THE ROLE of Gababreceptors in TEMPORAL LOBE EPILEPSY Kate Chandler, Bvetmed, MRCVS, Dipecvn Thesis Submitted for the Degree of D
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THE ROLE OF GABA b RECEPTORS IN TEMPORAL LOBE EPILEPSY Kate Chandler, BVetMed, MRCVS, DipECVN Thesis submitted for the degree of Doctor of Philosophy to the University of London (Submitted June, 2004) Department of Clinical and Experimental Epilepsy, Institute of Neurology, University College, London, Queen Square, London WC1N3BG 1 UMI Number: U602784 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U602784 Published by ProQuest LLC 2014. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract Temporal lobe epilepsy is the most common partial epilepsy syndrome seen in adult humans. The hippocampus is a key structure in the evolution of temporal lobe seizures. The axons of the dentate granule cells, the mossy fibres, constitute a major hippocampal excitatory input. Inhibitory phenomena at mossy fibre synapses may therefore prevent seizure propagation through the hippocampus. One such inhibitory phenomenon is heterosynaptic depression. In this thesis I studied the role of GABAb receptors in temporal lobe epilepsy. In particular I studied changes in GABAb receptor-mediated heterosynaptic depression at the mossy fibre synapse following status epilepticus. I have shown that status epilepticus, triggered by either perforant path stimulation or pilocarpine administration, was followed 24 hours later by a loss of GABAb receptor-mediated heterosynaptic depression among populations of mossy fibres. This was accompanied by a decrease in the sensitivity of mossy fibre transmission to the exogenous GABAb receptor agonist baclofen. Autoradiography revealed a reduction in GABAb receptor binding in stratum lucidum after status epilepticus. I then addressed the question: what is the source of the GABA that mediates heterosynaptic depression? I have also shown that the GABAb receptor agonist baclofen has antiepileptic properties. In addition, GABAb receptors do not appear to be involved in the function of the antiepileptic drug tiagabine. Failure of GABAb receptor-mediated modulation of mossy fibre transmission may contribute to the development of spontaneous seizures after status epilepticus. The GABAb receptor may be useful as a target for antiepileptic drugs. Acknowledgements I would like to thank my supervisors, Professor Dimitri Kullmann and Dr. Matthew Walker for their encouragement, guidance and support throughout the course of this work. I would also like to thank: The rest of the Kullmann laboratory for all their assistance and advice. Professor Norman Bowery and Dr. Alessandra Princivalle for their help with the autoradiography, and for making my time at the University of Birmingham so enjoyable and interesting. Dr. Ruth Fabian-Fine for performing the electron microscopy. Dr. Andrew Fisher and Hannah Sleven for their help with the in vivo experiments. Professor David Attwell for his support throughout my PhD. My family and friends for being extremely supportive and understanding when things were not going well. I am very grateful to the Wellcome Trust for funding this research. 3 Contents Abstract 2 Acknowledgements 3 List of Figures 7 Chapter 1: General Introduction 9 1.1 The study of epileptology 9 1.2 Social Impact 10 1.3 Modem definitions 11 Table 1.1 Seizure classification 13 Table 1.2 Epilepsy Classification 14 1.4 Temporal lobe epilepsy 16 1.5 Epileptogenesis 17 1.6 Synchronous activity 18 1.7 The hippocampus 20 Table 1.3 Key structures of the limbic system 21 Table 1.4 Components of the strata of the hippocampus 23 1.8 Hippocampal sclerosis 24 1.9 Animal Models 28 Table 1.5 Commonly used models of epilepsy, seizures, and epileptiform activity 30 1.9.1 Pilocarpine model 31 1.9.2 Perforant path stimulation 31 Table 1.6 : Development of kindled seizures after Racine (1972) 33 1.10 The mossy fibre - CA3 synapse 33 1.10.1 Mossy fibre pharmacology 35 1.10.2 Mossy fibre physiology 37 1.10.3 Long-term potentation (LTP) at the mossy fibre synapse 39 1.12 The CA3 network 41 1.13 Altered inhibition in epilepsy 42 1.14 GABA b receptors 44 1.14.1 Structure of the GABA b receptor 45 1.14.2 GABA b receptor effector systems 46 1.14.3 Modulation of epileptiform activity by GABA b receptor agonists and antagonists 46 1.14.3 Altered GABA b receptors in epilepsy 47 1.14.4 The role of GABA b receptors in absence epilepsy 48 1.15 Are other receptors altered in TLE? 50 1.15.1 Metabotropic glutamate receptors (mGluRs) 50 1.15.2 Other receptor types 51 1.16 Are GABAB receptors a useful target for antiepileptic drugs? 53 1.17 Tiagabine 54 4 1.18 Summary of introduction 56 Chapter 2: General Methods 57 2.1 Epilepsy Models 57 2.1.1 Perforant path stimulation 57 2.1.1.1 Electrode preparation 57 2.1.1.2 Anaesthesia 58 2.1.1.3 Electrode placement 58 2.1.2 The pilocarpine model 59 Table 2.1 Final protocol for pilocarpine model 63 2.2 Slice preparation 63 2.3 Electrophysiology 65 2.3.1 Field potentials 66 2.4 Immunogold labelling 67 2.5 Theory of autoradiography 68 Chapter 3: Is GABA b receptor mediated heterosynaptic depression present at the adult rat mossy fibre - CA3 synapse? 71 3.1 Introduction 73 3.2 Methods 74 3.3 Results 76 3.4 Discussion 86 Chapter 4: Is heterosynaptic depression altered after status epilepticus? 91 4.1 Introduction 91 4.2 Methods 91 4.3 Results 92 4.4 Discussion 96 Chapter 5: Is the GABA that causes heterosynaptic depression in control slices released from interneurons or mossy fibres? 99 5.1 Introduction 99 5.2 Methods 103 5.2.1 Immunogold labelling for GABA 103 5.2.2 Induction of long-term potentiation 106 5.3 Results 106 5.4 Discussion 112 Chapter 6: Is loss of heterosynaptic depression due to a reduction in interneuron recruitment or a change in GABA uptake? 116 6.1 Introduction 116 6.2 Methods 120 6.3 Results 122 6.4 Discussion 124 Chapter 7: Are GABAb receptors downregulated after status epilepticus? 127 7.1 Introduction 127 7.2 Methods 129 7.2.1 Autoradiography 130 7.2.2 Data analysis 131 7.3 Results 133 7.4 Discussion 138 Chapter 8: The role of GABAb receptor agonism in the treatment of status epilepticus 141 8.1 Introduction 141 8.2 Methods 144 Table 8.1 Scale used to measure sedation 145 8.3 Results 145 Table 8.2 Evolution of sedation after baclofen Administration 146 8.4 Discussion 153 Chapter 9: General Discussion and Further W ork 158 9.1 Summary of Findings 158 9.2 Limitations of this thesis 159 9.3 Possible mechanisms to explain my findings 163 9.4 Implications of my work 165 Table 9.1 : Summary of changes reported in GABA b receptors in TLE and experimental models 167 9.4 Further work 173 6 Figures 1.1 The hippocampus of the ratin situ 1.2 A cross-section of the hippocampus 1.3 The “trisynaptic” circuit of the hippocampus 2.1 Whole brain slicing method 3.1 Low power photomicrograph (x 30 magnification) of hippocampal slice 3.2 Recording mossy fibre field potentials 3.3 Identification of mossy fibre field potentials 3.4 Identifying two separate mossy fibre pathways using paired pulses 3.5 A schematic to show the slice stimulation protocol 3.6 Mossy fibre fEPSPs recorded during application of a train of 5 stimuli 3.7 The presynaptic fibre volley 3.8 Heterosynaptic depression in control slices is mediated by GABA b receptors 3.9 GABA b receptor mediated heterosynaptic depression is still present at physiological temperature 3.10 The fibre volley was unchanged in both shape and amplitude in the presence of the conditioning train 4.1 Mossy fibre fEPSPs in slices after pilocarpine-induced SE 4.2 GABA b receptor mediated heterosynaptic depression is absent after SE 4.3 There was no significant heterosynaptic depression after SE in both models 5.1 GABA immunogold-labelled mossy fibre terminals 5.2 The endocannabinoid agonist WIN 55,212-22 did not significantly change the magnitude of heterosynaptic depression 5.3 LTP induction 5.4 The post-LTP ratio was not significantly different from the pre-LTP ratio 6.1 The group II metabotropic glutamate receptor antagonist LY341495 at 500nM is sufficient to block the effects of the group II metabotropic glutamate receptor agonist DCG-IV on mossy fibre transmission 6.2 Heterosynaptic depression could not be rescued after SE by blocking group II metabotropic glutamate receptors or by blocking GABA uptake 6.3 Effect of N0711, LY341495 and increasing length of train 7.1 A concentration dependent reduction in amplitude of the fEPSP was seen when baclofen was applied 7.2 Mossy fibre fEPSPs were less sensitive to baclofen after SE 7.3 Reduction of binding of [3H]-CGP62349 to GABA b receptors in stratum lucidum after pilocarpine-induced SE 7.4 An example of an autoradiograph from an animal after pilocarpine induced SE 8.1 Seizure severity was significantly reduced two hours after treatment when compared to seizure severity before treatment 8.2 The mean level of sedation during baclofen treatment 8.3 Seizure severity as measured by the Racine scale was significantly reduced two hours after administration of baclofen 8.4 EEG traces taken from one experiment 8.5 Baclofen significantly reduced spike amplitude and frequency when compared to saline vehicle 8.6 Pretreating with the GABA b receptor antagonist SCH50911 did not affect the efficacy of the antiepileptic drug tiagabine 8 Chapter 1: General Introduction 1.1 The study of epileptology Throughout the ages people have been fascinated by the epileptic condition.