Calcium imaging in cone photoreceptor cell compartments using a genetically encoded ratiometric calcium indicator

Abstract

Purpose : Calcium dynamics greatly affect the kinetics of cone photoreceptor’s light sensitivity and adaptation, and calcium homeostasis is thought to be crucial for cone survival. Calcium regulation has been hypothesized to be compartmentalized in photoreceptors due to unique regulation of calcium entry and extrusion in different cell regions, and the presence of mitochondria that buffering Ca²⁺ between the outer and inner segment compartments. Whether or how alteration of [Ca²⁺]_i in one compartment can affect calcium homeostasis in adjacent compartments remains unknown. To address this, the ability to systemically determine Ca²⁺ level in each compartment of cones simultaneously is the key to answer the question.

Methods : Salsa6f transgenic reporter mouse line expresses a genetically encoded ratiometric calcium indicator (GCaMP6f-V5-tdTomato), which enables imaging and determination of [Ca²⁺]_i under steady state conditions. By crossbreeding Salsa6f mice with HRGP-CRE mice, we specifically target the expression of Salsa6f in cones. Live cell imaging of retinal slices was conducted using a two-photon microscope, allowing visualization of GCaMP6f and tdTomato signals.

Results : GCaMP6f and tdTomato fluorescence signals were clearly detected within individual cone compartments, and the ratio of these signals were used to determine relative and absolute [Ca²⁺]_i in different cone compartments. Calcium calibration curves have been established in each compartment (outer segment, inner segment, cell body and synaptic terminal), which will be further used to calculate absolute [Ca²⁺]. Curves were fitted with dose-response equation.The baseline [Ca²⁺]_i from dark- and light-adapted Salsa6f/HRGP-CRE cones was determined using the calibration curves, providing physiologic Ca²⁺ levels in different cone compartments.

Conclusions : Salsa6f/HRGP-CRE mice provide an effective approach for systemically imaging physiologic [Ca²⁺] in all cone photoreceptor compartments. Our live cell imaging system establishes a platform to explore calcium homeostasis in cones and presents an opportunity to examine [Ca²⁺]_i changes in cone degeneration models, and further investigate mitochondria dysfunction and calcium related cell death machinery in degenerating cones.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.