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The Journal of Neuroscience, December 5, 2007, 27(49):13468-13480; doi:10.1523/JNEUROSCI.3626-07.2007

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Behavioral/Systems/Cognitive
Light-Evoked Calcium Responses of Isolated Melanopsin-Expressing Retinal Ganglion Cells

Andrew T. E. Hartwick,¹ Jayne R. Bramley,¹ Jianing Yu,² Kelly T. Stevens,² Charles N. Allen,³ William H. Baldridge,² Patricia J. Sollars,¹ and Gary E. Pickard¹

¹Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523, ²Anatomy and Neurobiology, Ophthalmology and Visual Sciences, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5, and ³Center for Research on Occupational and Environmental Toxicology, Oregon Health and Science University, Portland, Oregon 97239

Correspondence should be addressed to Gary E. Pickard at the above address. Email: gpickard{at}lamar.colostate.edu

A small number (<2%) of mammalian retinal ganglion cells express the photopigment melanopsin and are intrinsically photosensitive (ipRGCs). Light depolarizes ipRGCs and increases intracellular calcium levels ([Ca²⁺]_i) but the signaling cascades underlying these responses have yet to be elucidated. To facilitate physiological studies on these rare photoreceptors, highly enriched ipRGC cultures from neonatal rats were generated using anti-melanopsin-mediated plate adhesion (immunopanning). This novel approach enabled experiments on isolated ipRGCs, eliminating the potential confounding influence of rod/cone-driven input. Light induced a rise in [Ca²⁺]_i (monitored using fura-2 imaging) in the immunopanned ipRGCs and the source of this Ca²⁺ signal was investigated. The Ca²⁺ responses were inhibited by 2-aminoethoxydiphenyl borate, SKF-96365 (1–2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole), flufenamic acid, lanthanum, and gadolinium, consistent with the involvement of canonical transient receptor potential (TRP) channels in ipRGC phototransduction. However, the contribution of direct Ca²⁺ flux through a putative TRP channel to ipRGC [Ca²⁺]_i was relatively small, as most (90%) of the light-induced Ca²⁺ responses could be blocked by preventing action potential firing with tetrodotoxin. The L-type voltage-gated Ca²⁺ channel (VGCC) blockers verapamil and (+)-cis-diltiazem significantly reduced the light-evoked Ca²⁺ responses, while the internal Ca²⁺ stores depleting agent thapsigargin had negligible effect. These results indicate that Ca²⁺ influx through VGCCs, activated after action potential firing, was the primary source for light-evoked elevations in ipRGC [Ca²⁺]_i. Furthermore, concurrent Ca²⁺ imaging and cell-attached electrophysiological recordings demonstrated that the Ca²⁺ responses were highly correlated to spike frequency, thereby establishing a direct link between action potential firing and somatic [Ca²⁺]_i in light-stimulated ipRGCs.

Key words: retinal ganglion cell; melanopsin; calcium; TRP channel; voltage-gated calcium channels; circadian rhythms

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Received Aug. 9, 2007; revised Sept. 26, 2007; accepted Oct. 22, 2007.

Correspondence should be addressed to Gary E. Pickard at the above address. Email: gpickard{at}lamar.colostate.edu

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