Alder, F.G.; (2011) Calcium regulation of spontaneous and evoked neurotransmitter release in small central synapses. Doctoral thesis , UCL (University College London).

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Abstract

Synaptic neurotransmitter release is mediated by presynaptic voltage-gated calcium channels (VGCCs) via a tightly controlled mechanism. Naturally occurring mutations in the CACNA1A gene, which encodes the pore forming α1-subunit of P/Q-type VGCCs, can disrupt synaptic neurotransmission and lead to neurological disorders such as migraine, ataxia and epilepsy. Here, we aimed to understand how mutations linked to a severe form of migraine – familial hemiplegic migraine type 1 (FHM1) – affects neurotransmitter release in small central synapses. The effects of FHM1 mutations on both VGCC function and transmitter release are currently controversial. It is widely agreed that FHM1 mutations cause a shift to more negative potentials in the voltage activation threshold of P/Q-type channels. However, it is less understood whether this leads to a gain- or loss- of function of neurotransmitter release. We studied the effects of two particular FHM1 mutations using a combination of genetic manipulations and fluorescent imaging methods. First, in order to overcome species-specific differences in subunit expression, we attempted to express human cDNA encoding mutant VGCCs in the human neuronal-like cell line, NT2. Second, we compared synaptic neurotransmission in neuronal hippocampal cultures prepared from mice harbouring FHM1 mutations, with wild-type controls. Our results revealed that synaptic transmission was highly heterogeneous among individual synapses. However, FHM1 mutations did not have a significant effect on synaptic vesicular release. We also investigated the role of VGCCs in triggering spontaneous neurotransmission. We found that approximately 25 % of miniature excitatory post-synaptic currents were dependent on the stochastic opening of presynaptic VGCCs. Further analysis showed that the slow Ca2+ buffer EGTA blocks VGCC-dependent spontaneous and evoked release to a similar extent, suggesting that the two types of neurotransmission may share the same release machinery. Taken together these data provide new insights into the regulation of evoked and spontaneous release by presynaptic VGCCs.

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