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Latrotoxin-evoked bursts of neurotransmitter release at the mouse neuromuscular synapse

University of British Columbia

Whole cell recordings were made at the mouse neuromuscular junction using sharp glass electrodes. Miniature end-plate potentials (minEPPs) and endplate potentials (EPPs) were recorded from muscle fibres as measures of quantal neurotransmitter release from motor nerve terminals. Effects of alphalatrotoxin application on such quantal release were assessed. Alpha-latrotoxin is a toxin found in black and brown widow spider venoms. The toxin binds irreversibly to a receptor specific to nerve terminal membranes and forms a channel or channels in the neuronal membrane. Latrotoxin channels open to admit excitatory cations into the nerve terminal which causes the release of multiple quanta, recorded as minEPPs, from the terminal. In the presence of external Ca²⁺, latrotoxin-evoked release was seen as discrete bursts of quanta. Evidence from previous literature suggests that these bursts originate from single release sites on the nerve terminal called active zones. The incidence of bursts was proportional to the concentration of external Ca²⁺. Burst intensity, measured as the number of recorded minEPPs per second (fm), varied in a stepwise fashion between 3.5 to 2100. Multimodal conductance states of single latrotoxin channels, previously described in the literature, might underlie the burst intensity sublevels now seen at the whole cell level. Indeed, single latrotoxin channel openings cooperate into bursts and vary with the external Ca²⁺, just like whole cell bursts do. It was also discovered that Ba²⁺ and Sr²⁺, like Ca²⁺, can support latrotoxin bursts. However, when latrotoxin was applied in the presence of Mg²⁺ and no other divalent cations, a steady rise in fm was seen without bursts. Release of quanta during intense and prolonged bursts was seen to decline over time; this decline might reflect depletion of "releasable" vesicles at the single active zone responsible for the burst. The application of 6% DMSO increased baseline fm 9.53 times, but burst fm was increased only 1.73 times by 6% DMSO. Thus it is probably true that release during the fastest bursts is not far removed from the maximal rate of release possible from a single active zone. This maximal rate of release might reflect the fundamental rate at which vesicles from the "releasable" pool can be induced to fuse with the subsynaptic membrane. The m, which reflects synchronous release of quanta from multiple active zones, is substantially increased when there is a burst. The burst-associated increase in m can be multiplied by 4% DMSO application to the same extent that baseline m can. Although release from other active zones is clearly increased during a single active zone burst, the contribution to burst fm made by these other active zones is tiny in proportion to the contribution made by the bursting active zone itself. The response of other active zones to the localized latrotoxin event is most probably due (a) to the entry of excitatory ions like Ca²⁺ and Na⁺ into the nerve terminal or (b) to the triggering of a second messenger cascade.

Thesis/Dissertation

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2009-02-17

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http://hdl.handle.net/2429/4649

Neuroscience

University of British Columbia

UBCV

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