Optical Measurements of Excitatory Synaptic Function in Retinal Circuitry

描述(申请人提供)：我们的长期目标是了解视网膜在特定细胞类型和突触水平上是如何处理视觉信息的。视网膜的加工在很大程度上依赖于一条垂直的、兴奋性的谷氨酸能通路，该通路来自光感受器双极细胞、神经节细胞(输出神经元)。已提出的双极细胞突触表达多种重要功能，包括：以高速率释放以维持高信噪比的能力；以突触或突触外释放从而向不同受体群体发出信号的能力；产生视觉的能力 通过依赖使用的可塑性进行适应；以及产生接受场特性的能力，包括方向选择性。所有已提出的功能都很难用全细胞膜片钳记录的常见方法来研究：膜片记录不能分辨树突树内的单个突触输入，也不能准确测量远端树突上的大电导或小信号。在这里，我们提出了一种新的方法组合，将产生对双极细胞突触传递的基本见解。我们将使用 新开发的基因编码的谷氨酸传感器(IGluSnFR)，与注射到眼睛玻璃体中的病毒一起传递，并在启动子的控制下表达，启动子驱动双极细胞突触后识别靶点的表达。当iGluSnFR与谷氨酸结合时，其荧光性质发生了变化，这些变化可以用双光子显微镜来测量。我们有一个工作中的双光子显微镜系统，该系统优化了在体外视锥感光细胞刺激过程中的荧光测量。目标1将表征谷氨酸在无长突细胞和神经节细胞上的释放，直到单个突触和小泡的水平。我们将在时间上将iGluSnFR信号与自发和光诱发的电事件相关联，在空间上与突触位置相关联。谷氨酸溢出将在谷氨酸释放增加或谷氨酸摄取有限的条件下进行成像。目标2将成像单个突触的突触抑制，并测试其在对比度适应中的作用。Aim 3将通过测量单个突触对方向选择性神经节细胞释放谷氨酸的方向a选择性，来测试在单个双极终端的输出上的并行处理。这些研究将对视网膜的功能组织产生基本的见解 突触水平，并将适用于研究衰老和疾病对视网膜功能的影响。

DESCRIPTION (provided by applicant): Our long-term goal is to understand how visual information is processed by the retina at the level of specific cell types and synapses. Retinal processing depends critically on a vertical, excitatory glutamatergic pathway, from photoreceptors  bipolar cells  ganglion cells (output neurons). Bipolar cell synapses have been proposed to express multiple important functions, including: the ability to release at high rates to maintain a high signal-to-noise ratio; the ability to release either synaptically or extrasynaptically and thereby signal different receptor populations; the ability to generate visual adaptation through use-dependent plasticity; and the ability to generate receptive field properties, including direction selectivity. All proposed functions have been difficult to study wih the common method of whole-cell patch clamp recording: patch recording cannot resolve individual synaptic inputs within the dendritic tree and cannot accurately measure large conductances or small signals on distal dendrites. Here, we propose a new combination of methods that will yield fundamental insights into bipolar cell synaptic transmission. We will use a newly-developed genetically encoded glutamate sensor (iGluSnFR), delivered with a virus injected into the vitreous of the eye and expressed under the control of promoters that drive expression in identified postsynaptic targets of bipolar cells. When iGluSnFR binds glutamate, its fluorescent properties change and these changes can be measured with two-photon microscopy. We have a working two-photon microscope system that is optimized for fluorescence measurements during cone photoreceptor stimulation in vitro. Aim 1 will characterize glutamate release onto amacrine and ganglion cells down to the level of individual synapses and vesicles. We will correlate iGluSnFR signals with spontaneous and light-evoked electrical events, in time, and with synaptic locations, in space. Glutamate spillover will be imaged under conditions with either elevated release or limited glutamate uptake. Aim 2 will image synaptic depression at individual synapses and test its role in contrast adaptation. Aim 3 will test for parallel processing at the output of single bipolar terminals by measuring directiona selectivity of glutamate release at individual synapses onto direction-selective ganglion cells. These studies will yield fundamental insights into the functional organization of the retina at the level of synapses and will be applicable to study the effects of aging and disease on retinal function.