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

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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.