTLE may affect decision-making under ambiguity (Labudda et al., 2009; Delazer et al., 2010; Delazer et al., 2011; Yamano et al., 2011;), which uses implicit information and requires processing of feedback and reward-based, adaptive learning (Brand et al., 2006). In these studies, decision-making under ambiguity was assessed by performance in the Iowa Gambling Task (IGT). The IGT is proposed to simulate decision-making in real-life situations in

256 234 which premises, outcomes, rewards or punishments are uncertain (Brand et al., 2006). In the IGT, MTLE-HS patients were reported as having shifted more often between advantageous and disadvantageous selections than controls, and had difficulties in establishing a consistent response pattern (Labudda et al., 2009; Delazer et al., 2010; Yamano et al., 2011; Delazer et al., 2011). The authors suggested that MTLE-HS patients selected decks randomly and were not capable of adapting their strategies from feedback. It has been suggested that decision-making abilities under ambiguity, as measured by the IGT, are separable from cognitive abilities, including executive functions and intelligence (Brand et al., 2006; Delazer et al., 2010; Toplak et al., 2010). For instance, recent studies compared decision-making under ambiguity and decision-making under risk in MTLE-HS patients. Risk refers to the form of uncertainty where probabilities are known, as opposed to ambiguity where outcome probabilities are incompletely known (Brand et al., 2006). While MTLE-HS patients showed severe deficits in decision-making under ambiguity in the IGT, they perform normally in decision-making under risk as assessed by the game of dice task (GDT) (Labudda et al., 2009; Delazer et al., 2010), or by the probability-associated gambling task (PAG) (Delazer et al., 2010), both of which offers explicit probabilities for gains and losses. Brand et al., (2006) suggested that decision-making under ambiguity and decision-making under risk both require a feedback route, in which experience in terms of gains and losses is processed. However, decision-making under risk also uses a cognitive route, in which executive functions use explicit information to plan and modify decision-making behaviour. This is supported by the fact that GDT performance has been shown to correlate with executive functions (Brand et al., 2006), but not with decision-making under ambiguity as assessed by the IGT (Brand et al., 2007; Brand et al., 2008). Likewise, Toplak et al., (2010) reviewed 43 studies and showed that the majority of these found no positive correlation with IGT performance and other cognitive abilities, including measures of inhibition, working memory, and set-shifting as indices of executive functions, as well as measures of verbal, nonverbal and full-scale IQ as indices of intelligence. Based on this literature, Labudda et al., (2009) and Delazer et al., (2010) suggested that brain regions underlying executive functions are preserved, and that this enabled MTLE-HS patients to make decisions based on explicit, but not implicit, information. Several lines of evidence suggest that impaired decision-making abilities in MTLE-HS patients are caused by lesions specific to the temporo-limbic system. First, a recent study showed that

257 235 IGT performance in patients with MTLE-HS was worse than patients with neocortical TLE (characterized by lesions in the lateral, basal or polar parts of the temporal lobe) (Delazer et al., 2011). Although patients with neocortical TLE and controls showed a significant learning effect over the blocks of the IGT, performance of patients with MTLE-HS showed no improvement from the first to the last block of trials. These results suggest that TLE patients with lesions associated with the mesial temporal lobe have greater deficits in decision-making compared to TLE patients with lesions associated with the neocrotex. (Delazer et al., 2011). Previous studies have similarly found impaired IGT performance in patients with MTLE who have lesions in the amygdalo-hippocampal complexes (Bonatti et al., 2009; Labudda et al., 2009; Delazer et al., 2010). Second, the IGT detected deficits with decision-making abilities in individuals with lesions in the amygdala and/or hippocampus (Bechara et al., 1999; Gutbrod et al., 2006; Gupta et al., 2010). Finally, a brain imaging study using functional connectivity analyses provided evidence that the amygdala and hippocampus are involved in reward-based learning, and the strength of the connection between these two structures predicts flexibility in adapting decisions (Cohen et al., 2008). In sum, we showed that rats after SE used suboptimal search strategies during spatial learning tasks in the MWM. This finding may be analogous to specific deficits in decision-making under ambiguity observed in human MTLE-HS. In both conditions, acquisition of behavioural (or decision-making) strategies relies on use of implicit information, and requires acquisition and adaption of behavioural strategies based on experience. Furthermore, the neural systems involved in acquisition and selection of behavioural strategies appear to rely on neural networks dissociable from cognitive abilities (e.g., spatial learning in rats; executive functions and intelligence in humans). Finally, human and rodent studies suggest that lesions within limbic structures may contribute to impaired use of behavioural strategies. 8.6 The temporal relationship between neuronal death, behavioural alterations and cognitive impairment following status epilepticus Because we conducted a detailed time-course evaluation of SE-induced neuronal death (see section 8.4), we were able to compare the progression of neuronal death with the occurrence of behavioural alterations in rats following SE. Our findings showed that neuronal loss occurs

258 236 within the hippocampus, amygdala, thalamus and piriform cortex as early as 3 to 12 hours following SE, with the majority of neuronal death present by 24 hours (see section 8.4). Although we demonstrated SE-induced behavioural changes in the hyperexcitability tests and in the open field within the first week after SE that persisted for at least 12 weeks (see chapter 6), previous studies have demonstrated the onset of behavioural alterations and cognitive impairment as early as 24 to 48 hours (see section 6.4.2). For example, pilocarpine-treated rats exhibited increased aggression to handling and increased hyperexcitability as early as 2 days after SE (Rice et al., 1998), and kainic acid-treated rats showed increased hyperreactivity in response to handling and increased activity in the open field 24-hours after SE (Milgram et al., 1988). In both studies, behavioural alterations persisted 6 to 8 weeks later (Milgram et al., 1988; Rice et al., 1998). Similar results were obtained in cognitive tasks (see Table A1-2). Chauvière et al., (2009) recently demonstrated impaired performance in the displaced object-recognition task at 4 days following pilocarpine-induced SE, the earliest time assessed, with deficits in spatial memory and object recognition still present at 40 days. Others reported impaired visualspatial learning in the MWM when testing was conducted 5 to 7 days following SE induced by either kainate or pilocarpine (Milgram et al., 1988; Rice and DeLorenzo, 1999; Cunha et al., 2009; Sun et al., 2009). Even though Hort et al., (2000) pre-trained rats for 8 days prior to SE onset, animals still exhibited impaired spatial memory 3 and 6 days following pilocarpineinduced SE (Hort et al., 1999). Because SE animals are in very poor physical condition immediately following SE, it is difficult to assess the behavioural and cognitive ability of rats less than 1 or 2 days following SE so that the time of the first appearance of impaired behavior remains unknown. In these studies, cognitive impairments persisted for up to 2 months, indicating no recovery of behavioural function (Milgram et al., 1988; Rice and DeLorenzo, 1999; Cunha et al., 2009; Sun et al., 2009). These findings, together with our data, indicate that neurodegeneration, behavioural alterations and cognitive impairment develop within the same timeframe and are consistent with a functional relationship between these processes.

259 The effect of neuroprotection on epileptogenesis, behavioural alterations, and cognitive impairment Neuroprotection within the hippocampus Effect of neuroprotection on epileptogenesis Human and animal studies suggest hippocampal sclerosis is involved in the development of TLE and in the genesis of behavioural and cognitive deficits (Engel et al., 1991; Engel, 1996; Mathern et al., 1996; Sloviter, 1999; Acharya et al., 2008). In chapter 5, we showed that tat-nr2b9c provided partial neuroprotection of hippocampal pyramidal cells (CA1, CA3 and CA4) when administered 3 hours following SE. This result allowed us to determine whether partial neuroprotection within the hippocampus reduced the incidence of SRSs, or modified behavioural and cognitive alterations in rats following lithium/pilocarpine-induced SE. In spite of its neuroprotective effect, tat-nr2b9c had no effect on the number of rats developing SRSs (discussed in section 6.4.3), on behaviour as assessed by the open field, elevated plus maze or hyperexictability tests (discussed in 6.4.3), or on MWM performance (see section 7.4.6). Our data show that partial neuroprotection of CA1, CA3 and CA4 pyramidal cells within the dorsal hippocampus was insufficient to prevent development of SRSs, or to modify behavioural and cognitive alterations in rats following SE. Table 8.1 provides a comparison of literature that investigated the effect of various neuroprotective drugs on the development and occurrence of SRSs, behavioural alterations, and learning and memory impairments in post-status epilepticus models. Numerous other studies are in agreement with our findings that partial neuroprotection of the hippocampus does not prevent the development of epilepsy (Rice et al., 1998; Halonen et al., 2001; Capella and Lemos, 2002; Ebert et al., 2002; Brandt et al., 2003b; Rigoulot et al., 2003; Rigoulot et al., 2004; dos Santos et al., 2005; Francois et al., 2006; Frisch et al., 2007; Chu et al., 2008). Even with nearly complete neuroprotection of the hippocampus, SRSs in rats following SE still occurred (Rice et al., 1998; Andre et al., 2007; Ebert et al., 2002; Brandt et al., 2006; Nehlig, 2007). In contrast to our findings and these studies, Bolanos et al., (1998) reported partial neuroprotection of the hippocampus and a decrease in the incidence of SRSs in rats treated with valproate (AED) for forty days following kainate-induced SE; however, because the occurrence of SRSs was monitored during the administration of valproate, it is difficult to determine whether the reduced

260 238 incidence of SRSs in these animals resulted from the partial neuroprotection of the hippocampus, or from the anticonvulsant effect of the drug. Jung et al., (2006) also reported that treatment of rats following lithium/pilocarpine-induced SE with celecoxib (a COX-2 inhibitor) offered partial neuroprotection of the hippocampus and reduced the incidence of SRSs; however, celecoxib also reduced the generation of ectopic granule cells in the hilus and reduced gliosis in the CA1, making it difficult to determine which if any of these drug effects were responsible for reducing the incidence of SRSs. The development of SRSs in the absence of overt brain damage in other seizure models further suggests that neuronal loss is not a prerequisite for epileptogenesis (see section ), and indicates that other factors such as synaptic reorganization (see section 1.5.2), reactive gliosis (see section 1.5.3) and/or neurogenesis (see section 1.5.4) may play a more important role in epileptogenesis. Although we did not assess the effect of tat-nr2b9c on neuronal loss within the dentate hilus, others have demonstrated that most neuroprotective agents either have no effect or result in only partial neuroprotection of this region (Halonen et al., 2001; Rigoulot et al., 2003; Chu et al., 2008; Pitkänen et al., 2004; Rigoulot et al., 2004; Pitkänen et al., 2005; Francois et al., 2006; Jung et al., 2006; Zhou et al., 2007), even if other hippocampal subfields (e.g., CA1, CA3 and CA4) are completely protected (Rice et al., 1998; Ebert et al., 2002; Brandt et al., 2003b; Andre et al., 2007) (see Table 8.1). These results indicate that neurodegeneration in the dentate hilus is more resistant compared with other hippocampal regions to neuroprotective agents (Löscher and Brandt, 2010). Loss of dentate hilus cells is a characteristic finding in most post-status epilepticus rodent models (Loscher, 2002; Martin and Pozo, 2006; Sharma et al., 2007; Löscher and Brandt, 2010), and is the most consistent cell loss reported in human MTLE-HS (Sloviter, 1994; Lowenstein, 2001; Nadler, 2003; Blümcke et al., 2007; Thom et al., 2009). Hilar cell loss involves both excitatory mossy cells and inhibitory peptide-containing interneurons (Sloviter, 1987). There are presently two controversial explanations for how hilar cell loss may result in hyperexictability of the dentate granule cells, which could be causal for increased seizure susceptibility or the development of SRSs. These explanations may also account for the lack of effect neuroprotection within other hippocampal subfields has on SRSs. 1. The dormant basket cell hypothesis postulates that degeneration of mossy cells results in reduction of afferent excitatory synaptic input onto insult-resistant inhibitory basket cells, rendering these cells dormant and granule cells hyperexcitable (Sloviter, 1987;

261 239 Sloviter, 1989; Sloviter et al., 1991; Sloviter et al., 2003). However, the validity of this theory has been widely challenged on both anatomical and physiological grounds (reviewed in: Bernard et al., 1998; Ratzliff et al., 2004; Dudek and Sutula, 2007). 2. Alternatively, loss of inhibitory interneurons in the hilus may lead to reduced GABAergic synaptic input to granule cells that could contribute to abnormal recurrent excitation of granule cells found in epileptic rats (Kobayashi and Buckmaster, 2003; Ratzliff et al., 2004; Sun et al., 2007). A few studies have suggested that epileptogenesis may develop independent of hilar cell loss in post-status epilepticus rodent models. Numerous studies have confirmed that partial reduction in SE-induced hilar cell loss by various neuroprotective agents had no effect on the development of epilepsy (Ebert et al., 2002; Pitkänen et al., 2004; Francois et al., 2005; Pitkänen et al., 2005; Francois et al., 2006; Jung et al., 2006; Chu et al., 2008) (see Table 9.1). Even though Brandt et al., (2006) reported complete neuroprotection of the hippocampus and hilus in rats treated with valproate following electrically-induced SE, this effect was still not sufficient to prevent genesis of SRSs Neuroprotection may offer disease-modifying effects Even though (partial) neuroprotection within the hippocampus did not prevent the development of chronic epilepsy, it may never the less offer disease-modifying effects. The literature on this, however, remains controversial. For instance, partial neuroprotection within the hippocampus has been shown to increase the latency period (Francois et al., 2005) or to reduce the frequency and severity of SRSs (Bolanos et al., 1998; Pitkänen et al., 2004; Francois et al., 2005; Jung et al., 2006; Chu et al., 2008) in rats following SE (see Table 8.1). In contrast, Pitkanen et al., (2005) and Ebert et al., (2002) showed that partial neuroprotection of the hippocampus increased the severity of epilepsy; more specifically, the number of surviving hilar cells positively correlated with the frequency of SRSs. Still, others failed to establish a relationship between partial (Capella and Lemos, 2002; Brandt et al., 2003b; Narkilahti et al., 2003b; Rigoulot et al., 2003; Rigoulot et al., 2004; Pitkänen et al., 2004; Francois et al., 2006; Frisch et al., 2007) or complete (Brandt et al., 2006) neuroprotection of the hippocampus and the severity of SRSs. A series of studies suggest that neuroprotection within the hippocampus may even play a role in the prognosis of treatment for epilepsy. These experiments showed that rats developing epilepsy

262 240 after an episode of SE induced by sustained electrical basolateral amygdala stimulation markedly differed in their response to phenobarbital (Brandt et al., 2004; Volk et al., 2006; Bethmann et al., 2008). While approximately 30% of rats did not respond to treatment, SRSs were suppressed in the other animals, resulting in two subgroups (i.e., responders and nonresponders). Nonresponders exhibited hippocampal damage, whereas most responders exhibited no overt brain damage, suggesting a causal relationship between neuronal damage and pharmacoresistance (Volk et al., 2006; Bethmann et al., 2008). This data is consistent with the clinical observation that hippocampal sclerosis is associated with poor prognosis of AED treatment in patients with TLE, but is a good indicator for a positive outcome to surgical treatment (Schmidt and Löscher, 2005) The effect of neuroprotection on behavioral alterations The effect of neuroprotection within the hippocampus on SE-induced cognitive and behavioural morbidity also remains ambiguous. Other studies are in agreement with our findings that partial neuroprotection within the hippocampus had no effect on SE-induced cognitive and/or behavioural morbidity (Cilio et al., 2001; Halonen et al., 2001; Narkilahti et al., 2003b; Pitkänen et al., 2004; Zhou et al., 2007). In contrast to these results, several studies have demonstrated that partial (Bolanos et al., 1998; Cilio et al., 2001; dos Santos et al., 2005; Frisch et al., 2007; Cunha et al., 2009; Jun et al., 2009) or almost complete neuroprotection (Rice et al., 1998; Brandt et al., 2006) of the hippocampus reduced behavioural alterations or improved learning and memory in rats after SE. The results of these studies are shown in Table 9.1, and are discussed in sections (behavioural alterations) or (impaired learning and memory) Effect of neuroprotection in extrahippocampal regions Although limbic seizures have often been attributed to hippocampal pathology, others have provided evidence that the parahippocampal regions, including the piriform and entorhinal cortices, may also play an important role. Francois et al., (2005) showed that simultaneous protection of the hippocampus, parahippocampal cortices, amygdala and thalamus increased the latency to SRSs; the latency to the first SRS was correlated with neuronal damage in the piriform and entorhinal cortices, but not in the hippocampus and amygdala. Similarly, André et al., (2003) showed that partial neuroprotection (by pregabatrin) in layer II of the piriform cortex and in layers III and IV of the entorhinal cortex, and without any neuroprotection within the

263 241 hippocampus, caused an increase in the latency to SRSs (see Table 8.1). In a separate study, André et al., (2007) demonstrated that only simultaneous protection of the hippocampus and the parahippocampal cortices (plus the amygdala and thalamus by carisbamate) was able to delay or completely block the occurrence of SRSs, whereas treatments protecting only CA1 and/or CA3 (by caffeine, topiramate, vigabatrin, and amygdala kindling) were not effective. Based on this set of experiments, the authors suggested that neurodegeneration in the piriform and entorhinal cortices is a critical factor early in the epileptogenic process, whereas the involvement of the hippocampus is delayed (Andre et al., 2007; Nehlig, 2007). Still, even with drugs like carisbamate that offered nearly complete neuroprotection of the hippocampal formation and parahippcampal cortices, some rats still developed epilepsy (Andre et al., 2007), suggesting that simply preventing neuronal loss may not be enough to prevent epileptogenesis. Our findings support the general consensus that neuroprotection, as narrowly defined as prevention of cell death, is not sufficient to prevent epilepsy (Sankar, 2005). Walker et al., (2007) argued that neuroprotection after SE should encompass not only the prevention of neuronal death, but also the preservation of neuronal and network function. In general, the majority of patients with MTLE-HS exhibit at least several epileptogenic structures (including not only the lesional site but also other localized and distant sites) that form complex seizure networks (Bartolomei et al., 2008b; Bartolomei et al., 2010), and the fact that SRSs can be induced in rats in the absence of neuronal death, argues against damage in any key or critical brain region as being a prerequisite for epileptogenesis. No drug has yet been found to prevent or modify the epileptogenic process induced by a brain insult in humans (Temkin, 2001; Sossa, 2006; Temkin, 2009). Clinical trials conducted have largely focused on the effect of conventional anti-epileptic drugs (AEDs), including phenytoin, phenobarbital, carbamazepine, or valproate, in the prevention of epilepsy or diseasemodification. Not surprisingly, AEDs have thus far failed for several proposed reasons (Temkin, 2001; Sossa, 2006; Temkin, 2009): (1) AEDs have been developed for symptomatic suppression of seizures and not for the prevention or modification of the epileptogenic process, and (2) the molecular mechanisms underlying epileptogensis and ictogenesis are fundamentally different (reviewed in: Loscher, 2002; Weaver, 2003). Further clinical trials are necessary to determine whether new anti-epileptogenic drugs, if any, developed in the laboratory are effective in humans.

264 242 Table 8.1 Consequences of neuroprotective drug treatment Drug Seizure model SE duration (limited by) Beginning of drug treatment Duration of drug treatment 1. Atipamezole (α 2 receptor antagonist) 2. Preconditioning by amygdala kindling Amygdala stimulation LDP protocol lithium/pilocarpine Exp 1: 3 h (diazepam); Exp 2: not limited 1 week after SE 9 weeks (minipump implant 100 μg/kg/h) 2 h (diazepam) Preconditioning with fully kindled amygdala seizures preceding SE N/A 3. Carbamazepine (AED) Kainate Not limited 1 day after SE 40 mg/kg, i.p., 3 times/day, for 56 days 4. Carbamazepine Pilocarpine (right dorsal hippocampus) 3 h (thiopental) 1 hr after 3 h SE 120 mg/kg, i.p., for 4 days 5. Carisbamate (RWJ ; neuromodulator) LDP protocol lithium/pilocarpine 2 hr (diazepam) Immediately after SE onset 60, 90, and 120 mg/kg, i.p., 2 times/day, for 7 days 6. Carisbamate LDP protocol lithium/pilocarpine 1 h (diazepam in controls) 1 h after SE onset 90 and 120 mg/kg, i.p., 2 times/day, for 6 days 7. Caffeine LDP protocol lithium/pilocarpine 8. Caffeine LDP protocol lithium/pilocarpine 2 h (diazepam) 14 days prior and 7 days after SE 2 h (diazepam) 15 days prior and 7 days after SE 0.3 g/l in drinking water, 14 days prior and 7 days after SE 0.3 g/l in drinking water, 14 days prior and 7 days after SE 9. Celecoxib (COX-2 inhibitor) LDP protocol lithium/pilocarpine 1 h (diazepam) 1 day after SE Oral administration, 20 mg/kg, for 14 days 10. Cycloheximide Pilocarpine 1.5 h (thionembutal) 20 min prior to SE onset 1mg/kg, s.c., for 1 dose 11. z-devd-fmk (caspase-3 inhibitor) 12. Diazepam (GABA A agonist) Amygdala stimulation 3 h (diazepam) 3 h after SE onset 6 µg/d/i.c.v., for 1 week Pilocarpine (right dorsal hippocampus) 3 h (thiopental) 1 hr after 3 h SE 20 mg/kg, i.p., for 4 days 13. Dizocilpine (NMDAR antagonist) Kainate 1.5 h (diazepam) Immediately after termination of SE 0.1 mg/kg, i.p., for 1 dose 14. Dizocilpine Kainate 1.5 h (diazepam) Immediately after termination of SE 0.1 mg/kg, i.p., for 1 dose Abbreviations: h, hour; LDP, N/A, not applicable; low-dose lithium/pilocarpine protocol; i.p., intraperitoneal injection; i.c.v., intracerebral injection; min, minutes; P, postnatal day; RLDP, repeated low-dose lithium/pilocarpine protocol; s.c., subcutaneous injection

265 243 Neuroprotective effects Latency to SRSs Table 8.1 (continued) Frequency, Incidence severity or of SRSs duration of SRSs Behavioural alterations Impairment of learning and memory References 1. Hilus (p) N.D. N.E. N.D. N.E. (Pitkänen et al., 2004) 2. CA1 (c), CA3 (c), amygdala (c), piriform cortex (c), layer II of entorhinal cortex (c), layers III/IV of entorhinal cortex (p), hilus (p) N.E. N.E. N.E. N.D. N.D. (André et al., 2007) 3. Hippocampus (p) N.D. N.E. N.D. N.D. (Capella and Lemos, 2002) 4. CA1 (p), CA4 (p), N.E. in CA3 N.D. N.D. N.D N.D. (Cunha et al., 2009) 5. Layer II piriform cortex (c), layers III-IV entorhinal cortex (c), CA1 (p; dose-related), N.E. in CA3 and hilus. Decreased (52-85 vs 16 days) N.E. N.D. N.D. (André et al., 2007) 6. CA1 (p), PC (p), EC (p), amygdala (p), thalamus (p) 7. CA1 (c), CA3 (p), layer III of piriform cortex, N.E. in hilus, 8. CA1 (c), hilus (p), layers III-IV PC, N.E. in EC 9. CA1 (p), CA3 (c), hilus (p) 10. Dorsal hippocampus (N.S.), (p) 11. CA1 (p), CA3 (p), hilus (p) 12. CA1 (p), CA3c (p), hilus (p), layers II- III PC (p), EC (p), amydala (p) 13. layers II-III PC (p), CA1 (c), CA3 (c), Hilus (p), EC (N.S.), amygdala (N.S.) Increased correlated with # of neurons in PC and EC N.E. N.D. N.D. N.D. (Francois et al., 2005) N.E. N.E. N.E. N.D. N.D. (André et al., 2007) N.E. N.E. N.E. N.D. N.D. (Rigoulot et al., 2003) N.D N.D. N.D. (Jung et al., 2006) N.D. N.E. N.E. N.E. (dos Santos et al., 2005) N.D. N.E. N.E. N.D. N.E. (Narkilahti et al., 2003b) N.D. N.E. Increased N.D. N.D. (Pitkänen et al., (positively correlated with # 2005) of hilar neurons) N.D. N.D. Increased (positively correlated with # of hilar neurons) N.D. N.D. (Ebert et al., 2002) 14. CA1 (c) N.D. N.E. N.D. (Rice et al., 1998) - behaviour (Mello et al., 1993) histopathology Abbreviations:, beneficial effect of drug treatment;, drug treatment exacerbates neuronal loss, (A) studies that are only available as abstracts; (c), complete neuroprotection; EC, entorhinal cortex; ND, not determined; NE, drug treatment is not effective; NS, not specified; NC, nearly complete neuroprotection (>90% of neurons remaining); (p), partial neuroprotection; PC, piriform cortex

266 244 Table 8.1 Consequences of neuroprotective drug treatment Drug Seizure model SE duration (limited by) Beginning of drug treatment 15. Dizocilpine Pilocarpine 1 h (diazepam) 20 min prior to pilocarpine injection Duration of drug treatment 0.1 mg/kg, i.p., for 1 dose 16. Erythropoietin (renal cytokine) LDP protocol lithium/pilocarpine 1 h (diazepam) 1 h after SE onset 5000 IU/kg in PBS, i.p., for 7 days 17. Erythropoietin LDP protocol 1 h (diazepam) 4 h before SE onset 10 U/g, i.p., for 1 dose lithium/pilocarpine 18. Gabapentin (AED) Kainic acid (P35) Not limited 1 day after SE 200 mg/kg, i.p., for 10 days 19. Ketamine (NMDAR antagonist) Pilocarpine (right dorsal hippocampus) 20. Lamotrigine Perforant path stimulation (AED) (PPS) 3 h (thiopental) 1 hr after 3 h SE 50 mg/kg, i.p., for 4 days 2 h after end of PPS (diazepam) 1 h after SE onset 12.5 mg/kg, i.p., 2 times/day, for 14 days 21. Levetiracetam (AED) 22. Phenytoin (AED) 23. Pregabalin (AED) 24. Pregabatrin (AED) 25.Retigabine (potassium channel opener) 26. Tat-NR2B9c (synthetic peptide) LDP protocol lithium/pilocarpine Pilocarpine (right dorsal hippocampus) LDP protocol lithium/pilocarpine LDP protocol lithium/pilocarpine 2 hour 24 h after SE onset 50 mg/kg, i.p., 2 (pentobarbital) times/day, for 2 weeks 3 h (thiopental) 1 hr after 3 h SE 60 mg/kg, i.p, for 4 days 2 hr (diazepam) Immediately after SE onset 50 mg/kg, for initial 7 days, 10 mg/kg for remaining days (until sacrificed) 2 h (diazepam) 20 min after pilocarpine 50 mg/kg, i.p., for 6 days (histology), 10 mg/kg, i.p., for remaining 55 days (monitoring SRSs) Kainate 1.5 h (diazepam) 0, 1 and 2 h after termination of SE RLDP protocol lithium/pilocarpine 3 mg/kg, i.p., for 3 doses 1 h (diazepam) 3 h after SE onset 3 nmol/g, i.p., for 1 dose 27. Topiramate (AED) LDP protocol lithium/pilocarpin 28. Topiramate LDP protocol lithium/pilocarpine 29. Topiramate LDP protocol lithium/pilocarpine 2 hr (diazepam) Immediately after SE onset 1 h (diazepam in controls) 1 h and 10 h after SE onset 2 h (diazepam) Topiramate at SE onset and 10 hr later, diazepam at 2 h and 10 h after SE onset Doses varying mg/kg, i.p., 2 times/day, for 7 days 10 mg/kg or 30 mg/kg, i.p. 2 times/day, for 7 days 10 mg/kg, 30 mg/kg, or 60 mg/kg, i.p., for 7 days Abbreviations: h, hour; LDP, N/A, not applicable; low-dose lithium/pilocarpine protocol; i.p., intraperitoneal injection; i.c.v., intracerebral injection; min, minutes; P, postnatal day; RLDP, repeated low-dose lithium/pilocarpine protocol; s.c., subcutaneous injection

267 245 Neuroprotective effects 15. anterior PC (c), posterior PC (p), CA1 (p), CA3 (p), thalamus (p), amygdala (p), N.E. in hilus, substantia nigra pars reticulata and EC 16. CA1(p), CA3 (c), Latency to SRSs Incidence of SRSs Table 8.1 (continued) Frequency, severity or duration of SRSs Behavioural alterations Impairment of learning and memory References N.D. N.E. N.E. N.D. N.D. (Brandt et al., 2003b) N.D. N.E. N.D. N.D. (Chu et al., 2008) hilus (p) 17. CA1 (p), CA3 (p) N.D. N.D. N.D. N.D. (Jun et al., 2009) 18. CA1 (p), CA3 (p) N.D. N.D. N.D. N.E. N.E. (Cilio et al., 2001) 19. CA1 (p), CA3 (p), N.E. in CA4 20. CA3 (p), hilus (p), PC (p), EC (p), amygdala (p), NE in CA1 21. CA1 (P), CA3 (p), hilus (p), 22. CA1 (p), CA3 (p), N.E. in CA4 23. Layer II PC (p), Layers III-IV EC (p), N.E. in hippocampus 24. Layer II PC (p), Layers III-IV EC (p), N.E. in hippocampus N.D. N.D. N.D N.D. (Cunha et al., 2009) N.D. N.E. N.D. N.D. N.E. (Halonen et al., 2001) N.D. N.E. N.D. N.D. N.E. (Zhou et al., 2007) N.D. N.D. N.D N.D. (Cunha et al., 2009) Increased N.E. N.E. N.D. N.D. (André et al., 2003) Increased N.E. N.E. N.D. N.D. (André et al., 2003) 25. layers II-III PC (p), N.E. in hippocampus, PC, EC and amygdala 26. CA1 (p), CA3 (p), CA4(p), N.E. in PC 27. CA3 (c), CA1 (p), N.E. in hilus, EC and PC 28. CA1 (p), CA3 (p) (only at 30 mg/kg drug dose), N.E. in hilus, DG, EC, and PC N.D. N.E. N.E. N.D. N.D. (Ebert et al., 2002) N.D. N.E. N.D. N.E. (results not published see thesis chapter 6) N.E. (results not published see thesis chapter 7) (Dykstra et al., 2009) N.E. N.E. N.E. N.D. N.D. (André et al., 2003) N.E. N.E. N.E. N.D. N.D. (Rigoulot et al., 2004) 29. CA1 (p), hilus (p), layers III-IV EC (p), N.E. in CA3 and PC N.E. N.E. N.E. N.D. N.D. (Francois et al., 2006) Abbreviations:, beneficial effect of drug treatment;, drug treatment exacerbates neuronal loss, (A) studies that are only available as abstracts; (c), complete neuroprotection; EC, entorhinal cortex; ND, not determined; NE,not effective; NS, not specified; (p), partial neuroprotection; PC, piriform cortex

268 246 Table 8.1 Consequences of neuroprotective drug treatment Drug Seizure model SE duration (limited by) Beginning of drug treatment Duration of drug treatment 30. Topiramate Pilocarpine 40 min (diazepam only in controls); 40 min (topiramate, 2 hr diazepam in experimental drug groups) Topiramate at 40 min after SE onset, diazepam at 2 hr after SE onset in experimental drug groups 20 mg/kg or 100 mg/kg, i.p., for 1 dose 31. Valproate (AED) Kainate (P35) Not limited in controls 24 h after SE 600 mg/kg, i.p., 2 times/day, for 30 days, tapered to 300 mg/kg, i.p., 2 times/day for final 10 days 32. Valproate Amygdala stimulation 4 h (diazepam) 4 hr after SE onset 4 weeks 33. Vigabatrin (inhibition of GABA transaminase) LDP protocol Lithium/pilocarpine 2 h (diazepam) Immediately after SE onset 400 mg/kg, i.p., for initial dose, 250 mg/kg, i.p., 3 times/day, for 45 days Abbreviations: h, hour; LDP, N/A, not applicable; low-dose lithium/pilocarpine protocol; i.p., intraperitoneal injection; i.c.v., intracerebral injection; min, minutes; P, postnatal day; RLDP, repeated low-dose lithium/pilocarpine protocol; s.c., subcutaneous injection

269 247 Neuroprotective effects 30. CA3 (p) and CA4 (p) at dose of 100 mg/kg TPM, N.E. with dose of 20 mg/kg TPM, N.E. in CA1 31. CA1 (c), CA3 (p) Latency to SRSs Incidence of SRSs Table 8.1 (continued) Frequency, severity or duration of SRSs Behavioural alterations Impairment of learning and memory N.D. N.E. N.E. N.D. with dose of 20 mg/kg TPM, N.E. with dose of 100 mg/kg TPM N.D. (during drug tapering) (during drug tapering) References (Frisch et al., 2007) improved cognitive function occurred at 20 mg/kg treatment of TPM, independent of neuroprotection within the hippocampus (Bolanos et al., 1998) 32. Hippocampus N.D. N.E. N.E. N.E. (Brandt et al., 2006) and hilus (c 1 out of 9 rats showed moderate damage in CA1) 33. CA3 (c), CA1 (p), layers II-IV of entorhinal cortex, N.E. hilus N.E. N.E. N.E. N.D. N.D. (André et al., 2003) Abbreviations:, beneficial effect of drug treatment;, drug treatment exacerbates neuronal loss, (A) studies that are only available as abstracts; (c), complete neuroprotection; EC, entorhinal cortex; ND, not determined; NE,not effective; NS, not specified; (p), partial neuroprotection; PC, piriform cortex

270 Conclusion This present thesis demonstrates that the lithium/pilocarpine RLDP procedure for the induction of SE reliably produces neurodegeneration and behavioural alterations in rats, and effectively models the main features of human MTLE-HS (section 1.1). However, the effectiveness of using this protocol is strain-dependent as it reduced mortality in Wistar, but not in LEH, rats (see chapter 3). We showed that the majority of neuronal loss and behavioural alterations were caused by SE as the IPI, and with the possible exception of neuronal loss in the thalamic somatosensory nuclei (see section 4.4.2), these processes were unaffected by development of SRSs at the 3 months recovery time. Stereological analysis of neurons (stained for the neuronal specific marker [NeuN]) at various times (1 to 3 months) following SE showed regional variability in the evolution of neuronal loss within the hippocampus, amygdala, thalamus and piriform cortex, with the majority of neuronal death present by 24 hrs of recovery (see chapter 4). SE resulted in decreased exploratory behavior as assessed in the open field test, increased aggression to handling, increased hyperreactivity as assessed in the touch respone test, and anxiolytic effects as measured in the elevated-plus maze test (see chapter 6). Furthermore, deficits in search strategies use, as well as impaired spatial learning and memory, contributed to poor performance in the Morris water maze (see chapter 7). Overall, our results do not support the general hypothesis that genesis of SRSs, cognitive impairment and behavioural alterations are caused by SE-induced neuronal death. To assess the relationship between these processes, we first compared the progression of neuronal death and behavioural alterations in rats after SE. As discussed in section 8.6, our data and comparison of results from other studies indicate that the majority of neuronal death and behavioural alterations develop within the first several days after SE. Despite this, a detailed analysis assessing onset of behavioural changes at even earlier times (i.e., 1, 3, 6 and 12 hours after SE) is required to more thoroughly assess the temporal relationship between these processes. However, SE animals are often in poor physical condition immediately following SE, and this precludes assessement of behavioural alterations in rats less than 1 or 2 days of recovery. Interestingly, Castro et al., (2010) demonstrated that when compared to systemic injection of pilocarpine (320 mg/kg), SE in rats induced by intrahippocampal injection of pilocarpine (total 2.4 mg) recovered almost immediately following cessation of SE. Because this model also results in hippocampal neuronal death (Castro et al., 2010; Furtado et al., 2011), it offers an alternative to other SE models in

271 249 assessing the temporal relationship between seizure-induced neurodegeneration and behavioural alterations. As discussed in section 8.7, we next assessed the effect of partial neuroprotection within the hippocampus (by tat-nr2b9c) on development of SRSs and behavioural alterations in rats following SE. Our data showed that reducing pyramidal cell loss in the hippocampus had no effect on the number of rats developing SRSs or on behavioural alterations, and argues against a causal relationship between neurodegeneration within this region, genesis of SRSs and behavioural morbidity. As discussed in section 9.3, further studies are necessary to assess the connection between different patterns of neurogeneration, epileptogenesis and behavioural alterations caused by SE in rats.

272 250 Chapter 9 Future directions 9.1 Cell death mechanisms contributing to differential rates of neuronal loss following SE Previous literature Previous studies on the pilocarpine model showed that neuronal death occurs by necrosis and/or apoptosis (Sloviter et al., 1996; Fujikawa et al., 1999; Fujikawa et al., 2000b; Bengzon et al., 2002; Weise et al., 2005; Henshall, 2007; Henshall and Murphy, 2008; Fujikawa et al., 2010). In contrast to early neuronal loss, which occurs in the first 24 to 48 hours following a neurological insult and is predominately necrotic, delayed or secondary neuronal death occurring at later times has been identified to be mainly apoptotic (Kermer et al., 1999; Snider et al., 1999; Weise et al., 2005). Different cell death mechanisms have been reported to occur in different brain regions (Sloviter et al., 1996; Lopez-Meraz et al., 2010), or within the same neuronal population in rats following SE (Narkilahti et al., 2003a; Weise et al., 2005). Numerous studies have described the extent and severity of SE-induced neurodegeneration in the pilocarpine and lithium/pilocarpine models (Turski et al., 1983a; Turski et al., 1983b; Honchar et al., 1983; Fujikawa, 1996; Motte et al., 1998; Covolan and Mello, 2000; Peredery et al., 2000; Poirier et al., 2000). Still, our understanding on how SE-induced neuronal death in different brain regions progresses over time remains unclear. Previous studies have been limited by the semi-quantitative assessment of neuronal damage and by the limited number of recovery times assessed (see sections and 4.4.1). The effect of SE on neuronal loss is further discussed in section Summary of our findings In chapter 4, we extended these findings by conducting a detailed, quantitative time course comparison of SE-induced neuronal death in 19 brain regions within the hippocampus, amygdala, thalamus and piriform cortex. Neuronal death was assessed by stereological analysis of neurons (stained for the neuronal specific marker [NeuN]) at ten different intervals after SE (from 1 hour to 3 months). Our results showed that depending upon the brain region, neuronal death occurred as early as 1 hour after SE, with the majority of neuronal death in all brain regions present by 24 hours. While specific regions within the hippocampus (dorsal and ventral

273 251 CA1) and amygdala (LaDL, LaVM, BLP, BMP) showed additional neuronal loss between 1 and 14 days after SE, the somatosensory thalamic nuclei (VPM, VPL) were the only areas with additional neuronal death detected after 2 weeks. In general, we demonstrated that different brain regions exhibit differential rates of neuronal loss following lithium/pilocarpine induced SE. These findings are further discussed in sections 4.4 and Proposed studies The cell death mechanisms involved in neuronal death following lithium/pilocarpine-induced SE remain unclear. While some studies showed that neuronal death in this model is predominately necrotic ( Fujikawa et al., 1999; Fujikawa et al., 2000b; Fujikawa, 2005; Fujikawa et al., 2010; Kotariya et al., 2010), others have provided evidence that delayed neuronal loss is apoptotic (Bengzon et al., 2002; Narkilahti et al., 2003a; Weise et al., 2005; Henshall, 2007; Wang et al., 2008) (see section 4.4.4). As discussed in section , specific signalling cascades contributing to apoptotic and necrotic morphologies have been reported. For instance, the expression and activation of calpain was detected in early degenerating necrotic neurons following SE (Araújo et al., 2008; Wang et al., 2008), whereas caspase-3 was found in delayed degenerating neurons exhibiting apoptotic features ( Narkilahti et al., 2003a; Weise et al., 2005; Wang et al., 2008). Other studies, however, have failed to find caspase-3 activation following prolonged seizures (Fujikawa et al., 2002; Wang et al., 2008). The detailed time-course analysis of neuronal death we completed in chapter 4 establishes a framework in which further studies can investigate whether differential rates of neuronal loss reflect regional differences in cell death mechanisms. We hypothesize that necrotic and apoptotic cell death mechanisms will be present in regions exhibiting early ( 1 day) and delayed (>1day) neuronal loss, respectively. In view of the fact that a large proportion of neuronal death occurs by 24 hours after SE (see section 9.1.2), we expect that necrosis will be the dominate cell death morphology. However, because the dorsal and ventral CA1 subfields of the hippocampus, the lateral (LaDL and LaVM) and basalateral amydaloid nuclei (BLP and BMP), and the thalamic somatosensory nuclei showed delayed neuronal loss (see section 9.1.2), we expect that a proportion of degenerating neurons within these regions will exhibit apoptotic features. An initial study can focus on hippocampal subfields that exhibit fast (CA4) and relatively slow (CA1) neuronal loss. Different cell death

274 252 morphologies can be detected by use of electron and light microscopy. Brain sections can also be co-stained with Fluoro-jade B (FJB, a marker for degenerating neurons), for NeuN (neurons), and for specific apoptotic (e.g., caspase-3, caspase-8, p38, ERK) and necrotic (e.g., calpain, JNK) signalling cascades at various times (1hr 7 days) following SE. Analysis by confocal microscopy will permit the identification of degenerating neurons (FJB+/NeuN+) that co-stain for specific cell-death signalling markers. Although evidence for apoptotic neuronal death has previously been assessed by the presence of TUNEL staining and DNA laddering, these morphological changes have also been reported in necrotic neurons (Fujikawa et al., 1999; Fujikawa et al., 2000b; Fujikawa et al., 2002). Therefore, a combination of experimental approaches is often required, with confocal microscopy offering the best confirmation on cell death phenotypes. Several extensive reviews describing apoptotic and necrotic cell death mechanisms are available (Saraste, 1999; Saraste and Pulkki, 2000; Manning and Zuzel, 2003; Goldstein and Kroemer, 2007; Vanlangenakker et al., 2008). 9.2 Specific cognitive alterations in rats following SE Previous literature As deficits in cognition and memory are commonly observed in human MTLE-HS (Boro and Haut, 2003; Devinsky, 2003; Motamedi and Meador, 2003; Devinsky, 2004a; Gaitatzis et al., 2004; Vingerhoets, 2006; Garcia-Morales et al., 2008), causing reduced quality of life for the patient (Cramer, 2002; Gilliam, 2002; Cramer et al., 2003; Boylan et al., 2004; Johnson et al., 2004), a major goal in experimental epilepsy studies is to identify and evaluate cognitive and behavioural dysfunction (Stafstrom, 2006). Previous studies have demonstrated that rats after SE exhibit elevated escape latencies compared to controls when evaluated in the hidden platform MWM task (see section ). Although elevated search times are often interpreted as impaired spatial learning, others have argued that this deficit may be more attributable to impairment in behavioural strategies (Schenk and Morris, 1985; Morris, 1989; Whishaw, 1989; Cain, 1997). This idea is substantiated by observations that rats following SE often exhibit a thigmotaxic response in the MWM, and that this behaviour occurs in the absence of sensory or motor impairment (Milgram et al., 1988; Hort et al., 1999; Kubova et al., 2004; McKay and Persinger, 2004; Groticke et al., 2008; Jun et al., 2009) (see section ). The effect of SE on cognitive and behavioural morbidity is further discussed in section 1.6.

275 Summary of our findings In chapter 7, we extended previous findings by performing a detailed analysis on the effect of SE in rats on search strategy use in the hidden platform MWM task. Swim paths were analyzed according to a categorization developed by Graziano et al., (2003), which is a seven category qualitative analysis of prototypical behaviour in the MWM: thigmotaxis, circling, random searching, scanning, self-orienting, approaching target, and ability to directly locate the target. The search strategies are organized on a continuum of difficulty and efficiency where thigmotaxis is considered the least efficient way of solving the hidden-platform MWM task, while direct finding of the hidden platform is the most efficacious (Graziano et al., 2003). These swim paths were divided into 3 broader categories that included spatial strategies, systematic non-spatial strategies, and repetitive looping-based strategies (Brody and Holtzman, 2006) (see section ). In general, we found that suboptimal use of search strategies, along with impaired spatial learning and memory, contributed to poor MWM performance in rats following SE. Furthermore, we detected between animal differences in the ability to improve performance over extended training. Our results showed that 22% of rats after SE exhibited no behavioural impairment, with performance in the MWM and spatial probe tests indistinguishable from controls. Of the animals that showed MWM impairment compared to controls, 44% acquired more efficient search strategies, and the increased use of these strategies accounted for their improved performance. In contrast, 34% of rats showed no improvement in escape latencies or shift in strategy use Proposed studies Previous studies have shown that spatial learning, behavioural strategy learning, and the ability to switch between behavioural strategies are dissociable from one another (Morris, 1984; Whishaw and Tomie, 1987; Whishaw and Petrie, 1988; Morris, 1989; Whishaw, 1989; Day and Schallert, 1996; Day et al., 1999), and that each of these components rely to some extent on different neural systems (see sections , 7.4.4, and ). Our results showed impaired spatial memory and search strategy use in rats following SE. Future studies are necessary to determine whether spatial learning, behavioural strategy learning, and/or the ability to switch between behavioural strategies are preserved to verify the specificity of cognitive alterations in

276 254 these animals. Because we were able to separate rats following SE into three different groups based on MWM performance (see section 9.2.2), our first hypothesis is that these group differences can be accounted for by selective differences in cognitive alterations. By using different testing paradigms in the MWM (described below), future studies can specifically assess the effect of SE on spatial learning, behavioural strategies learning, and the pliability of switching between different search strategies. McKay and Persinger (2004) demonstrated that different patterns of brain damage following SE can differentially affect MWM performance (see section 7.4.5). Because spatial learning and behavioural strategies learning involve different neuronal networks, our second hypothesis is that different patterns of neuronal loss are responsible for the group differences we observed in rats following SE. This hypothesis can be assessed by quantifying neuronal loss in brains regions that underlie specific cognitive functions, including spatial learning (e.g., dorsal hippocampus, posterior parietal cortex), behavioural strategies learning (e.g., prefrontal cortex, striatum, cerebellum, medial thalamus), and the ability to switch between behavioural strategies (e.g., striatum, hippocampus, amygdala) (see sections and ) Spatial learning and memory Rats treated with anticholinergic drugs (Whishaw and Tomie, 1987; Whishaw, 1989; Day and Schallert, 1996), following traumatic brain injury (Brody and Holtzman, 2006; Thompson et al., 2006), or with lesions to specific brain structures (e.g., hippocampus, caudate-putamen) (Whishaw and Petrie, 1988; Day et al., 1999) tend to perseverate in thigmotaxic responding for many more trials than controls. Our findings have demonstrated a similar thigmotaxic response after SE in rats. The authors suggested the animal s focus on trying to locate an escape around the walls of the pool by using repetitive-looping based strategies prevented the ability to acquire spatial information (Day and Schallert, 1996; Day et al., 1999; Thompson et al., 2006); therefore, these studies initially trained rats in the MWM with procedures that deterred the animal s thigmotaxic response. In the shrinking platform task, training begins with an escape platform that occupies nearly the entire pool. The area to which the rats could escape is made smaller by substituting smaller platforms as training progresses. The use of repetitive looping-based strategies is discouraged in the first several trials by placing a block between the submerged platform and the walls of the pool. Although rats following traumatic brain injury still exhibited

277 255 impaired spatial learning in this task (Thompson et al., 2006), rats treated with anticholinergic drugs and with hippocampal lesions exhibited similar performance to controls in finding the submerged platform, and preferentially searched the goal quadrant on the probe trials (Day and Schallert, 1996; Day et al., 1999). Our results indicate that rats after SE were capable of acquiring spatial memory by the fact that they (1) showed an increase in use of spatial strategies during extended MWM training, and (2) exhibited higher search times when the submerged platform was moved diagonally from its original location (see section 7.4.4). Because the shrinking platform task discourages the use of repetitive-looping based strategies, we hypothesize that rats following SE and tested with this procedure will acquire spatial information and show a preference to the platform quadrant in the probe tests. This task will confirm whether impaired performance in rats after SE assessed using the spatial acquisition MWM task is caused by impaired use of spatial information Acquisition and retention of behavioural-strategies Acquisition of behavioural strategies can be assessed with a nonspatial training paradigm (Morris, 1989; Saber and Cain, 2003). In this task, the submerged platform is moved to a different quadrant of the pool on successive trials, thus preventing the learning of a place response. We hypothesize that although a majority of rats after SE will acquire more efficient search strategies in the nonspatial training paradigm, a subset of these animals will not show improved performance. This is based on the fact that we observed no improvement in performance with a subset of SE rats when assessed in the spatial acquisition MWM task (see section 9.2.2). This task will confirm the percentage of rats following SE that exhibit deficits in acquisition of search strategies Pliability of search strategy use A number of different testing paradigms can be used to assess an animal s ability to efficiently alter search strategies. The shift learning paradigm has been used in rats treated with atropine (Day and Schallert, 1996), with hippocampal lesions (Day et al., 1999), or following traumatic brain injury (Thompson et al., 2006). In this paradigm, the animal is first trained to learn the position of a hidden submerged platform using the shrinking platform task (see section ). After acquisition of spatial search strategies, the platform is shifted diagonally across from the

278 256 old location in a probe trial to determine the animal s ability to effectively switch search strategies. Sham animals will efficiently switch from use of spatial strategies to non-spatial, systematic strategies (Day and Schallert, 1996; Day et al., 1999; Thompson et al., 2006). In contrast, rats treated with atropine or with hippocampal lesions were demonstrated to initially use spatial strategies, but then revert to use of repetitive looping-based strategies (Day and Schallert, 1996; Day et al., 1999). Rats subjected to traumatic brain injury showed deficits in spatial learning (see section ) and persisted in using repetitive looping-based strategies in the shift learning paradigm (Thompson et al., 2006). Day et al., (1999) additionally tested rats with hippocampal lesions on an alternative version of the shift learning paradigm. In this task, animals were first trained to use spatial strategies using the shrinking platform task (see section ). When training was complete, the submerged platform was randomly shifted around the walls of the pool so that rats were required to switch to use of repetitive looping-based strategies. Although hippocampal-lesioned rats showed an initial preference for responding thigmotaxically, once spatial search strategies were acquired in the shrinking platform task, these animals required more trials compared to controls to switch back to a thigmotaxic response in the shift-training paradigm (Day et al., 1999). Even with spatial learning deficits, this procedure can still be used since animals would be required to switch from use of systematic, non-spatial strategies, to use of repetitive looping-based strategies. Because the majority of rats after SE were capable of acquiring spatial strategies but continued to select less efficient search strategies (see section ), we hypothesize that these animals will show deficits in the ability to alter between search strategies. This task will confirm the percentage of rats following SE that are capable of acquiring search strategies (see section ), but exhibit behavioural inflexibility.

279 The causal relationship between neurodegeneration, genesis of SRSs and behavioural alterations Previous literature Different patterns of hippocampal neuronal loss are observed in human MTLE (Thom et al., 2010; Thom et al., 2005; Blümcke et al., 2007; Mueller et al., 2009; de Lanerolle et al., 2003) and this correlates with the age of an IPI (Thom et al., 2010; Blümcke et al., 2007; Van Paesschen et al., 1997), or is predictive of response to AED treatment and postsurgical outcome (Thom et al., 2010; Thom et al., 2005; Blümcke et al., 2007; Mueller et al., 2009; de Lanerolle et al., 2003) (see section 8.3.1). Extrahippocampal neurodegeneration is observed in patients with MTLE (Hudson et al., 1993; Du et al., 1993; Wolf et al., 1997; Juhász et al., 1999; Yilmazer- Hanke et al., 2000; Bernasconi et al., 2003; Natsume et al., 2003; Bernasconi et al., 2005; Dawodu and Thom, 2005; Thivard et al., 2005); however, futher studies are required to determine whether different patterns of neuronal loss occur in extrahippocampal regions and if so, investigate if this is correlated with the severity and distribution of hippocampal neuronal loss, and the contribution (if any) this damage has on disease outcome. In fact, several studies reported extrahippocampal neuronal death in the absence of HS (Bernasconi et al., 2001; Bernasconi et al., 2003). Animal models of epilepsy have demonstrated a correlation between the pattern of neuronal loss and severity of the IPI or disease outcome. For instance, several studies showed that adult rats that were electrically stimulated in the amygdala exhibited different types of self-sustaining status epilepticus (SSSE). Neuronal loss was found to be more restricted in animals that displayed ambulatory SSSE (with facial automatisms, neck myoclonus and concomitant ambulatory behavior) compared with typical SSSE (with facial automatisms, neck and forelimb myoclonus, rearing and falling, and tonic-clonic seizures) (Tilleli et al., 2005). These findings indicate that severity of the IPI (i.e., different types of SE) leads to different patterns of neuronal loss. Brandt et al., (2003) showed that the type of SSSE not only results in different patterns of neuronal loss, but also affects the percentage of rats developing epilepsy. In this study, three different types of SSSE were induced in adult rats by electrical stimulation of the amygdala. In rats with partial (type I) SSSE, neurodegeneration was restricted to the ipsilateral amygdala,

280 258 endopiriform nucleus, mediodorsal thalamus and piriform and entorhinal cortices. In rats exhibiting a partial SSSE with generalized seizures (type II), neurodegeneration was more marked and less variable compared to rats with type I SSSE, and also extended to include less severe neuronal loss in the contralateral hemisphere. Variable damage was also detected in the CA1 and CA3a sectors of the hippocampus. In rats with generalized (type III) SSSE, extensive bilateral damage was observed and reported to be similar to the pattern of neuronal loss detected following pilocarpine and kainate-induced SE. Only 33% of rats with type I SSSE developed SRSs, compared to >90% after type II or type III SSSE. Furthermore, epileptic rats following type I SSSE exhibited a longer latency to development of epilepsy, and exhibited less severe and frequent SRSs. A series of experiments showed rats developing epilepsy after an episode SE markedly differed in their response to phenobarbital (Brandt et al., 2004; Volk et al., 2006; Bethmann et al., 2008). While phenobarbital suppressed SRSs in rats with no overt brain damage, rats with hippocampal pyramidal cell loss did not respond to treatment. These results are further discussed in section McKay and Persinger (2004) showed that different patterns of neuronal loss (e.g., amygdala, hippocampus, thalalmus) in rats after SE can differentially affect performance in the MWM. These results are further discussed in section Summary of our findings Our findings showed that in adult rats following lithium/pilocarpine-induced SE, partial neuroprotection (by tat-nr2b9c) within the hippocampus had no effect on the incidence of SRSs, or on behavioural alterations. In contrast, others demonstrated that neuroprotection of specific brain regions can affect disease outcome and mitigate behavioural morbidity. These results are extensively discussed in section 8.7 and compared in Table 8.1. The differential effects of various neuroprotective strategies on disease outcome may be related to differences in the pattern of neuroprotection conferred, or in the pattern of neuronal loss acquired by different models of SE (see table 8.1).

281 Proposed studies A prominent question that arises from our work and previous literature is how different patterns of seizure-induced neuronal loss affect (1) the development and severity of SRSs, (2) the type of behavioural alterations displayed, and (3) the effectiveness of different neuroprotective strategies. Several animal models of epilepsy offer the opportunity for future studies to explore how restricted versus widespread, or how hippocampal versus extrahippocampal neuronal loss affect these processes. In general, we hypothesize that different patterns of SE-induced neuronal loss in adult rats will result in differences in the severity of epilepsy, the specificity of behavioural deficits displayed, and the effectiveness of different neuroprotective strategies used. This work can allow us to investigate whether (1) different animal models of epilepsy more clearly represent different subcategories of neuropathology observed in human MTLE, and (2) identify specific brain regions or neuronal networks contributing to the severity of epilepsy or underlying specific behavioural alterations. A comparison of different neuroprotective strategies effective in selective brain regions can also be used as a tool to assess the causal relationship between preserved neuronal networks, severity of disease outcome (e.g., severity or frequency of SRSs) and behavioural changes. For example, as shown in Table 8.1, different neuroprotective drugs were found to be effective in selective brain regions. Future studies can therefore use a combination of neuroprotective strategies and different seizure models to characterize the relationship between different patterns of neuronal loss and disease morbidity SE models result in different patterns of neuronal loss In chapter 4, we showed SE induced by the RLDP lithium/pilocarpine procedure results in widespread neurodegeneration within the hippocampus, thalamus, amygdala and piriform cortex. This model also results in behavioural and cognitive alterations (see chapters 6 and 7). Castro et al., (2010) showed that when compared to systemic injection of pilocarpine (320 mg/kg), SE induced by intrahippocampal injection of pilocarpine (total 2.4 mg, referred to as the H-PILO model) resulted in significantly less extrahippocampal neuronal death. The H-PILO and lithium/pilocarpine models offer an opportunity for future studies to assess how differences in the severity of extrahippocampal neuronal death affect disease outcome and the effectiveness of different neuroprotective strategies. For instance, even though we showed that partial neuroprotection in the hippocampus (by tat-nr2b9c) had no effect on seizure development and

282 260 behavioural alterations in the RLDP lithium/pilocarpine model, it may have a beneficial effect in the H-PILO model since damage is more restricted to the hippocampus. As previously described in section 9.3.1, several studies have demonstrated that different types of SSSE induced by electrical stimulation of the basolateral amygdala also produce differences in the pattern of neurodegeneration (Brandt et al., 2003; Tilleli et al., 2005). These differences include (1) neuronal death restricted to the ipsilateral side of seizure onset versus bilateral neuronal loss, and (2) neuronal death restricted to extrahippocampal regions versus extrahippocampal and hippocampal neuronal loss. Thus, this SE model also offers the opportunity to investigate the causal relationship between different patterns of neuronal loss, development of epilepsy and behavioural morbidity.

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