Channeling Vision: Voltage-Gated Calcium Channels of Rods and Cones
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2018-11-27
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Abstract
Congenital stationary night blindness (CSNB) is a set of inherited diseases characterized by defects in neurotransmission from photoreceptors to second-order neurons in the retina. CSNB2A, specifically, is caused by mutations in Cacna1f – the pore-forming sub-unit of the voltage-gated calcium channel (VGCC), CaV1.4, which is responsible for calcium-mediated glutamate release at photoreceptor ribbon synapses. In this work, we initially expanded the characterization of the Cacna1f-KO mouse retina with a particular focus on cones using our own Cacna1fG305X mouse line. We have comprehensively characterized cone morphology and viability throughout the murine lifespan, and also verified several findings reported in an alternative Cacna1f-KO model which suggest possibility of late-stage rescue. We also identified ectopic, synapse-like cone-rod bipolar cell contacts, which have been described in no other model of retinal disease. We then sought to investigate whether late-stage rescue of retinal morphology and function is feasible in the Cacna1f-KO retina. To this end, we have designed a strategy employing transgenic mice that, with appropriate gene combinations, will allow for inducible expression of Cacna1f. We have characterized several transgenic mice relevant to these experimental aims, and provided some proof-of-concept for future experiments which may provide insight into the plausibility of therapeutic interventions. We also sought to establish an alternative model of CSNB2A in the post-embryonic chick. The relative lack of tools for genetic manipulation of this model led to us developing a novel means – avian adeno-associated viral (A3V) vectors. Using A3V vectors we were able to achieve highly efficient local transduction of photoreceptors following sub-retinal injection, thus providing us with a new tool for investigation of chicken retinal circuitry. Finally, in an attempt to replicate CSNB2A in the chicken retina, we sought to characterize VGCC expression to definitively establish the target for gene knockdown. We were able to definitively prove expression of a Cacna1f orthologue in the chicken retina, and establish its sequence and mRNA expression patterns. Unfortunately, shRNA-encoding A3Vs were unable to achieve significant knockdown, but we have established a powerful framework for future investigations in this model.
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Waldner, D. (2018). Channeling Vision: Voltage-Gated Calcium Channels of Rods and Cones (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/34540