NEUROPROTECTION OF RAT RETINAL GANGLION CELLS MEDIATED THROUGH ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTORS

Glutamate-induced excitotoxicity is thought to play an important role in several neurodegenerative diseases in the central nervous system (CNS). In this study, neuroprotection against glutamate-induced excitotoxicity was analyzed using acetylcholine (ACh), nicotine and the alpha 7 specific nicotinic acetylcholine receptor (alpha 7 nAChR) agonist, N-[(3R)-1-azabicyclo[2.2.2]oct-3-y1]-4-chlorobenzamide hydrochloride (PNU-282987), in cultured adult rat retinal neurons. Adult Long Evans rat retinas were dissociated and retinal ganglion cells (RGCs) were isolated from all other retinal tissue using a two-step panning technique. Once isolated, RGCs were cultured under various pharmacological conditions to demonstrate excitotoxicity and neuroprotection against excitotoxicity. After 3 days, RGCs were immunostained with antibodies against the glycoprotein, Thy 1.1, counted and cell survival was assessed relative to control untreated conditions. 500 AM glutamate induced excitotoxicity in large and small RGCs in an adult rat dissociated culture. After 3 days in culture with glutamate, the cell survival of large RGCs decreased by an average of 48.16% while the cell survival of small RGCs decreased by an average of 42.03%. Using specific glutamate receptor agonists and antagonists, we provide evidence that the excitotoxic response was mediated through alpha-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid (AMPA)/kainic acid (KA) and N-methyl-D-aspartate (NMDA) glutamate receptors through an apoptotic mechanism. However, the excitotoxic effect of glutamate on all RGCs was eliminated if cells were cultured for an hour with 10 ILM ACh, 100 trAl nicotine or glutamate insult. Inhibition studies using 10 nM methyllycaconitine (MLA) or oc-bungarotoxin (alpha-Bgt) supported the hypothesis that neuroprotection against glutamate-induced excitotoxicity on rat RGCs was mediated through alpha a nAChRs. In immunocytochemical studies, double-labeled experiments using antibodies against Thy 1.1 and alpha a nAChR subunits demonstrated that both large and small RGCs contained alpha a nAChR subunits. The data presented in this study support the hypothesis that ACh and nicotinic acetylcholine receptor (nAChR) agonists provide neuroprotection against glutamate-induced excitotoxicity in adult rat RGCs through activation of alpha 7 nAChR subunits. These studies lay the groundwork required for analyzing the effect of specific alpha 7 nAChR agonists using in vivo models of excitotoxicity. Understanding the type of ACh receptors involved in neuroprotection in the rat retina could ultimately lead to therapeutic treatment for any CNS disease that involves excitotoxicity. (C) 2013 IBRO. Published by Elsevier Ltd. All rights reserved.