Purpose: Compelling evidence suggests that alpha2 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 alpha2 adrenergic system in the retina is not well understood. A major goal of this work was to explore the role of the alpha2 adrenergic system in the modulation of cytosolic Ca(2+) signaling at retinal synaptic layers, particularly the inner plexiform layer (IPL), where communication between RGCs and their presynaptic cells takes place.
Methods: Functional Ca(2+) 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 Ca(2+) were monitored with the fluorescent Ca(2+) dye fluo-4. The Ca(2+) 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 Ca(2+) increase at the IPL and OPL. In both cases, this Ca(2+) signal was eliminated by nimodipine, a selective L-type voltage-gated Ca(2+)-channel blocker, or when the extracellular Ca(2+) in the Ringer was replaced with equal molar EGTA. At IPL, the Ca(2+) signal was also suppressed in a dose-dependent manner by brimonidine and other alpha2 receptor agonists, such as medetomidine. The suppressive action of brimonidine and medetomidine was completely blocked by classic alpha2 receptor antagonists, such as yohimbine, rauwolscine, and atipamezole. Interestingly, the alpha2 receptor agonists had no effect on the high K(+) Ringer-elicited cytosolic Ca(2+) signal at OPL. Blocking the N-methyl-d-aspartate (NMDA) type of ionotropic glutamate receptor with D-AP5 attenuated this high K(+)-elicited Ca(2+) signal by approximately 20% at IPL. D-AP5 had no effect on the Ca(2+) signal at OPL.
Conclusions: These findings provide the first direct evidence of alpha2 receptor-mediated modulation of L-type Ca(2+) channel activity in the CNS (the retina is part of the CNS). This alpha2 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 alpha2 system is the regulation of synaptic transmission at IPL and that brimonidine and other alpha2 agonists may protect RGCs under disease conditions by preventing abnormal elevation of cytosolic free Ca(2+) either in RGCs, in their presynaptic cells, or in both.