Probing Ion Channel Function in Drosophila Motoneurons with Targeted Genetic Manipulation

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The ion channel complement of neurons determines not only membrane excitability, but also key aspects of synaptic function. At chemical synapses, multiple essential presynaptic functions, including synaptic vesicle (SV) release, cycling of SVs between different vesicle pools, short-term plasticity, and the regulation of SV endocytosis are affected by Ca2+ influx through voltage gated calcium channels (VGCCs). Electrical synapse function is not as static as long assumed, and it can be affected by the membrane properties of the coupled neurons. Therefore, addressing the specific functions of different ion channels and their strategic subcellular localization is critical to understanding synapse and neuronal network function in the healthy and the diseased brain. This project will combine the genetic power of Drosophila with electro- and optophysiological recordings to address ion channel function in the regulation of Ca2+ dependent presynaptic functions at chemical synapses and in the tuning of electrical synapse function. We recently discovered unexpected roles of the Cav1 homolog, Dmca1D, at the presynaptic terminal of larval Drosophila motoneurons. First, in contrast to Cav2 channels, which localize to presynaptic active zones and are required for evoked synaptic vesicle (SV) release, Cav1 localizes outside actives zones and is not required for SV release. Instead, Ca2+ influx through Cav1 augments SV recycling and affects short-term plasticity. Second, in the presynaptic terminal, the two parallel action potential triggered Ca2+ signals through Cav2 and Cav1 are functionally separated by a membrane bound calcium buffer, the plasma membrane calcium ATPase, PMCA. Therefore, the strategic localization of Cav2, Cav1, and PMCA allows the separate regulation of SV release and recycling by activity dependent Ca2+ influx. Building on our findings, we now propose to study the interplay of the Cav1/Cav2/PMCA functional triad with synaptotagmin 7 in the control of asynchronous release, short term plasticity, and replenishment of the readily releasable pool of SVs (aim 1). In addition, we have gathered evidence for novel, unexpected roles of voltage gated ion channels in tuning the function of electrical synapses. We found that the central pattern generator (CPG) for Drosophila flight is comprised of a small network of electrically coupled motoneurons. Our data suggest that the same electrical synapses can synchronize or desynchronize network activity, depending on electrical coupling strength and the membrane excitability profiles of the coupled neurons. We will use targeted manipulation of voltage gated ion channels in the CPG component motoneurons to probe the role of membrane excitability in tuning electrical synapse function and the resulting consequences for network firing coordination (aim 2). We expect novel insight into the dynamic regulation of neuronal network function by chemical and electrical synapses.