Probing light responses of ON bipolar and AII amacrine cells with calcium imaging

项目总结/摘要 视网膜双极细胞是连接光感受器和神经节细胞的关键。一种双极细胞类型， 双极细胞，在夜间传递昏暗的光信号，而大约10种锥双极细胞在夜间传递昏暗的光信号。 白天视觉图像的详细信息。因为视觉图像包含来自各种不同的信息， 特征（对比度、空间、时间、颜色等），每种锥双极类型提取某些特征并传输 他们最佳。双极细胞中最大的一类，ON类，传达了积极的对比， 是由一个转导级联介导的。当全细胞修补时，它们的光反应迅速减弱。 因此，有关不同ON锥体双极细胞类型的生理特性的信息很少。 最近，一种新的钙指示蛋白（GCaMP 3）被开发出来，它可以特异性地靶向ON 双极细胞（在mGluR 6启动子的控制下）或紧密连接的AII无长突细胞（在控制下 mGluR 1启动子）。我们在这里建议用双光子显微镜对该指示器进行成像，并将其 用电生理学来研究这些细胞的生理和视觉贡献。目标1将调查 视杆双极细胞的适应机制主要依赖于钙的积累，以降低视杆细胞的存活率。 响应增益视网膜将用递增的光强度刺激，钙信号将记录在视网膜中。 杆状双极树突和轴突终末。输入输出函数将决定钙的量， 导致适应。钙的来源将通过排空钙储存、阻断 细胞内钙通道或阻断TRPM 1转导通道。目标2将决定 ON锥双极细胞类型之间的生理差异在两个方面。首先，视网膜将 用具有不同强度的闪烁或时间调制的正弦光刺激， 不同锥双极类型的反应将通过对位于所有ON中的轴突终末进行成像来记录。 内丛状层的层。第二，AII细胞将被去极化，并且其与细胞的耦合强度将被改变。 锥双极型将通过钙成像测量。为了揭示成像的细胞类型身份， 终端，在记录会话结束时，将用微电极将染料注入多个细胞中。目的 3将测量在不同光强度下AII网络内的耦合和噪声的动态， 两种互补的方法。首先，AII无长突细胞将被与GFP融合的通道视紫红质感染; 所有II细胞将以全细胞构型进行修补;通道视紫红质在距离修补的细胞不同距离处 将刺激细胞;并记录细胞中产生的电压。第二，所有的无长突细胞都将 感染了GCaMP 3;电流将被注入一个细胞，是全细胞补丁;和产生的钙 将测量相邻AII细胞中的响应。堵塞间隙后将重复这些实验 连接和/或Na+通道。这些实验将极大地促进我们对视网膜的理解 电路和并行处理，他们将有助于将这些知识应用于恢复视力的努力。

Project Summary/Abstract Retinal bipolar cells are the key link between photoreceptors and ganglion cells. One bipolar cell type, the rod bipolar cell, transmits the dim light signal at night, while about 10 types of cone bipolar cells transmit the detailed information of the visual image in daylight. Because the visual image contains information from various features (contrast, spatial, temporal, color, etc.), each cone bipolar type extracts certain features and transmits them optimally. The largest class of bipolar cells, the ON class, conveys positive contrast with responses that are mediated by a transduction cascade. When whole-cell patched, their light responses runs down rapidly. Consequently, information about the physiological properties of different ON cone bipolar cell types is scarce. Recently, a new calcium indicator protein (GCaMP3) was developed, and it can specifically be targeted to ON bipolar cells (under control of mGluR6 promoter) or to the closely connected AII amacrine cells (under control of mGluR1 promoter). We here propose to image this indicator with two-photon microscopy and combined it with electrophysiology to investigate the physiology and visual contribution of these cells. Aim 1 will investigate the rod bipolar cell's adaptation mechanism that critically depends on calcium accumulation to lower the response gain. Retinas will be stimulated with ascending light intensities and calcium signal will be recorded in rod bipolar dendrites and axon terminals. Input-output functions will determine the amount of calcium that causes adaptation. The source of calcium will be determined by either emptying calcium stores, blocking intracellular calcium channels, or blocking TRPM1 transduction channels. Aim 2 will determine the physiological differences among the types of ON cone bipolar cells in two ways. First, the retina will be stimulated with flashing or temporally modulated sinusoidal light with varying intensities, and the calcium responses of different cone bipolar types will be recorded by imaging axon terminals that reside in all ON layers of the inner plexiform layer. Second, an AII cell will be depolarized, and the strength of its coupling to the cone bipolar types will be measured by calcium imaging. In order to reveal the cell type identity of the imaged terminals, at the end of the recording session, dye will be injected into multiple cells with a microelectrode. Aim 3 will measure the dynamics of coupling and noise within the AII network under different light intensities using two complementary methods. First, AII amacrine cells will be infected with channelrhodopsin fused to GFP; an AII cell will be patched with whole cell configuration; channelrhodopsin at various distances from the patched cell will be stimulated; and the resulting voltage in the cell will be recorded. Second, AII amacrine cells will be infected with GCaMP3; current will be injected into a cell that is whole-cell patched; and the resulting calcium response in neighboring AII cells will be measured. These experiments will be repeated after blocking gap junctions and/or Na+ channels. The proposed experiments will greatly facilitate our understanding of retinal circuits and parallel processing and they will help apply this knowledge to efforts in restoring vision.