Huston, Elaine; (1995) Involvement of voltage activated calcium channels in neurotransmitter release and its modulation in cultured rat cerebellar granule neurones. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The aim of this project was to examine the exocytotic release of glutamate, newly synthesized from [3H]-glutamine, from cultured rat cerebellar granule neurones, upon K+-induced depolarisation. Primarily the study examined the involvement of voltage- dependent calcium channels both in the release of glutamate and in its modulation by the GABAB receptor agonist (-)-baclofen. Various methods of depolarising neurones in order to stimulate neurotransmitter release have been examined and discussed. 50mM K+-induced depolarisation stimulated cerebellar granule neurones to release [3H]-glutamate in a Ca2+-dependent manner. The mechanisms involved in this K+-stimulated release process were examined in detail and discussed. Release of glutamate was not supported by non-specific increase in cytosolic Ca2+, as induced by the presence of the Ca2+ ionophore, ionomycin. K+-stimulated release could, however, be supported by either Ca2+ or Ba2+. Release was also completely abolished by the presence of the non-specific, high voltage Ca2+ channel blocker, Cd2+. These results suggested that K+-stimulated release of glutamate from cerebellar granule neurones was normally dependent on the entry of Ca2+ through high voltage activated calcium channels. The study investigated the role of individual subtypes of voltage dependent calcium channels in the K+-stimulated release of [3H]-glutamate and found multiple subtypes of Ca2+ channels, including the L, N, and P/Q-types, to be involved in transmitter release under various conditions of stimulus. The study indicated that L- and non-L-type channels mediate separate components of release, and that subtypes of non-L-type channels coexist in the pre-synaptic terminals of cerebellar granule neurones and contribute to glutamate release with some degree of co-operativity. K+-stimulated release of [3H]-glutamate was inhibited by the GABAB receptor agonist (-)-baclofen, and this inhibition was partially antagonised by the GABAB receptor antagonist phaclofen, but not 2-hydroxy-saclofen. This modulation of release by (-)-baclofen was mediated via a pertussis toxin sensitive GTP-binding protein which appeared to modulate exocytotic release via inhibition of voltage dependent calcium channels. The subtypes of calcium channels involved in this modulatory mechanism were investigated. Blockade of the dihydropyridine-sensitive calcium channel had no effect on modulation of release, but block of the ωCgTx GVIA- and ωAga IVA- sensitive calcium channels together or block of ωCTx MVIIC-sensitive Ca2+ channels effectively abolished inhibition of release by (-)-baclofen.

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