Abstract Cyclic GMP and calcium (Ca2+) are effectors of phototransduction and maintenance of their homeostasis is critical for photoreceptor cell health and function. Indeed, perturbance of this homeostasis is thought to be a primary driver of cell death in different forms of retinal degenerations. For example, mutations affecting phototransduction genes leading to elevated cGMP is expected to cause toxic Ca2+ influx into the outer segment via the cGMP-gated channels. Although a reasonable hypothesis, it is difficult to reconcile with 1) the cell death machinery is localized in the inner segment, and 2) the current dogma that the outer and inner segments Ca2+ are compartmentalized and insulated from each other. This raises an important and unanswered question: how does [Ca2+] buildup in the outer segment alter [Ca2+] in the inner segment to activate cell death? Our central hypothesis is that altered Ca2+ homeostasis at the outer segment leads to mitochondrial stress at the inner segment. This, in turn, allows Ca2+ to equilibrate with the proximal cell compartments where the cell death machinery resides. To test this hypothesis, we will use mouse models that express genetically encoded, ratiometric Ca2+ indicators in retinal rods and cones. To prevent activating the photoreceptor cells, we will use 1) a multiphoton microscope equipped with a super-sensitive HyD detector that images under extremely low photon flux and 2) mouse models with attenuated phototransduction to reduce Ca2+ feedback. Using retinae obtained from both male and female mice, we will image [Ca2+]i from different cellular compartments simultaneously, in both dark adapted and light exposed conditions, in healthy and degenerating photoreceptors, to visualize how changes in [Ca2+] in one cellular compartment may affect [Ca2+] in another compartment. The proposed experiments should provide a better understanding of the role of Ca2+ homeostasis in health and disease.

Project Narrative Photoreceptor cells utilize a prototypical G-protein signaling cascade, called phototransduction, to convey the presence of light. Genetic mutations and environmental factors that affect the performance of phototransduction cause many different forms of blinding diseases through mechanisms that are poorly understood. Investigation of these mechanisms will help in the design of treatment options for visual disorders arising from dysfunction of the phototransduction cascade.