Light-Evoked Calcium Responses of Isolated Melanopsin- Expressing Retinal Ganglion Cells

Authors

Andrew T. E. Hartwick, Colorado State University
Jayne R. Bramley, Colorado State University
Jianing Yu, Dalhousie University
Kelly T. Stevens, Dalhousie University
Charles N. Allen, Oregon Health and Science University
William H. Baldridge, Dalhousie University
Patricia J. Sollars, University of Nebraska-LincolnFollow
Gary E. Pickard, University of Nebraska-LincolnFollow

Document Type

Article

Date of this Version

12-5-2007

Citation

The Journal of Neuroscience, December 5, 2007 • 27(49): pp. 13468 –13480. DOI:10.1523/JNEUROSCI.3626-07.2007

Comments

Copyright © 2007 Society for Neuroscience. Used by permission.

Abstract

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 lightstimulated ipRGCs.