Valproate and 4-methyloctanoic acid, an analogue of valproate, in animal models of epilepsy Pi-shan Chang Thesis submitted to University College London for the degree of Doctor of Philosophy September 2009 Department of Clinical and Experimental Epilepsy Institute of Neurology University College London 1 Declaration I, Pi-shan Chang, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. The wireless telemetric recording system presented in chapter 9 was developed in collaboration with opensource instruments, MA, USA. All experiments were conducted by me. 2 Abstract Valproic acid (VPA) is a commonly used drug for the treatment of epilepsy, bipolar disorder and migraine, yet its mechanisms of action are unknown. The neuroprotective effect of VPA has been hypothesized to be secondary to inhibition of the cAMP/protein kinase A (PKA) pathway. Here, the result show that VPA (1mM) inhibited mossy fibre long-term potentiation induced (LTP) by application of high frequency stimulation in dentate gyrus. Furthermore, VPA (1mM) inhibited enhancement of mossy fibre responses induced by application of forskolin (50 μM), consistent with an effect on the PKA pathway. Using biochemical assays, it was further demonstrated that this was not due to a direct effect on PKA, but resulted from inhibition of adenylyl cyclase. The results further show using in vitro seizure models (Pentylenetetrazole model and low- Mg2+ model) that this mechanism cannot fully explain VPA’s anti-seizure effect, but rather, by modifying synaptic plasticity, it may be more important for VPA’s antiepileptogenic and neuroprotective action. VPA therefore has distinct mechanisms of action that contribute to its diverse biological activity. In hippocampi from epileptic rats (following pilocarpine-induced status epilepticus), but not in control tissue, VPA affects short-term plasticity, indicating that VPA may have specific effects in epileptic rather than control animals. Using in vitro seizure models (Pentylenetetrazole model and low-Mg2+ model) and an in vivo status epilepticus model (the perforant pathway stimulation model), 4- methyloctanoic acid is further established that it is a more potent antiepileptic drug than VPA and provides neuroprotective effects which are similar to VPA. Furthermore, 4- methyloctanoic acid (1mM) inhibited enhancement of mossy fibre responses induced by application of forskolin (50 μM), indicating that 4-methyloctanoic acid shares the same effect as VPA on modulation of PKA. 3 Contents Declaration ........................................................................................................................2 Abstract .............................................................................................................................3 Figure list ..........................................................................................................................8 Table list..........................................................................................................................13 Abbreviations ..................................................................................................................16 Acknowledgements.........................................................................................................19 Chapter 1: Introduction ...................................................................................................20 1.1 Epilepsy.................................................................................................................21 1.1.1 Temporal lobe epilepsy ..................................................................................23 1.2 Hippocampus formation........................................................................................24 1.2.1 Intrinsic input from cells in same sector ........................................................26 1.2.2 Intrinsic input from other sectors ...................................................................27 1.2.3 Brain-stem afferent.........................................................................................28 1.2.4 Efferents from the hippocampal.....................................................................29 1.3 Long term potentiation (LTP) ...............................................................................29 1.3.1 NMDA receptor mediated LTP ......................................................................31 1.3.2 Non-NMDA receptor mediated LTP..............................................................32 1.4 Epileptogenesis .....................................................................................................34 1.5 Animal models of epileptogenesis ........................................................................35 1.5.1 Animal models of temporal lobe epilepsy......................................................35 1.5.2 The kindling model ........................................................................................37 1.5.3 The status epilepticus models.........................................................................37 1.5.3.1 Pilocarpine model.....................................................................................38 1.5.3.2 Kainic acid model ....................................................................................40 1.5.3.3 Electrical stimulation model ....................................................................41 1.6 Mechanisms of epileptogenesis.............................................................................42 1.6.1 Excitatory systems in epileptogenesis............................................................42 1.6.1.1 Glutamate receptors in epileptogenesis....................................................43 1.6.1.2 NMDA receptor in epileptogenesis..........................................................43 1.6.1.3 AMPA receptor activation in epileptogenesis..........................................44 1.6.1.4 Kainate receptor activation in epileptogenesis.........................................45 1.6.1.5 Metabotropic glutamate receptor in epileptogenesis................................46 1.6.2 Inhibitory systems in epileptogenesis ............................................................48 1.6.2.1 GABA(A) receptors in epileptogenesis ...................................................48 1.6.2.2 GABA(B) receptors in epileptogenesis....................................................49 1.6.3 Non-synaptic mechanisms in epileptogenesis................................................50 1.6.4 Cell loss and epileptogenesis .........................................................................52 1.6.4.1 Apoptosis..................................................................................................52 1.6.4.2 Mitochondrial dysfunction in epileptogenesis .........................................54 1.6.5 Nitric oxide.....................................................................................................54 1.6.6 Neural reorganization in epileptogenesis.......................................................55 1.6.6.1 Neurogenesis in epileptigenesis ...............................................................55 1.6.6.2 Growth factors in epileptogenesis............................................................56 1.6.6.3 Mossy fibre sprouting in epileptogenesis.................................................57 1.7 Interventions to prevent epileptogenesis...............................................................59 1.7.1 Initial insult modification...............................................................................60 1.7.2 Neuroprotection to prevent epileptogenesis...................................................61 1.7.2.1 Neuronal survival signalling pathways ....................................................61 4 1.7.2.2 Antiepileptic drugs provide neuroprotective effects during epileptogenesis .....................................................................................................64 1.8 Antiepileptogenesis ...............................................................................................65 1.9 Valproic acid..........................................................................................................67 1.9.1 Mechanisms of action ....................................................................................68 1.9.1.2 The action of VPA on cAMP/PKA signalling system.............................69 1.9.1.3 VPA’s effect on PKA-cAMP can also influence the extracellular signal- regulated kinase (ERK) pathway. ........................................................................71 1.9.1.4 VPA’s effect on PKA-cAMP can also influence the AMPA receptor.....72 1.9.2 The side effect of VPA ...................................................................................73 1. 10 Summary ............................................................................................................74 1.11 Aims ....................................................................................................................77 Chapter 2: General materials and methods .....................................................................78 2.1 Introduction...........................................................................................................79 2.2 In vivo experiments ...............................................................................................80