Endogenous acetylcholine rescues NMDA-induced long-lasting hippocampal cell damage via stimulation of muscarinic M1 receptors: Elucidation using organic hippocampal slice cultures

Abstract

This study aimed to investigate a recuing role of cholinergic systems in the excitotoxicity-induced hippocampal cell damage. Organotypic hippocampal slice cultures (OHSCs) were prepared from 7-day-old mice and exposed to N-methyl-d-aspartate (NMDA) for 24h. After washing out the NMDA, OHSCs were incubated in medium containing test drugs for 0–6 days. Hippocampal cell damage was evaluated by propidium iodide staining, immunofluorescence, and Western blotting. NMDA (1–10μM) dose-dependently damaged hippocampal cells. The toxic effect of 3μM NMDA was also observed at 3–6 days, even after washing out NMDA, and was blocked by MK-801 from day 3 to day 6. Post-treatments with tacrine, donepezil, and galantamine reduced the NMDA-induced long-lasting hippocampal cell damage. The effect of tacrine was induced in a manner dependent on the incubation period after NMDA treatment and was confirmed by Nissl staining and immunostaining with NeuN, a marker of mature neurons. The effect of tacrine was attenuated by scopolamine and a muscarinic M1 receptor antagonist, pirenzepine, but not by a muscarinic M3 receptor antagonist, darifenacin, or a nicotinic receptor antagonist, mecamylamine. The protein kinase C inhibitor Ro-31-8220 abolished the effect of tacrine. The pretreatment with 3μM NMDA had no effect on the expression level of presynaptic cholinergic markers, choline acetyltransferase and vesicular acetylcholine transporter, in OHSCs. These results suggest that a low concentration of NMDA causes long-lasting hippocampal cell damage and that endogenous acetylcholine plays, via muscarinic M1 receptor, a rescuing role in the excitotoxicity-induced long-lasting hippocampal cell damage.