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Abstract

Calcium mediates various neuronal functions. The complexity of neuronal Ca2+ signaling is well exemplified by retinal cone photoreceptors, which, with their distinct compartmentalization, offer unique possibilities for studying the diversity of Ca2+ functions in a single cell. Measuring subcellular Ca2+ signals in cones under physiological conditions is not only fundamental for understanding cone function, it also bears important insights into pathophysiological processes governing retinal neurodegeneration. However, due to the proximity of light-sensitive outer segments to other cellular compartments, optical measurements of light-evoked Ca2+ responses in cones are challenging. We addressed this problem by generating a transgenic mouse (HR2.1:TN-XL) in which both short-and middle-wavelength-sensitive cones selectively express the genetically encoded ratiometric Ca2+ biosensor TN-XL. We show that HR2.1:TN-XL allows recording of light-evoked Ca2+ responses using two-photon imaging in individual cone photoreceptor terminals and to probe phototransduction and its diverse regulatory mechanisms with pharmacology at subcellular resolution. To further test this system, we asked whether the classical, nitric oxide (NO)-soluble guanylyl-cyclase (sGC)-cGMP pathway could modulate Ca2 in cone terminals. Surprisingly, NO reduced Ca2+ resting levels in mouse cones, without evidence for direct sGC involvement. In conclusion, HR2.1:TN-XL mice offer unprecedented opportunities to elucidate light-driven Ca2+ dynamics and their (dys) regulation in cone photoreceptors.