Patterns of Hippocampal Neuronal Loss and Axon Reorganization of the Dentate Gyrus in the Mouse Pilocarpine Model of Temporal Lobe Epilepsy ZHANG SI (MBBS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ANATOMY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2008 Acknowledgements I am greatly indebted to my supervisor, Dr Tang Feng Ru, Head & Principal Investigator of Epilepsy Research Lab of National Neuroscience Institute of Singapore, Adjunct Associate Professor of National University of Singapore, Adjunct Professor of Xi’an Jiao Tong University of P.R China, for his invaluable guidance, patience, encouragement, and criticism throughout this study I cannot manage without his full support during my Ph.D training I would like to express my sincere gratitude to my co-supervisor, Associate Professor Sanjay Khanna, Department of Physiology, National University of Singapore, for his constant support and encouragement, as well as valuable suggestions I am deeply indebted to Professor Ling Eng Ang, Professor Bay Boon Huat, and Associate Professor Tay Sam Wah Samuel of Department of Anatomy, National University of Singaprore, for their generous and constant supports, which are indispensable for my Ph.D study I am very grateful to all staff members and fellows of Department of Anatomy of National University of Singaprore, and National Neuroscience Institute, especially to Ms Chia Schwn Chin and Mrs Yee Gek Tan for their excellent technical assistance Finally, I would like to acknowledge the self-giving support from my wife and my mother Repaying the forever debt to them is my lifetime thesis i Table of Contents TITLE PAGE ACKNOWLEGEMENT…………………………………………………………… i TABLE OF CONTENTS……………………………………………………………ii LIST OF FIGURES……………………………………………………………… vii LIST OF TABLES…………………………………………………………………viii LIST OF ABBREVIATIONS………………………………………………………ix LIST OF PUBLICATIONS……………………………………………………… x SUMMARY………………………………………………………………………….xi CHAPTER 1: INTRODUCTION………………………………………………… 1.1 Neuroanatomy of the dentate gyrus ………………………….………………… 1.1.1 Major cell types in the dentate gyrus………………………………………3 1.1.1.1 The granule cells (GC)…………………………………………… 1.1.1.2 The mossy cells…………………………………………………….6 1.1.1.3 The pyramidal basket cells…………………………………………7 1.1.1.4 Other interneurons of the dentate gyrus……………………………8 1.1.2 Associational/commissural connections of the dentate gyrus………… 11 1.1.3 Afferent of the dentate gyrus……………………………… ……………14 1.1.3.1 Afferent from the entorhinal cortex…………………………….…14 1.1.3.2 Afferent from the septal nuclei……………………… ….……….14 1.1.3.3 Afferent from the supramammillary and other hypothalamic nuclei………………………………………………………………………… …….15 1.1.3.4 Afferent from the brainstem… ………………………………….15 ii 1.2 The dentate gyrus and epileptogenesis………………………………………… 16 1.2.1 Filtering and gating properties of the dentate gyrus… ………………….16 1.2.2 Repeated activation of the dentate gyrus can promote propagation of seizures into the hippocampus .… 18 1.3 Relationship among patterns of hippocampal neuronal loss, severity of epileptic attacks and responsiveness to anti-epileptic drugs in the temporal lobe epilepsy (TLE): correlation between neuroanatomical classification and epileptogenesis 19 1.4 Hypotheses of epileptogenesis for temporal lobe epilepsy (TLE)…………… 21 1.4.1 Animal models of temporal lobe epilepsy……………………………… 21 1.4.1.1 Kindling model……………………………………………………21 1.4.1.2 SE model………………………………………………………….22 1.4.2 Hypotheses of epileptogenesis from previous studies……………………22 1.4.2.1 The “dormant basket cell” hypothesis…………………………….22 1.4.2.2 Loss of interneurons and its association with hyperexcitability… 24 1.5 Hypotheses and aims of the present study……………………………………….26 CHAPTER 2: MATERIALS AND METHODS………………………………… 29 2.1 Pilocarpine Treatment………………………………………………………… 30 2.1.1 Animals………………………………………………………………… 30 2.1.2 Materials………………………………………………………………….30 2.1.3 Procedure…………………………………………………………………30 2.2 Iontophoretical injection of phaseolus vulgaris leucoagglutinin (PHA-L) or cholera toxin subunit B (CTB)……………………………………………………….31 2.2.1 Principle………………………………………………………………… 31 2.2.2 Materials………………………………………………………………….33 iii 2.2.3 Procedure…………………………………………………………………33 2.3 PHA-L or CTB single immunocytochemistry and Cresyl violet acetate (CVA) counterstaining……………………………………………………………………….35 2.3.1 Principle………………………………………………………………… 35 2.3.2 Materials………………………………………………………………….36 2.3.3 Procedure…………………………………………………………………37 2.4 PHA-L and CB, CR or PV double immunocytochemistry…………………… 38 2.4.1 Principle………………………………………………………………… 38 2.4.2 Materials………………………………………………………………….39 2.4.3 Procedure…………………………………………………………………40 2.5 CTB and CB, CR, PV double labeling………………………………………… 41 2.5.1 Principle………………………………………………………………… 41 2.5.2 Materials………………………………………………………………….41 2.5.3 Procedure…………………………………………………………………42 2.6 NeuN immunocytochemistry……………………………………………………43 2.6.1 Principle………………………………………………………………… 43 2.6.2 Materials………………………………………………………………….43 2.6.3 Procedure…………………………………………………………………44 2.7 Long-term EEG and video camera monitoring………………………………….44 2.7.1 Materials………………………………………………………………….44 2.7.2 Procedure…………………………………………………………………45 2.8 Transmission electron microscopic study of PHA-L immunostaining in CA3 area of the hippocampus………………………………………………………………… 46 2.8.1 Materials………………………………………………………………….46 2.8.2 Procedure…………………………………………………………………47 iv 2.9 Two-dimension (anterior-posterior) measurement of distribution of PHA-L immunopositive fibers in CA3 area and the dentate gyrus………………………… 48 2.10 Data Analysis………………………………………………………………… 48 2.10.1 Materials……………………………………………………………… 48 2.10.2 Procedure……………………………………………………………… 49 CHAPTER 3: RESULTS………………………………………………………… 51 3.1 NeuN immunocytochemistry……………………………………………………52 3.2 PHA-L Immunocytochemistry, and PHA-L and CB, CR, PV double labeling…54 3.2.1 PHA-L immunopositive fibers in CA3 area of the hippocampus……… 55 3.2.1.1 Iontophoretical injection of PHA-L into the septal part of the dorsal DG………………………………………………………………………….55 3.2.1.2 Iontophoretical injection of PHA-L into the temporal part of the dorsal DG………………………………………………………………… 57 3.2.1.3 Iontophoretical injection of PHA-L into the ventral DG…………58 3.2.2 PHA-L immunopositive fibers in CA1 area of the hippocampus……… 60 3.2.3 PHA-L immunopositive fibers in ipsi- and contra-lateral DG of the hippocampus…………………………………………………………………… 62 3.2.3.1 Iontophoretical injection of PHA-L into the septal part of the dorsal DG………………………………………………………………………….62 3.2.3.2 Iontophoretic injection of PHA-L into the temporal part of the dorsal DG………………………………………………………………… 63 3.2.3.3 Iontophoretic injection of PHA-L into the ventral DG………… 64 3.3 CR immunocytochemistry……………………………………………………….66 3.4 CTB immunochemistry and CB, CR, PV double labeling………………………68 v 3.4.1 Iontophoretical injection of CTB into CA3 area, CTB and CB, CR or PV double labeling…………………………………………………………68 3.4.2 Iontophoretical injection of CTB into DG, CTB and CB, CR or PV double labeling…………………………………………………………… 68 3.5 Electron microscopic study of PHA-L immunopositive fibers in CA3 area…….69 3.6 Long-term EEG (Telemetry) and video monitoring…………………………… 70 CHAPTER 4: DISCUSSION……………………………………………………….72 4.1 Linkage between pathological changes of hippocampus and frequency of epileptic attacks in patients and animal model of temporal lobe epilepsy………… 73 4.2 Associational/commissural connections of the dentate gyrus in the experimental mice at months after PISE and their roles in epileptogenesis…………………… 75 4.3 Reorganized connections from DG to CA1 and CA3 areas…………………… 78 4.3.1 Reorganized connection from DG to CA3 area………………………….78 4.3.2 Reorganized connection from DG to CA1 area………………………….79 4.4 Eileptic attacks in mice with two patterns of neuronal loss and axon reorganization……………………………………………………………………… 79 4.5 Limitations of the present study…………………………………………………81 CHAPTER 5: CONCLUSIONS……………………………………………………82 REFERENCES…………………………………………………….……………… 86 APPENDIX……………………………………………………………………… 93 FIGURES AND FIGURE LEGENDS…………………………………………… 105 TABLES…………………………………………………………………………… 143 vi List of Figures Figure 1: Diagrammatic representation of iontophoretic microinjection into the dentate gyrus at septal part, temporal part part of dorsal hippocampus, and ventral hippocampus………………………………………………………105 Figure 2: Illustration of Telemetry study on the mice after Pilocarpine-induced status epilepticus…………………………………………………………………107 Figure 3: NeuN immunocytochemistry in the hippocampi of experimental mice and neuronal distribution in control mice…….…………………………… 109 Figure 4: Histogram of quantitative study on NeuN immunocytochemistry in the hippocampi of experimental and control mice………………………… 111 Figure 5: Mossy fiber projections from the detent gyrus in CA3 area at septal part of the dorsal hippocampus by PHA-L immunopositive staining in experimental and control mice………………………………………………………… 113 Figure 6: Mossy fiber projections from the detent gyrus in CA3 area at temporal part of the dorsal hippocampus by PHA-L immunopositive staining in experimental and control mice……………………………………………115 Figure 7: Mossy fiber projections from the detent gyrus in CA3 area at the ventral hippocampus by PHA-L immunopositive staining in experimental and control mice……………………………………………………………….117 Figure 8: Mossy fiber projections from the detent gyrus in CA1 area of the dorsal hippocampus by PHA-L immunopositive staining in experimental mice 119 Figure 9: Mossy fiber projections from the detent gyrus in the ventral hippocampus shown by PHA-L immunopositive staining in experimental mice…… 121 Figure 10: Histogram of quantitative study on the changes of the anterior-posterior span of PHA-L immunopositive fibers from the septal part, temporal part of dorsal hippocampus, and ventral hippocampus in the experimental groups compared to the control group………………………… …… 123 Figure 11: Mossy fiber projections from the detent gyrus in ipsi- and contra-lateral hippocampus from DG in the septal part of the dorsal hippocampus by PHA-L immunopositive staining in experimental and control mice … 125 vii Figure 12: Mossy fiber projections from the detent gyrus in ipsi- and contra-lateral hippocampus from DG in the temporal part of the dorsal hippocampus by PHA-L immunopositive staining in experimental and control mice… 127 Figure 13: Mossy fiber projections from the detent gyrus in ipsi- and contra-lateral hippocampus from DG in the ventral hippocampus by PHA-L immunopositive staining in experimental and control mice… 129 Figure 14: Calretinin immunostaining in the dentate gyrus at the septal, temporal (E) parts of the dorsal hippocampous, and in the ventral hippocampus of experimental and control mice………………………………………….131 Figure 15: CTB retrogradely labeling and its CB colocalizing study in the dentate gyrus of the hippocampus………………….……………………………133 Figure 16: CTB retrogradely labeling and its CB colocalizing study in the dentate gyrus of the ventral hippocampus………………………………………135 Figure 17: Transmsion electron microscopic study on PHA-L immunopositive axon terminals in CA3 area of the temporal part of the dorsal hippocampus in the experimental mice with Type neuronal loss and control mice… 137 Figure 18: Hisotgram of long-term Telemetry study on the sponteanous recurrent seizures in experimental mice at months after pilocarpine induced status epilepticus……………………… …………………………………… 139 Figure 19: Diagrammatic representation on reorganization of associational/commissural projections and mossy fiber projections of the dentate gyrus………………………………………………………….…141 List of Tables Table 1: Coordinates for PHA-L or CTB injection…………………………….….143 Table 2: The sizes of PHA-L or CTB injection sites and the number of mice used for double immunostaining………………………………………….……….144 Table 3: Patterns of hippocampal neuronal loss, axon reorganization in the dentate gyrus in mice with Type 1, Type neuronal loss and their comparison with the control mice…… ……………………………………… ………… 145 viii List of Abbreviations ABC, avidin– biotin complex CB, calbindin CBP, calcium-binding protein CBPs, calcium-binding protein immunopositive neurons CR, calretinin CTB, cholera toxin subunit CVA, crystal violet acetate DAB, 3,3’-diaminobenzidine DG, dentate gyrus GC, granule cell of dentate gyrus GCL, granule cell layer of dentate gyrus Hi, hilus of dentate gyrus HIP, hippocampal formation IML, inner molecular layer of dentate gyrus MC, mossy cell of the dentate hilus ML, molecular layer of dentate gyrus; MTLE, mesial temporal lobe epilepsy N/A, not applicable NeuN, Neuronal Nuclei PB, phosphate buffer; PBS, phosphate buffered saline PHA-L, Phaseolus vulgaris leucoagglutinin PISE, pilocarpine induced status epilepticus PV, parvalbumin SE, status epilepticus SOLRLM: stratum oriens, stratum lucidum, stratum radiatum, and stratum lacunosum moleculare SORLM, stratum oriens, stratum radiatum, and stratum lacunosum moleculare SRS, spontenaous recurrent seizure TBS, Tris-buffered saline TLE, temporal lobe epilepsy ix Appendix Figure 14: Calretinin immunostaining shows dense CR immunopositive band in the inner molecular layer of the dentate gyrus in the septal (A) and temporal (E) parts of the dorsal hippocampous and in the ventral hippocampus (H) In the hippocampus with Type neuronal loss, Calretinin immunostaining becomes weaker in the septal (B), temporal (F) parts of the dorsal DG, and in the ventral DG (I) In the hippocampus with Type neuronal loss, Calretinin immunostaining almost disappears due to the loss of CR immunopositive neuron in the ventral hilus (C, G, D) 131 Appendix Figure 15: When CTB is injected into the CA3 to retrogradely label CB neurons in the GCL of the hippocampus, double-labeled CTB and CB immunopositive neurons are demonstrated in both the control mice (A brown and B yellow) and epileptic mice of with either type neuronal loss or Type neuronal loss (C brown and D yellow) 133 Appendix Figure 16: When CTB is injected into the dorsal DG to retrogradely label CR neurons in the hilus of the ventral hippocampus, double-labeled CTB and CR immunopositive neurons are demonstrated in both the control mice (A yellow) and mice with type neuronal loss (B yellow), but never found in mice with Type neuronal loss 135 Appendix Figure 17: Electron microscopic photograph shows PHA-L immunopositive axon terminals in CA3 area of the temporal part of the dorsal hippocampus in the control (A, B, C) and experimental mice with Type neuronal loss (D, E, F) Note that in the control mice, typical large PHA-L immunopositive axon terminals (A, B) surrounded by multilobulated spines with asymmetric synapses, are found in CA3 area Whereas in the same area they almost disappear in experimental mice with Type neuronal loss In this group of mice, only small PHA-L immunopositive axon terminals with asymmetric axons are found (D, E) 137 Appendix Figure 18: Long-term EEG and video monitoring indicate that the frequency of epilepsy occurrence in mice at months after pilocarpine induced status epilepticus was 0.71±1.01/per day in Type and 0.43±0.49/per day in Type (P>0.05; Fig 6A) No significant difference is observed However, when the ratio of SRS occurring day to the total recording days was calculated, mice with Type neuronal loss show significantly more days (ratio=0.40±0.13) to have epilepsy than that in Type (ratio= 0.23±0.10) (P