Synaptic transmission in the rod pathway of the mammalian retina

 描述(由申请人提供)：我们研究计划的广泛目标是了解神经回路功能如何依赖于组成细胞和突触的内在属性。这项提议的具体目标是确定视杆细胞在视网膜中的视觉处理如何依赖于视杆双极(RB)细胞与突触后AII无长突细胞突触的信号传递时间。Rb→AII突触的传递由内在(例如，突触特有的囊泡释放和再循环动力学)和外部(例如，抑制 RBS通过神经元间回路)因子。这项应用包括两个特定的目标，整合了对Rb→AII传递的突触和电路水平的分析。特异性目标1验证了这一新的假设，即钙离子进入Rb末端通过两种钙调蛋白(CaM)依赖的机制来调节突触传递的强度和时间：一种是通过调节钙离子非依赖性磷脂酶A2(IPLA2)来调节膜脂组成；另一种是影响自发释放的结构性活性刹车。传输的调制是通过电生理实验来研究的，时间编码是在RBS缺乏一种快速的胞吐方式：复杂蛋白(Clpx)3敲除的视网膜中被检测到的。特定的目的2决定了平行抑制通路在Rb上的组织和生理功能。扫描块面电子显微成像(SBEM)重建确定抑制性无长突细胞(ACS)突触前与RBS。本研究采用多种电生理技术对AC-→RB突触进行了功能研究，包括AC-RB记录、AC的光发生刺激和双极细胞的光发生刺激--BC-突触前。与公共健康相关：了解视网膜突触如何实施时间编码有助于视网膜假体的设计和人类视网膜疾病动物模型的研究。视觉研究的一个目标是发展基于基因的治疗光感受器变性所致失明的方法，而一种有前景的治疗方法是通过病毒介导的光门控阳离子通道通道视紫红质-2(ChR2)的表达来产生视网膜间神经元的光敏感性。我们将在中间神经元中表达ChR2，以研究视网膜回路中的突触相互作用，从而产生关于视网膜工作范围的关键信息，其中ChR2是唯一的光传感器。我们在国家眼科和视力研究计划中提出了视网膜疾病计划的三个目标：1)确定治疗视网膜色素变性的潜在治疗策略，2)增加了解 光感受器后适应(即兴奋性和抑制性突触之间的相互作用)，以及3)增加对神经网络中细胞间相互作用如何产生可解释为视觉图像的信号的理解。

 DESCRIPTION (provided by applicant): The broad goal of our research program is to understand how neural circuit function depends on the intrinsic properties of component cells and synapses. The specific goal of this proposal is to determine how rod-mediated visual processing in the retina depends on the timing of signaling at rod bipolar (RB) cell synapses with postsynaptic AII amacrine cells. Transmission at the RB→AII synapse is shaped by intrinsic (e.g. synapse-specific dynamics of vesicle release and recycling) and extrinsic (e.g. inhibition of RBs by interneuron circuits) factors. This application comprises two specific aims that integrate synapse- and circuit-level analyses of RB→AII transmission. Specific Aim 1 tests the novel hypothesis that Ca2+ influx into the RB terminal regulates the strength and timing of transmission at the synapse by two calmodulin (CaM)-dependent mechanisms: one modulates membrane lipid composition by regulating a Ca2+-independent phospholipase A2 (iPLA2); the second affects a constitutively active brake on spontaneous release. Modulation of transmission is studied by electrophysiological experimentation, and temporal coding is examined in a retina in which RBs lack a fast mode of exocytosis: the complexin (clpx) 3 knockout. Specific Aim 2 determines the organization and physiological function of parallel inhibitory pathways converging on the RB. Scanning block face electron micrographic (SBEM) reconstruction identifies inhibitory amacrine cells (ACs) presynaptic to RBs. AC→RB synapses are studied functionally using various electrophysiological techniques: paired AC-RB recording, optogenetic stimulation of ACs, and optogenetic stimulation of bipolar cells-BCs-presynaptic to the ACs. Relevance to Public Health: Understanding how temporal coding is implemented by retinal synapses informs the design of retinal prosthetics and the study of animal models of human retinal diseases. A goal of vision research is the development of gene-based therapies for treating blindness caused by photoreceptor degeneration, and a promising therapy is the generation of light sensitivity in retinal interneurons by virally-mediated expression of channelrhodopsin-2 (ChR2), a light-gated cation channel. We will express ChR2 in interneurons to study synaptic interactions in retinal circuits and thereby generate critical information about the operating range of a retina in which ChR2 is the only light sensor. We address three goals of the Retinal Diseases Program in the National Plan for Eye and Vision Research: 1) determining potential therapeutic strategies for treatment of retinitis pigmentosa, 2) increasing understanding of post-photoreceptor adaptation (i.e. interactions between excitatory and inhibitory synapses), and 3) increasing understanding of how inter-cellular interactions in neural networks generate signals that are interpretable as visual images.