We investigated the mechanism of facilitation at the crayfish inhibitory neuromuscular junction before and after blocking P-type Ca2+channels. P-type channels have been shown to be closer to releasable synaptic vesicles than non-P-type channels at this synapse. Prior to the block of P-type channels, facilitation evoked by a train of 10 action potentials at 100 Hz was increased by application of 40 mM [Mg2+]o, but decreased by pressure-injected EGTA. Blocking P-type channels with 5 nM ω-Aga IVA, which reduced total Ca2+influx and release to levels comparable to that recorded in 40 mM [Mg2+]o, did not change the magnitude of facilitation. We explored whether this observation could be attributed to the buffer saturation model of facilitation, since increasing the Ca2+channel-vesicle separation could potentially enhance the role of endogenous buffers. The characteristics of facilitation in synapses treated with ω-Aga IVA were probed with broad action potentials in the presence of K+channel blockers. After Ca2+channel-vesicle separation was increased by ω-Aga IVA, facilitation probed with broad action potential was still decreased by EGTA injection and increased by 40 mM [Mg2+]o. EGTA-AM perfusion was used to test the impact of EGTA over a range of concentration in ω-Aga IVA-poisoned preparations. The results showed a concentration dependent decrease in facilitation as EGTA concentration rose. Thus, probing facilitation with EGTA and reduced Ca2+influx showed that characteristics of facilitation are not changed after the role of endogenous buffer is enhanced by increasing Ca2+channel-vesicle separation. There is no clear indication that buffer saturation has become the dominant mechanism for facilitation after ω-Aga IVA poisoning. Finally, we sought correlation between residual Ca2+and the magnitude of facilitation. Using fluorescence transients of a low affinity Ca2+indicator, we calculated the ratio of fluorescence amplitude measured immediately before test pulse (residual Ca2+) to that evoked during action potential (local Ca2+). This ratio provides an estimate of relative changes between residual Ca2+and local Ca2+important for release. There is a significant increase in the ratio when Ca2+influx is reduced by 40 mM [Mg2+]o. The magnitude of facilitation exhibited a clear and positive correlation with the ratio, regardless of separation between Ca2+channels and releasable vesicles. This correlation suggests the importance of relative changes between residual and local Ca2+and lends support to the residual Ca2+hypothesis of facilitation. © 2008 IBRO.
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