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

项目摘要/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环节。一种双极细胞类型，杆状 双极电池，在夜间传输微弱的光信号，而大约10种锥形双极电池传输 白天的视觉图像的详细信息。因为可视图像包含来自各种类型的信息 特征(对比度、空间、时间、颜色等)，每个锥体双极类型提取特定的特征并传输 他们是最好的。双极细胞中最大的一类，即ON类，与 都是由转导级联反应介导的。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On 双极细胞(受mGluR6启动子控制)或紧密连接的AII无长突细胞(受控制 MGluR1启动子)。我们在这里建议用双光子显微镜成像这个指示器，并将它结合起来 用电生理学来研究这些细胞的生理和视觉贡献。AIM 1将调查 杆状双极细胞的适应机制，关键依赖于钙的积累来降低 响应增益。视网膜将受到上升光强度的刺激，钙信号将被记录在 杆状双极树突和轴突终末。投入产出函数将决定所需的钙量 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TRPM1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有两种方式。首先，视网膜将是 用不同强度的闪光或时间调制的正弦光刺激，钙离子 不同锥体双极类型的反应将通过驻留在All On上的轴突终末成像来记录 内层网状层。其次，AII细胞将被去极化，其与 锥体双极类型将通过钙成像进行测量。为了揭示被成像人的细胞类型身份 在终端，在记录过程结束时，将用微电极将染料注入多个细胞。目标 3将测量在不同光强度下AII网络内的耦合和噪声的动力学 两种互补的方法。首先，所有的无长突细胞都将被融合到GFP的视紫红质通道感染； 所有细胞都将以完整的细胞配置进行修补；在与修补后的视紫红质不同的距离处 电池将被激励；电池中产生的电压将被记录下来。第二，所有的无长突细胞 感染了GCaMP3；电流将被注入全细胞贴片的细胞中；产生的钙 将测量邻近AII细胞的反应。这些实验将在封堵间隙后重复进行 结点和/或Na+通道。建议的实验将极大地促进我们对视网膜的了解。 电路和并行处理，它们将有助于将这些知识应用到恢复视力的努力中。

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.