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。其中一种双极细胞--杆状双极细胞在夜间传递微弱的光信号，而大约10种锥形双极细胞在白天传递视觉图像的详细信息。由于视觉图像包含来自各种特征(对比度、空间、时间、颜色等)的信息，每个锥体双极类型提取特定的特征并最佳地传输它们。最大的一类双极细胞，即ON类，与由转导级联介导的反应形成了积极的对比。当全细胞贴片时，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(GCaMP3)被开发出来，它可以特异性地靶向双极细胞(受mGluR6启动子控制)或紧密相连的AII无长突细胞(受mGluR1启动子控制)。我们建议用双光子显微镜对这一指示器成像，并将其与电生理学相结合，以研究这些细胞的生理和视觉贡献。目的1将研究杆状双极细胞的适应机制，该机制关键依赖于钙的积累来降低反应增益。视网膜将受到上升光强度的刺激，并在视杆双极树突和轴突终末记录到钙信号。投入产出函数将决定导致适应的钙量。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将通过两种方法确定On锥双极细胞类型之间的生理差异。首先，视网膜将被不同强度的闪光或时间调制的正弦光刺激，不同锥体双极类型的钙反应将通过成像驻留在内丛状层所有层的轴突终末来记录。其次，AII细胞将被去极化，其与锥体双极类型的耦合强度将通过钙成像进行测量。为了揭示成像终端的细胞类型身份，在记录过程结束时，将染料注入带有微电极的多个细胞。目标3将使用两种互补的方法测量不同光强下AII网络内的耦合和噪声的动力学。首先，将与GFP融合的通道视紫红质感染AII无长突细胞；AII细胞将以完整的细胞构型贴片；刺激距离贴片细胞不同距离的通道视紫红质；并记录细胞内产生的电压。其次，将GCaMP3感染AII无长突细胞；将电流注入全细胞贴片的细胞；并测量相邻AII细胞中由此产生的钙反应。在阻断缝隙连接和/或钠通道后，将重复这些实验。拟议中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，它们将有助于将这些知识应用于恢复视力的努力。 与公共卫生相关：我们的目标是对双极细胞和AII无长突细胞上的光诱发钙反应进行成像，这将对该领域产生重大影响，因为这些记录仍然是新的，它们有望为从视网膜的特定细胞室进行有效记录铺平道路。这将相对较快地产生重要的新信息，并将更好地理解夜间和白天视觉的视觉处理原理。这种理解反过来将有助于为不断发展的基因治疗工具设计更优化的方法。

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.