Mapping Protein Distribution in the Canine Photoreceptor Sensory Cilium and Calyceal Processes by Ultrastructure Expansion Microscopy.

Purpose: Photoreceptors are highly polarized sensory neurons, possessing a unique ciliary structure known as the photoreceptor sensory cilium (PSC). Vertebrates have two subtypes of photoreceptors: rods, which are responsible for night vision, and cones, which enable daylight vision and color perception. Despite the identification of functional and morphological differences between these subtypes, ultrastructural analysis of the PSC molecular architecture between rods and cones is still lacking. This study employed ultrastructure expansion microscopy (U-ExM) to characterize the PSC molecular architecture in canine retina. Methods: Canine neuroretinas (5-mm punches) were fixed in paraformaldehyde solution for either short or long durations. Additionally, 20-µm-thick cryosections from frozen archival retinal tissues fixed using the longer protocol were analyzed. A U-ExM protocol previously developed for mouse retina was adapted to these canine tissues with a battery of specific antibodies that label the various compartments of the PSC. Results: We demonstrated that U-ExM is applicable to both non-frozen and cryopreserved retinal tissues processed with standard paraformaldehyde fixation. Using this validated U-ExM protocol, we revealed the molecular localization of numerous ciliopathy-related proteins in canine photoreceptors. Furthermore, we identified significant architectural differences in the PSC, ciliary rootlet, and calyceal processes between canine rods and cones. Conclusions: U-ExM is a powerful tool for studying the PSC molecular architecture using frozen archival retinas that are processed following standard paraformaldehyde fixation and embedding protocols. The findings gained from this study pave the way for a better understanding of alterations in the molecular architecture of the PSC in canine models of retinal ciliopathies.