Acetylcholine neuroprotection against glutamate-induced excitotoxicity in adult pig retinal ganglion cells is partially mediated through α4 nAChRs

In the mammalian retina, excess glutamate release has been shown to be involved in retinal ganglion cell (RGC) death associated with various diseases. Recent studies have determined that activation of alpha 7 nicotinic acetylcholine receptors (nAChRs) partially protect isolated RGCs from glutamate-induced excitotoxicity. In this study, we further classify the types of nAChRs involved in neuroprotection against glutamate-induced excitotoxicity using isolated adult pig RGCs. Cells were isolated with a modified two-step immunoselective panning technique designed to isolate RGCs from other retinal neurons. Once isolated, nAChR subunits were identified using a combination of pharmacological and immunocytochemical techniques. In cell culture experiments, a variety of alpha 4 nAChR specific agonists were found to have a partial neuroprotective against glutamate-induced excitotoxicity. This neuroprotection was abolished in the presence of the alpha 4 nAChR antagonist, dihydro-beta-erythroidine (DHOE). Immunocytochemical results localized several nAChR subunits on isolated adult pig RGCs; in particular alpha 4, alpha 7 and beta 2 nAChR subunits. Large RGCs exclusively immunostained with antibodies against alpha 7 nAChR subunits whereas alpha 4 and beta 2 subunits exclusively immunostained only small RGCs. Double label experiments provided evidence that alpha 4 and beta 2 subunits co-localize on small RGCs. Knowledge of the receptor subtypes responsible for neuroprotection may lead to treatments associated with glutamate-induced excitotoxicity. (c) 2006 Elsevier Ltd. All rights reserved.