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

描述（申请人提供）：视网膜双极细胞是连接光感受器和神经节细胞的关键环节。一种双极细胞类型，杆状双极细胞，在夜间传输微弱的光信号，而大约10种锥状双极细胞在白天传输视觉图像的详细信息。由于视觉图像包含各种特征（对比度、空间、时间、颜色等）的信息，每个锥双极类型提取某些特征并以最佳方式传输它们。双极细胞中最大的一类，ON类，通过转导级联介导的反应传递阳性对比。当全细胞修补后，它们的光反应会迅速下降。因此，关于不同ON锥双极细胞类型的生理特性的信息是稀缺的。最近，一种新的钙指示蛋白（GCaMP3）被开发出来，它可以特异性靶向ON双极细胞（受mGluR6启动子控制）或紧密相连的AII无分泌细胞（受mGluR1启动子控制）。我们建议用双光子显微镜成像这个指示器，并结合电生理学来研究这些细胞的生理和视觉贡献。目的1将研究杆双极细胞的适应机制，该机制主要依赖于钙积累来降低响应增益。视网膜将受到上升光强度的刺激，在杆状双极树突和轴突终末记录钙信号。输入输出函数将决定引起适应的钙的量。钙的来源将通过排空钙储存、阻断细胞内钙通道或阻断TRPM1转导通道来确定。目的2将以两种方式确定ON锥双极细胞类型之间的生理差异。首先，用不同强度的闪烁或暂时调制的正弦光刺激视网膜，并通过成像位于内丛状层所有ON层的轴突终端记录不同锥双极类型的钙响应。其次，AII细胞将去极化，其与锥双极型的耦合强度将通过钙成像来测量。为了显示成像终端的细胞类型，在记录结束时，将用微电极将染料注入多个细胞。Aim 3将使用两种互补的方法测量不同光强下AII网络内耦合和噪声的动态。首先，用融合GFP的通道视紫红质感染所有的无毛细胞；AII单元将被修补为全单元配置；距离贴片细胞不同距离的通道视紫质将受到刺激；电池中产生的电压将被记录下来。第二，所有的腺分泌细胞都会被GCaMP3感染；电流将被注入一个全细胞修补的细胞；从而测量相邻AII细胞对钙的反应。这些实验将在阻断间隙连接和/或Na+通道后重复进行。所提出的实验将极大地促进我们对视网膜回路和并行处理的理解，并将有助于将这些知识应用于恢复视力的努力。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Our goal of imaging light-evoked calcium responses in the ON bipolar cells and the AII amacrine cells will have a substantial impact on the field because these recordings are still novel and they promise to pave the way for efficient recordings from specific cell compartments in the retina. These will yield important new information relatively fast, and will gain greater understanding of the principle of visual processing in night and day vision. This understanding in turn will help design more optimal approaches for the ever developing tools of genetic therapy.