Optical Measurements of Excitatory Synaptic Function in Retinal Circuitry

视网膜回路兴奋性突触功能的光学测量

• 批准号: 8425741
• 负责人: Bart Gerard Borghuis
• 金额: $ 20.78万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-12-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8425741
• 关键词: AMPA Receptors; Affinity; Aging; Amacrine Cells; Binding; Cells; Complement; Custom; Dendrites; Disease; Distal; Event; Eye; Fluorescence; Glaucoma; Glutamates; Goals; Heterogeneity; Image; Imaging technology; In Vitro; Individual; Ischemia; Knowledge; Label; Life; Light; Location; Measurement; Measures; Mental Depression; Methods; Microscope; Microscopy; Modeling; Monitor; Neurons; Noise; Optic Nerve; Optics; Output; Pathway interactions; Photoreceptors; Physiological; Population; Presynaptic Terminals; Process; Property; Reporter; Retina; Retinal; Retinal Cone; Retinal Diseases; Retinitis Pigmentosa; Role; Signal Transduction; Site; Stimulus; Synapses; Synaptic Transmission; System; Testing; Time; Tissues; Tomatoes; Trees; Variant; Vertebrate Photoreceptors; Vesicle; Virus; Vision; Visual; Whole-Cell Recordings; Work; age effect; base; cell type; design; ganglion cell; genetic technology; insight; light intensity; neurotransmitter release; normal aging; novel strategies; parallel processing; patch clamp; postsynaptic; prevent; promoter; public health relevance; receptive field; receptor; research study; response; retinal neuron; sensor; synaptic depression; synaptic function; two-photon; uptake; visual adaptation; visual information

DESCRIPTION (provided by applicant): Our long-term goal is to understand how visual information is processed by the retina at the level of specific cell types and synapses. Retinal processing depends critically on a vertical, excitatory glutamatergic pathway, from photoreceptors  bipolar cells  ganglion cells (output neurons). Bipolar cell synapses have been proposed to express multiple important functions, including: the ability to release at high rates to maintain a high signal-to-noise ratio; the ability to release either synaptically or extrasynaptically and thereby signal different receptor populations; the ability to generate visual adaptation through use-dependent plasticity; and the ability to generate receptive field properties, including direction selectivity. All proposed functions have been difficult to study wih the common method of whole-cell patch clamp recording: patch recording cannot resolve individual synaptic inputs within the dendritic tree and cannot accurately measure large conductances or small signals on distal dendrites. Here, we propose a new combination of methods that will yield fundamental insights into bipolar cell synaptic transmission. We will use a newly-developed genetically encoded glutamate sensor (iGluSnFR), delivered with a virus injected into the vitreous of the eye and expressed under the control of promoters that drive expression in identified postsynaptic targets of bipolar cells. When iGluSnFR binds glutamate, its fluorescent properties change and these changes can be measured with two-photon microscopy. We have a working two-photon microscope system that is optimized for fluorescence measurements during cone photoreceptor stimulation in vitro. Aim 1 will characterize glutamate release onto amacrine and ganglion cells down to the level of individual synapses and vesicles. We will correlate iGluSnFR signals with spontaneous and light-evoked electrical events, in time, and with synaptic locations, in space. Glutamate spillover will be imaged under conditions with either elevated release or limited glutamate uptake. Aim 2 will image synaptic depression at individual synapses and test its role in contrast adaptation. Aim 3 will test for parallel processing at the output of single bipolar terminals by measuring directiona selectivity of glutamate release at individual synapses onto direction-selective ganglion cells. These studies will yield fundamental insights into the functional organization of the retina at the level of synapses and will be applicable to study the effects of aging and disease on retinal function.

描述（由申请人提供）：我们的长期目标是了解视网膜在特定细胞类型和突触的层面上如何处理视觉信息。视网膜加工严重取决于受感受器双极细胞神经节细胞（输出神经元）的垂直兴奋性谷氨酸能途径。已经提出了双极细胞突触来表达多个重要功能，包括：以高速释放以保持高信噪比的能力；能够释放突触或突触外释放，从而向不同的受体群体发出信号；产生视觉的能力 通过使用依赖性可塑性适应；以及产生接受场特性的能力，包括方向选择性。所有提出的功能都难以研究全细胞贴片夹记录的常见方法：贴片记录无法解析树突树内的单个突触输入，并且无法准确测量远端树突上的大电导率或小信号。在这里，我们提出了一种新的方法组合，这些方法将产生对双极细胞突触传播的基本见解。我们将使用一个 新发达的遗传编码的谷氨酸传感器（IGLUSNFR），并用病毒注射到眼睛的玻璃体中，并在启动子的控制下表达，这些启动子在鉴定出鉴定的双相电细胞的突触后靶标中驱动表达。当Iglusnfr结合谷氨酸时，其荧光性能会发生变化，并且可以通过两光子显微镜测量这些变化。我们有一个有效的两光子显微镜系统，该系统在体外锥形感受器刺激期间对荧光测量进行了优化。 AIM 1将表征谷氨酸释放到悬氨酸和神经节细胞上，直至单个突触和囊泡的水平。我们将在空间中将iglusnfr信号与自发和诱发的电气事件以及突触位置相关联。在释放升高或有限的谷氨酸摄取的条件下，将成像谷氨酸溢出。 AIM 2将在单个突触中对突触抑郁症进行图像，并测试其在对比度适应中的作用。 AIM 3将通过测量在单个突触上的谷氨酸释放方向上释放到方向选择性神经节细胞的方向，以测试单双极末端的输出。这些研究将产生对视网膜功能组织的基本见解 突触水平，将适用于研究衰老和疾病对视网膜功能的影响。