α2 adrenergic receptor-mediated modulation of cytosolic Ca++ signals at the inner plexiform layer of the rat retina

PURPOSE. Compelling evidence suggests that alpha 2 agonists, such as brimonidine, protect retinal ganglion cells (RGCs) from injury in a wide range of animal models. However, the mechanism of action for this protection and the physiological role of the alpha 2 adrenergic system in the retina is not well understood. A major goal of this work was to explore the role of the alpha 2 adrenergic system in the modulation of cytosolic Ca2+ signaling at retinal synaptic layers, particularly the inner plexiform layer (IPL), where communication between RGCs and their presynaptic cells takes place. METHODS. Functional Ca2+ imaging at the inner plexiform layer (IPL) and outer plexiform layer (OPL) of living rat retinal slices was conducted with a high-speed confocal system. The relative changes of cytosolic free Ca2+ were monitored with the fluorescent Ca2+ dye fluo-4. The Ca2+ signal was elicited by membrane depolarization produced by a high K+ (40 mM) Ringer solution that was delivered rapidly and briefly to the test regions of the retinal slice by a custom-made multichannel local perfusion system. RESULTS. A brief application (8 seconds) of high K+ Ringer elicited a robust cytosolic Ca2+ increase at the IPL and OPL. In both cases, this Ca2+ signal was eliminated by nimodipine, a selective L-type voltage-gated Ca2+-channel blocker, or when the extracellular Ca2+ in the Ringer was replaced with equal molar EGTA. At IPL, the Ca2+ signal was also suppressed in a dose-dependent manner by brimonidine and other alpha 2 receptor agonists, such as medetomidine. The suppressive action of brimonidine and medetomidine was completely blocked by classic alpha 2 receptor antagonists, such as yohimbine, rauwolscine, and atipamezole. Interestingly, the alpha 2 receptor agonists had no effect on the high K+ Ringer-elicited cytosolic Ca2+ signal at OPL. Blocking the N-methyl-i)-aspartate (NMDA) type of ionotropic glutamate receptor with D-AP5 attenuated this high K+-elicited Ca2+ signal by approximately 20% at IPL. D-AP5 had no effect on the Ca2+ signal at OPL. CONCLUSIONS. These findings provide the first direct evidence of alpha 2 receptor-mediated modulation of L-rype Ca2+ channel activity in the CNS (the retina is part of the CNS). This alpha 2 modulation appears to occur at the IPL but not at the OPL of the retina. These findings suggest that a physiological function of the retinal alpha 2 system is the regulation of synaptic transmission at IPL and that brimonidine and other alpha 2 agonists may protect RGCs under disease conditions by preventing abnormal elevation of cytosolic free Ca2+ either in RGCs, in their presynaptic cells, or in both.