Regulation of Photoreceptor Neurotransmission

描述(申请人提供)：光感受器的光反应通过视网膜中第一突触传递，通过正在进行的谷氨酸填充囊泡释放的变化。目标1建议确定限制锥体在明暗条件下持续释放的速率的机制。持续释放的限速步骤是钙依赖的，因此通过钙进入的电压依赖变化，将光引起的膜电位变化与释放速率联系起来。模拟、成像和电生理学将被用来检验这一假说，即钙调蛋白和钙调蛋白激酶II的作用通过加速突触小泡的分子启动或通过增强囊泡在带状活动区的附着来调节持续释放。实验还测试了缓释速率是否会受到PRIO囊泡融合事件后释放部位功能状态恢复的速率的限制。先前的释放可能限制随后的释放部位功能的一种方式是通过扰乱钙通道和释放部位之间的紧密空间关系。与仅在带状活动区发生释放的锥体不同，从棒中缓慢持续释放的大部分发生在活动区以外的异位位置。这种异位的、非带状的释放是由内质网中储存的钙的释放驱动的。目的2验证一种假说，即在黑暗中，内质网钙离子的持续释放驱动杆状突触的释放，这种细胞内库的钙释放是由钙离子通过内质网从核周持续向突触终末输送维持的。每个锥体都有多达50个带状活动区。AIM 3询问锥体中单个带的钙变化是否与其电压依赖性不同，从而促进突触的异质性，或者它们是否像单个分布的带一样运作。总之，这些实验旨在确定在视网膜第一个突触形成视觉的关键过程，对于了解与疾病相关的突触活动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.