Regulation of Photoreceptor Neurotransmission

描述（由申请人提供）：光感受器的光反应通过持续释放谷氨酸囊泡的变化在视网膜的第一突触上传递。目的1提出确定在光照和黑暗条件下限制锥细胞持续释放速率的机制。连续释放的限速步骤是钙依赖的，因此将光诱发的膜电位变化与钙进入的电压依赖变化的释放速率联系起来。建模、成像和电生理学将用于验证钙调蛋白和钙调蛋白激酶II的作用通过加速突触囊泡的分子启动或通过增强带状活动区的囊泡附着来调节持续释放的假设。实验还测试了持续释放的速率是否可能受到释放位点功能状态在prio泡融合事件后恢复的速率的限制。先前释放可能限制随后释放位点功能的一种方式是破坏钙通道和释放位点之间的密切空间关系。与锥细胞的释放只发生在带状活跃区不同，杆状细胞的大多数缓慢持续释放发生在活跃区以外的异位。这种异位，非带状释放是由储存在内质网的钙释放驱动的。目的2验证了从内质网持续释放钙驱动黑暗中杆状细胞突触释放的假设，并且这种细胞内储存的释放是通过钙离子通过内质网从核周到突触末端的连续隧道而持续的。每个锥体有多达50个带状活动区。目的3询问视锥细胞中单个带的钙变化是否因其电压依赖性而不同，从而促进了突触的异质性，或者它们是否像单个分布带一样运作。总之，这些实验旨在确定视网膜第一突触形成视觉的关键过程，对于理解突触活动疾病相关变化的后果以及通过治疗干预恢复正常视觉功能至关重要。

DESCRIPTION (provided by applicant): Light responses of photoreceptors are transmitted across the first synapse in the retina by changes in the ongoing release of glutamate-filled vesicles. Aim 1 proposes to identify the mechanisms that limit rates of ongoing release from cones in light and dark. The rate-limiting steps in continuous release are calcium-dependent, thus linking light-evoked changes in membrane potential to release rates through voltage-dependent changes in calcium entry. Modeling, imaging, and electrophysiology will be used to test the hypothesis that the actions of calmodulin and calmodulin kinase II regulate sustained release by speeding molecular priming of synaptic vesicles or by enhancing vesicle attachment at the ribbon-style active zone. Experiments also test whether the rate of sustained release may be limited by the rate at which the functional status of release sites can be restored after a prio vesicle fusion event. One way in which prior release might restrict subsequent release site function is by disrupting the close spatial relationship between calcium channels and release sites. Unlike cones where release occurs only at ribbon-style active zones, most of the slow sustained release from rods occurs at ectopic sites outside the active zone. This ectopic, non-ribbon release is driven by release of calcium stored in the endoplasmic reticulum. Aim 2 tests the hypothesis that sustained release of calcium from the endoplasmic reticulum drives synaptic release from rods in darkness and that this release from intracellular stores is sustained by the continuous tunneling of calcium ions through endoplasmic reticulum from perikaryon to synaptic terminal. Cones have as many as 50 ribbon-style active zones apiece. Aim 3 asks whether calcium changes at individual ribbons in a cone differ in their voltage-dependence, thus promoting synaptic heterogeneity, or whether they operate like a single distributed ribbon. Together, these experiments are designed to identify key processes that shape vision at the first synapse in the retina and are essential for understanding the consequences of disease-related changes in synaptic activity as well as for restoring normal visual function by therapeutic interventions.