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Quantification and timing of processes involved in stimulus-secretion coupling at the mouse neuromuscular junction Bain, Allen Ian


At the mammalian neuromuscular junction, perhaps the most studied synapse, many aspects of neurotransmitter release and stimulation-induced enhancement of release are still poorly understood. Central hypotheses include: about release, the Ca²⁺ hypothesis (del Castillo and Katz, 1954), and the Ca²⁺-voltage hypothesis (Parnas and Parnas, 1988), and about enhancement, the residual Ca²⁺ hypothesis (Katz & Miledi, 1968). In the present work, these hypotheses were tested by analysis of the magnitude and timing of release with the technical advantages of computer-assisted analysis of data for large numbers of stimuli and responses and an emphasis on the relative magnitude of phasic and non-phasic release components. In mouse nerve-diaphragm in vitro, phasic neurotransmitter release evoked by action potentials grew with r«0.1 ms and decayed with r«0.3 ms, consistent for Ca²⁺, Sr²⁺ and Ba²⁺. Non-phasic release decayed, with a polyphasic time course that varied with the divalent cation. The time course of the opening of voltage-dependent presynaptic divalent cation channels underlying the release process was assessed using "tails" of raised MEPP frequency induced by trains of "direct" pulses (TTX present) in Ba²⁺- containing solution. Pulses exceeding 50 ms duration were nearly equi-effective (by integral) to more brief pulses,indicating that this Ca²⁺ channel undergoes little inactivation. In the presence of Sr²⁺ or Ba²⁺, short term stimulation-induced enhancement of release was consistent with a simple "residual ion" model, with 'cooperativity' of 4, and decay of putative intracellular ion with r«200 ms or r«3 to 5s, respectively. In Ca²⁺, facilitation (short term enhancement) was inconsistent with a residual ion model but could be resolved into two components: a multiplicative component seen as an about two-fold parallel increase in m and fm for short trains (decay r«80 ms), dependent on intracellular Ca²⁺ and Ca²⁺ influx, plus an additive component (decay r«200 ms) consistent with the residual ion model. Potentiation (long term enhancement) was found to consist primarily in parallel of a parallel multiplication of phasic and non-phasic release with r<20 s. It was absent when tetrodotoxin was present ('direct' stimulation), suggesting dependence upon Na+ influx and accumulation. With prolonged tetani, non-phasic release increased further, in a manner consistent with gradually accumulating Ca²⁺.

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