MELANOPSIN AND PACAP IN INTRINSICALLY PHOTOSENSITIVE RETINAL GANGLION CELLS REGULATE THE DEVELOPMENT OF RETINOFUGAL PROJECTIONS IN MICE

Abstract

In the retina, a small subset of retinal ganglion cells are intrinsically photo-sensitive (ipRGCs) and express the photopigment melanopsin and the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP). These cells project to brain nuclei to mediate a variety of visual behaviors involved in both non-image and image-forming functions, such as circadian photoentrainment and conscious visual perception. ipRGCs have been implicated in a number of developmental processes such as cone photoreceptor development, patterning of retinal vasculature, and synapse formation and maturation. However, whether or not these molecules are involved in development of retinofugal projections to the suprachiasmatic nucleus (SCN) and refinement of eye-specific segregation in the dorsal lateral geniculate nucleus (dLGN) have not been extensively determined. To answer these questions, I used tract tracing with eye injections and mesoscale imaging to assess ipRGC projections to the SCN and dLGN of both juvenile melanopsin (Opn4cre/cre) and conditional ipRGC-specific PACAP (Opn4Cre/+ / Adcyap1fl/fl) knockout mice. I found that in melanopsin knockout mice, axonal projections to the SCN were altered, and eye-specific segregation in the dLGN was impaired. In PACAP knockout mice, axonal projections to the SCN are unaltered, but eye-specific segregation in the dLGN is impaired. These results suggest that melanopsin and PACAP play differential roles in the proper development of retinofugal projections in mice. In parallel, I applied volumetric super-resolution fluorescence imaging using Expansion Microscopy (ExM) to study synaptic connectivity of ipRGCs in the mouse retina. This strategy utilizes hydrogel chemistry to physically expand brain and retina samples, enabling nanoscale analysis of synaptic properties. I report an experimental approach for studying ipRGC synaptic connectivity in the mouse retina that combines immunohistochemical synaptic protein staining, cellular labeling of RGCs, and ExM imaging to achieve an ∼ 4x isotropic increase in spatial resolution, and describe the challenges that I encountered.