Adenosine A 1 -Receptor Modulation of Glutamate- Induced Calcium Influx in Rat Retinal Ganglion Cells

PURPOSE. Although adenosine receptors (A 1 -Rs and A 2 -Rs) have been identified in the mammalian retina, the role of adenosine in this tissue is not fully understood. The purpose of this work was to investigate the action of adenosine on glutamate-induced calcium influx in rat retinal ganglion cells (RGCs) and to determine whether adenosine modulates RGC voltage-gated calcium channels. METHODS. Purified RGC cultures were generated from neonatal rats with a two-step panning procedure. Isolated RGCs were loaded with the ratiometric calcium-indicator dye fura-2, and the effect of adenosine (and related agonists and antagonists) on intracellular calcium levels ([Ca 2ϩ ] i ) during exposure to glutamate (10 M with 10 M glycine) was assessed. The effect of adenosine on calcium channel currents was also studied in isolated RGCs with whole-cell patch-clamp techniques. In addition, the effect of adenosine on [Ca 2ϩ ] i was investigated in fura dextran-loaded RGCs in an intact adult rat retina preparation. RESULTS. In isolated RGCs, adenosine (10 and 100 M) significantly reduced the glutamate-induced increase in [Ca 2ϩ ] i (ϳ30%). The effect of adenosine was blocked by the A 1 -R antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), but not by the A 2 -R antagonist 3,7-dimethyl-1-propargylxanthine (DMPX). Adenosine (10 M) inhibited calcium channel currents by 43%, and again this effect was blocked by DPCPX, but not DMPX. Adenosine (100 M) also significantly reduced the elevation of [Ca 2ϩ ] i in RGCs in the intact retina during exposure to N-methyl-D-aspartate (NMDA; 100 M). CONCLUSIONS. Adenosine can inhibit glutamate-induced calcium influx and voltage-gated calcium currents in rat RGCs through A 1 -R activation. This work supports a role for adenosine as a neuromodulator of mammalian RGCs. (Invest Ophthalmol Vis Sci

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

A small number (\u3c2%) 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

Small molecule antagonists of melanopsin-mediated phototransduction

Melanopsin, expressed in a subset of retinal ganglion cells, mediates behavioral adaptation to ambient light and other non-image forming photic responses. This has raised the possibility that pharmacological manipulation of melanopsin can modulate several CNS responses including photophobia, sleep, circadian rhythms and neuroendocrine function. Here we describe the identification of a potent synthetic melanopsin antagonist with in vivo activity. Novel sulfonamide compounds inhibiting melanopsin (opsinamides) compete with retinal binding to melanopsin and inhibit its function without affecting rod/cone mediated responses. In vivo administration of opsinamides to mice specifically and reversibly modified melanopsin-dependent light responses including the pupillary light reflex and light aversion. The discovery of opsinamides raises the prospect of therapeutic control of the melanopsin phototransduction system to regulate light-dependent behavior and remediate pathological conditions

Glutamatergic calcium dynamics and deregulation of rat retinal ganglion cells

A rise in intracellular calcium levels ([Ca2+]i) is a key trigger for the lethal effects of the excitatory neurotransmitter glutamate in various central neurons, but a consensus has not been reached on the pathways that mediate glutamate-dependent increases of [Ca2+]i in retinal ganglion cells (RGCs). Using Ca2+ imaging techniques we demonstrated that, in the absence of external Mg2+, the Ca2+ signal evoked by glutamate was predominantly mediated by NMDA-type glutamate receptors (NMDA-Rs) in immunopanned RGCs isolated from neonatal or adult rats. Voltage-gated Ca2+ channels and AMPA/kainate-Rs contributed a smaller portion of the Ca2+ response at saturating concentrations of glutamate. Consistent with NMDA-R involvement, extracellular Mg2+ inhibited RGC glutamate responses, while glycine had a potentiating effect. With Mg2+ present externally, the effect of AMPA/kainate-R antagonists was enhanced and both NMDA- and AMPA/kainate-R antagonists greatly reduced the glutamate-induced increases of RGC [Ca2+]i. This finding indicates that the primary contribution of AMPA/kainate-Rs to RGC glutamatergic Ca2+ dynamics is through the depolarization-dependent relief of the Mg2+ block of NMDA-R channels. The effect of glutamate receptor antagonists on glutamatergic Ca2+ signals from RGCs in adult rat retinal wholemounts yielded results similar to those obtained using immunopanned RGCs. Additional experiments on isolated RGCs revealed that during a 1 h glutamate (10–1000 μm) exposure, 18–28% of RGCs exhibited delayed Ca2+ deregulation (DCD) and the RGCs that underwent DCD were positive for the death marker annexin V. RGCs with larger glutamate-evoked Ca2+ signals were more likely to undergo DCD, and NMDA-R blockade significantly reduced the occurrence of DCD. Identifying the mechanisms underlying RGC excitotoxicity aids in our understanding of the pathophysiology of retinal ischaemia, and this work establishes a major role for NMDA-R-mediated increases in [Ca2+]i in glutamate-related RGC death