Whalley, Ben; (2004) Electrophysiological investigation of some mechanisms underlying muscarinic agonist-induced epileptiform activity observed in immature rat olfactory cortical brain slices, in vitro. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

The properties and mechanisms underlying the muscarinic acetylcholine receptor (mAChR) agonist-induced epileptiform bursting phenomenon seen in immature rat piriform cortex slices in vitro were investigated using 'sharp' intracellular recording. In accordance with a previous report, mAChR agonist-induced epileptiform activity occurred regularly in immature slices, but never in adult preparations; moreover, the magnitude of paroxysmal depolarising shifts, burst duration and incidence of bursts induced in the presence of the muscarinic agonist oxotremorine-M (OXO-M; 10 ?M) were found to be inversely related to animal age. Burst incidence was also higher at the anterior and posterior (compared with median) regions of the immature piriform cortex. A range of anti-epileptic drugs were screened for their ability to abolish the OXO-M- induced bursting behaviour. Based on their postsynaptic effect, the descending order of potency was: topiramate >carbamazepine >felbamate =gabapentin >lamotrigine >phenobarbitone; the order based on their ability to reduce afferent synaptic transmission was: felbamate >lamotrigine >topiramate >gabapentin >phenobarbitone. The ability of the acetylcholinesterase inhibitor neostigmine to mimic the effects of OXO-M in immature slices was also assessed; although epileptiform bursting was not induced, several changes in neuronal and synaptic excitability were observed (blocked by atropine) that suggest a possible role for the endogenous cholinergic system in epileptogenesis in this brain area. A significant presynaptic endogenous 'cholinergic tone' within the slices was also confirmed using paired-pulse synaptic analysis. The presynaptic effects of OXO-M on excitatory synaptic potentials (EPSPs) evoked from adult and immature slices by afferent (lateral olfactory tract: LOT) or intrinsic fibre stimulation were also examined and the results confirmed with paired- pulse analysis. Significant differences between the responsiveness of adult and immature preparations, pertinent to epileptogenesis, were found: e.g. in immature slices, LOT afferent-evoked EPSPs in OXO-M showed a prolonged depolarizing phase, all or nothing behaviour and superimposed spike firing (GABABR-mediated inhibition was also reduced), while intrinsic fibre-evoked excitatory synaptic transmission showed only inhibition', this inhibition was mediated by M1-type mAChRs in the adult, but M2- type mAChRs in immature slices. Synaptic responses showing epileptiform characteristics were also found to occur more frequently in the anterior and posterior regions of the piriform cortex; the median region thus appears to act as a buffer to overexcitability. Possible differences in GABAaR-mediated inhibitory postsynaptic potentials (IPSPs) and their sensitivity to OXO-M in adult and immature slices that could contribute to muscarinic epileptiform activity were also examined. Thus, isolated IPSPs evoked in immature preparations were smaller than those evoked in adults, and mAChR-mediated IPSP suppression was also greater in immature slices; additionally, adult and immature IPSPs evoked in the anterior and posterior regions of the slice were smaller than those evoked in the median region. As with EPSPs, presynaptic muscarinic inhibition was mediated by M2 mAChRs in immature slices, but Ml mAChRs in adults; results were confirmed using paired-pulse analysis. The effect of rostro-caudal lesions between piriform cortical layers I and II (apical) or layer III and the endopiriform nucleus (basal) were examined in adult and immature slices. Neither lesion had any effect upon evoked EPSPs, depolarizing effects of OXO-M or epileptiform bursting. However, the slow post-stimulus afterdepolarization (sADP), normally induced in OXO-M, was absent in adult but not immature lesioned slices. Interestingly, the gap junction blockers carbenoxolone and octanol disrupted muscarinic epileptiform activity and also diminished the evoked sADP in immature slices. In conclusion, the present work has demonstrated that a number of critical physiological, pharmacological and regional differences exist between the excitatory and inhibitory synaptic systems, presynaptic muscarinic sensitivity and gap junction connectivity of adult and immature piriform cortex, which contribute towards the increased susceptibility of this brain area to epileptiform events in the immature animal.

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