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）：视网膜双极细胞是感光细胞和神经节细胞之间的关键联系。一种双极细胞类型（杆双极细胞）在晚上传输昏暗的光信号，而大约10种类型的锥双极细胞在白天在视觉图像中传递了视觉图像的详细信息。由于视觉图像包含来自各种特征的信息（对比度，空间，时间，颜色等），因此每种锥双极类型提取某些特征并最佳地传输它们。最大的双极细胞（上类）表达了正与由转导级联介导的响应的阳性对比。全细胞修补时，它们的光反应会迅速降低。因此，关于锥双极细胞类型上不同生理特性的信息很少。最近，开发了一种新的钙指示剂蛋白（GCAMP3），并且可以专门针对双极细胞（在MGLUR6启动子的控制下）或紧密连接的AII amacrine细胞（在MGLUR1启动子的控制下）。我们在这里建议用两光子显微镜对该指标进行成像，并将其与电生理学结合使用，以研究这些细胞的生理和视觉贡献。 AIM 1将研究杆双极细胞的适应机制，该机制严重取决于钙的积累以降低响应增益。视网膜将通过上升的光强度刺激，钙信号将记录在杆双极树突和轴突末端中。输入输出功能将确定引起适应性的钙量。钙的来源将通过排空钙存储，阻断细胞内钙通道或阻止TRPM1转导通道来确定。 AIM 2将通过两种方式确定锥双极细胞上的锥双极细胞类型之间的生理差异。首先，将通过具有不同强度的闪烁或时间调制的正弦光刺激视网膜，并且通过成像轴突端子的成像轴突端子在所有丛状层的层上都会记录不同锥双极类型的钙反应。其次，AII细胞将被去极化，其耦合到锥双极类型的强度将通过钙成像测量。为了揭示成像终端的细胞类型身份，在记录会话结束时，染料将用微电极注入多个细胞。 AIM 3将使用两种互补方法在不同的光强度下测量AII网络中耦合和噪声的动力学。首先，AII无链氨酸细胞将被融合到GFP的通道旋转感染； AII单元格将用全单元格配置修补；将刺激距离修补细胞的各个距离的通道Rhopopsin；并且将记录电池中产生的电压。其次，AII裁活细胞将被GCAMP3感染；电流将注入全细胞修补的单元格中；并且将测量相邻AII细胞中产生的钙反应。阻止间隙连接和/或NA+通道后，将重复这些实验。提出的实验将极大地促进我们对视网膜电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。 公共卫生相关性：我们对双极细胞和AII无大细胞中光诱发的钙反应进行成像的目标将对该领域产生重大影响，因为这些记录仍然是新颖的，并且他们承诺为视网膜特定细胞舱的有效记录铺平道路。这些将相对较快地产生重要的新信息，并将对白天和日视视觉处理原理有更深入的了解。反过来，这种理解将有助于为开发的基因治疗工具设计更佳的方法。