Characterization of action potential -induced calcium domains at single nerve terminals

Neurotransmitter release at fast synapses is triggered by calcium ion (Ca2+) entry into the nerve terminal during action potential (AP) depolarization. The goal of this thesis is to characterize AP-mediated Ca2+entry into nerve terminals and the spatio-temporal profile of its associated intracellular Ca2+ concentration ([Ca 2+]) change. The cultured Xenopus neuromuscular junction (NMJ) made it possible to study the presynaptic conductances, as well as directly measuring single presynaptic AP-induced [Ca2+] changes in a fast synaptic preparation.;Using whole-cell voltage clamp of nerve terminals, we found that both N-type Ca2+ and Ca2+-activated potassium (K Ca) channels are functionally colocalized at the nerve terminal. Nonstationary current fluctuation analysis provided estimates of the number and single channel conductance of Ca2+ and KCa channels.;In order to measure AP-induced [Ca2+] changes within single nerve terminals, we employed a confocal spot detection method. Only a low affinity fluorescent Ca2+ indicator enabled the measurement of fast AP-induced fluorescence transients that preceded neurotransmitter release. These transients exhibited a rapid rising phase (∼1 ms), followed by a rapid (∼2 ms) and a slow decay phase (∼50 ms). Using a high-resolution stage-scan device in conjunction with the confocal spot-detection system we demonstrated that the amplitude, rise time, and the rapid decay phase of these transients are very sensitive to their recording location within the nerve terminal. Plots of fluorescence transients versus spot displacement revealed the presence of AP-induced fluorescence domains that dissipated within ∼7 ms. From model simulations of measured fluorescence domains, we conclude that their width represents the size of the underlying Ca2+ entry site. Model simulations and experimental data also provided insight into the Ca2+ binding properties of endogenous buffers.;To determine if nerve terminal Ca2+ entry sites were spatially associated with sites of neurotransmitter release, we examined whether the distribution of the synaptic vesicle membrane protein SV2 coincided with sites where fast fluorescence transients were measured. We found a contact-dependent enhancement of synaptic vesicle aggregation at Ca2+ entry sites. Furthermore, many of these sites colocalized with postsynaptic receptors suggesting that presynaptic Ca2+ channel dusters, and hence Ca 2+ domains, are localized to functional synaptic contacts.