Probing Ion Channel Function in Drosophila Motoneurons with Targeted Genetic Manipulation

神经元的离子通道补体不仅决定膜的兴奋性，而且决定突触功能的关键方面。在化学突触，多种基本的突触前功能，包括突触小泡(SV)的释放，SVS在不同囊泡池之间的循环，短期可塑性，以及SV内吞的调节都受到钙离子通过电压门控钙通道(VGCC)内流的影响。电突触功能并不像长期假设的那样是静态的，它可以受到耦合神经元的膜特性的影响。因此，解决不同离子通道的特定功能及其战略性的亚细胞定位对于了解健康和疾病大脑中的突触和神经元网络功能至关重要。这个项目将结合果蝇的遗传力和电和光生理记录来研究离子通道在调节化学突触的钙依赖的突触前功能和调节电突触功能方面的作用。我们最近发现了Cav1同源基因Dmca1D在果蝇幼虫运动神经元突触前末端的意想不到的作用。首先，与Cav2通道不同，Cav2通道定位于突触前活动区，是诱发突触小泡(SV)释放所必需的，而Cav1定位于活动区外部，SV释放不需要。相反，钙离子通过Cav1的内流增加了SV的循环，并影响了短期可塑性。第二，在突触前终末，通过Cav2和Cav1的两个平行动作电位触发的钙信号被膜结合的钙缓冲液--质膜钙ATPase PMCA功能性地分开。因此，Cav2、Cav1和PMCA的战略定位允许通过活性依赖的钙内流单独调节SV的释放和再循环。在我们研究结果的基础上，我们现在建议研究Cav1/Cav2/PMCA功能三联体与突触素7在控制SVS的异步释放、短期可塑性和补充容易释放的SVS池(目标1)中的相互作用。此外，我们还收集了电压门控离子通道在调节电突触功能方面的新的、意想不到的作用的证据。我们发现，果蝇飞行的中央模式发生器(CPG)是由一个电子耦合的运动神经元组成的小网络。我们的数据表明，相同的电突触可以同步或去同步网络活动，这取决于电耦合强度和耦合神经元的膜兴奋性曲线。我们将利用对CPG成分运动神经元的电压门控离子通道的靶向操作来探讨膜兴奋性在调节电突触功能中的作用以及由此导致的网络放电协调的后果(目标2)。我们期望对化学和电突触对神经元网络功能的动态调节有新的见解。

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.