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Date of Award
Anatomy and Cell Biology
Neurons communicate by fusing synaptic vesicles (SV) packed with neuronal transmitters with the presynaptic membrane and releasing them into the synaptic cleft. The release of neurotransmitters occurs at morphological specializations termed active zones (AZs). There are two known forms of neurotransmitter release, evoked and spontaneous release. Spontaneous transmitter release is stochastic, and this property is thought to be fundamental for information transfer. However, the rules governing neuronal spontaneous transmitter release at individual AZs are not well understood. It is known that spontaneous release is critical for normal neuronal development and homeostasis. Extensive evidence suggests that the spontaneous release can be regulated by the state of the vesicle fusion machinery known as the SNARE complex. Additionally, spontaneous release can be regulated by Ca2+ influx via spontaneous openings of voltage-gated calcium channels (VGCC). Moreover, presynaptic Ca2+ transients also have been shown to regulate the release of neurotransmitters that occurs spontaneously. However, it is still debated how this regulation occurs. We took advantage of genetically encoded calcium indicator (GCaMP) tagged postsynaptically at Drosophila neuromuscular junction. This marker enables optical monitoring of single vesicle fusion events at individual AZs. To address this question, we combined optical and electrophysiology detection of spontaneous release events. This technique allowed us to couple the spatial resolution of microscopy with the temporal resolution of electrophysiology. Using these techniques, we found that the AZs ensemble is composed of two distinct populations. One population with a low release probability fits Poissonian statistics, termed low activity AZs (LAZ). The second, smaller population of AZs, possesses a higher release activity and deviates from Poissonian distribution. This population of high activity AZs termed (HAZ) had an elevated state of transient activity driven by two temporal components, one that lasts on the scale of minutes and the other on the sub-second scale. We developed a three-state model for the AZs states of activity tested using Monte Carlo (MC) simulations. MC simulations produced an excellent fit for the activities and latency distributions. To test this model, we examined Complexin (Cpx) deficient fly lines. One of the roles of Cpx is to prevent the spontaneous fusion of SVs. Cpx deficiency promoted the high activity (HA) state while its overexpression largely abolished it. Furthermore, a mutation in the SNARE protein Syntaxin-1A, which mimics the spontaneous release phenotype of Cpx deficiency, also promoted the HA state. We tested the effects of presynaptic Ca2+ on the spontaneous transmitter release properties. We either promoted or blocked Ca2+ influx via VGCCs, and the results showed that Ca2+ transient promoted the sub-second state of activity. Tests inducing release from internal stores promoted bursts of multiquantal spontaneous events at AZ clusters. Altogether, our results demonstrate that spontaneous transmitter release is highly heterogeneous. That AZs can transition between a LA, HA, and SB state. These transitions can be regulated by the protein Cpx, the state of the SNARE complex, and Ca2+ transients.
Astacio, Herson Rene, "Spontaneous Transmitter Release At Individual Active Zones" (2021). Wayne State University Dissertations. 3531.