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

用钙成像探测 ON 双极和 AII 无长突细胞的光反应

• 批准号: 8030207
• 负责人: Robert G Smith
• 金额: $ 23.16万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8030207
• 关键词: Address; Amacrine Cells; Calcium; Calcium Channel; Calcium Signaling; Cells; Color; Coupled; Coupling; Dendrites; Dyes; Electrical Synapse; Electrophysiology (science); Fluorescent Dyes; Frequencies; Gap Junctions; Goals; Heparin; Image; Inner Plexiform Layer; Knowledge; Light; Link; Maps; Measures; Meclofenamic Acid; Mediating; Methods; Microelectrodes; Microscopy; Morphology; Neuromodulator; Neurons; Noise; Ocular Physiology; Output; Photons; Photoreceptors; Physiological; Presynaptic Terminals; Process; Property; Proteins; Retina; Retinal; Retinal Cone; Running; Ryanodine; Ryanodine Receptor Calcium Release Channel; Signal Transduction; Source; Stimulus; Stratification; Synapses; Testing; Thapsigargin; Vision; Visual; calcium indicator; cell type; computerized data processing; design; ganglion cell; gene therapy; light intensity; novel; parallel processing; promoter; receptor; research study; response; retinal rods; stem; tool; two-photon; visual information; visual process; visual processing; voltage

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.

描述（由申请人提供）：视网膜双极细胞是光感受器和神经节细胞之间的关键联系。其中一种双极细胞，即杆状双极细胞，在夜间传递微弱的光信号，而大约10种锥状双极细胞在白天传递视觉图像的详细信息。因为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，每种锥体双极类型都会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，传达了与由转导级联介导的反应的正对比。当全细胞修补时，它们的光反应迅速减弱。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（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mGluR 1启动子的控制下）。在这里，我们建议用双光子显微镜成像这个指标，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研究视杆双极细胞的适应机制，即视杆双极细胞依赖钙离子的积累来降低反应增益。视网膜将用递增的光强度刺激，并且钙信号将记录在视杆双极树突和轴突终末中。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TRPM 1转导通道来确定。目的2从两个方面确定视锥双极细胞类型之间的生理差异。首先，将用具有不同强度的闪烁或时间调制的正弦光刺激视网膜，并且将通过对驻留在内丛状层的所有ON层中的轴突末梢进行成像来记录不同锥双极类型的钙响应。第二，AII细胞将被去极化，并且其与锥双极类型的耦合强度将通过钙成像来测量。为了揭示成像终端的细胞类型身份，在记录阶段结束时，将用微电极将染料注入多个细胞中。目标3将使用两种互补的方法测量不同光强度下AII网络内的耦合和噪声的动态。首先，AII无长突细胞将被与GFP融合的通道视紫红质感染; AII细胞将以全细胞构型进行修补;将刺激距离修补细胞不同距离的通道视紫红质;并记录细胞中产生的电压。其次，AII无长突细胞将被GCaMP 3感染;电流将被注入到一个全细胞补丁的细胞中;并测量相邻AII细胞中产生的钙反应。在阻断间隙连接和/或Na+通道后重复这些实验。拟议的实验将大大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。 公共卫生关系：我们的目标成像光诱发的钙反应在ON双极细胞和AII无长突细胞将产生重大影响的领域，因为这些记录仍然是新颖的，他们承诺铺平道路，有效的记录从特定的细胞室在视网膜。这些将相对较快地产生重要的新信息，并将更好地了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。