Ultrastructural analysis of melanopsin-containing retinal ganglion cells using a novel approach

项目摘要 连接体是神经回路中突触连接的综合图。连接组学分析 神经回路为理解其组织和具体功能提供了基础。然而，在这方面， 构建完全映射的连接体是一项困难的任务。技术进步导致了一代 连接组映射的新工具。将连续切片电子显微照片编译成三维 体积产生一个数字化的组织片充满细胞类型和微电路探索。然而，使用此 提出有针对性的问题的方法仍然具有挑战性。到目前为止，连接组学的一个主要障碍是， 研究缺乏可靠的电子显微镜遗传标记。最后，我们成功地 生产这种工具。我在生产和验证创新工具方面发挥了主要作用， 对遗传特异性神经元的靶向连接组学分析。我们的工具使用cre-lox技术来标记 靶向细胞具有在光镜和电子显微镜水平下可见的稳健标记。荧光标记物 彻底改变了在光水平上对神经回路的研究，我们的新工具将这些相同的优势带到了 超微结构水平。我的试验数据显示，在光照和光照条件下， 电子显微镜水平，提示靶向连接组学分析的可行性。前进，我的目标 是1）测试这种方法在映射神经回路中的有效性，2）利用我们的工具来阐明 本发明涉及视网膜和脑中的固有光敏神经节细胞（ipRGC）的连接性。ipRGC是一个 视网膜神经节细胞（RGC）的特殊类别与传统的RGC不同，在它们的反应 属性和轴突终止。虽然大多数RGC发送快速，瞬态信号编码图像形成 在这些特征中，ipRGC发送编码辐照度或全局光强度的缓慢、持续的信号。ipRGC轴突 终止于大脑的非图像形成区域，在那里辐射信号用于调节昼夜节律 节律和瞳孔扩张。虽然ipRGC的一般解剖学和生理学已有很好的文献记载，但我们仍不清楚ipRGC的结构和功能。 缺乏对其连接性的详细描述。我打算用我们的新工具进行连接组分析 ipRGC电路的。这个工具的使用将阐明辐照度编码背后的结构连通性 电路，并阐明非规范ipRGC输入的图像形成视觉通路的功能。 总的来说，这项建议将验证我们的工具，有针对性的连接组学，之前，它在科学传播 社区，并提供有价值的洞察处理和调制的感官信息，通过 神经回路

PROJECT SUMMARY A connectome is a comprehensive map of the synaptic connections in a neural circuit. Connectomic analysis of a neural circuit provides a foundation for understanding its organization and specific functions. However, constructing a fully-mapped connectome is a difficult task. Technological advances have led to the generation of new tools for connectomic mapping. Serial-section electron micrographs compiled into a 3-dimensional volume produce a digitized piece of tissue full of cell-types and micro-circuits to explore. However, using this approach to ask a targeted question remains challenging. Up to this point, a major barrier in connectomic research was the lack of a dependable, genetic marker for electron microscopy. Finally, we have succeeded in producing this tool. I have held a primary role in the production and validation of an innovative tool enabling targeted connectomic analysis of genetically-specified neurons. Our tool uses cre-lox technology to label targeted cells with robust markers visible at both the light and electron microscopic level. Fluorescent markers revolutionized the study of neural circuits at the light level, and our novel tool brings these same advantages to the ultrastructural level. My pilot data show expected patterns of cell-type-specific labeling at both the light and electron microscopic level, suggesting feasibility of targeted connectomic analysis. Moving forward, my goals are to 1) test the efficacy of this approach in mapping neural circuits and 2) exploit our tool to elucidate the connectivity of intrinsically photosensitive ganglion cells (ipRGCs) in the retina and brain. ipRGCs are a specialized class of retinal ganglion cells (RGCs) differing from conventional RGCs in both their response properties and axonal terminations. While most RGCs send fast, transient signals encoding image forming features, ipRGCs send slow, sustained signals encoding irradiance, or global light intensity. ipRGC axons terminate in non-image forming regions of the brain where irradiance signals are used to regulate circadian rhythms and pupil dilation. Although the general anatomy and physiology of ipRGCs is well documented, we lack a detailed description of their connectivity. I plan to use our novel tool to conduct a connectomic analysis of ipRGC circuitry. The use of this tool will illuminate the structural connectivity underlying irradiance coding circuits and elucidate the function of the non-canonical ipRGC inputs to the image forming visual pathway. Overall, this proposal will validate our tool for targeted connectomics, prior to its dispersal in the scientific community, and provide valuable insight into the processing and modulation of sensory information through neural circuits.