The NO-cGMP-PKG pathway plays an essential role in the acquisition of ethanol resistance by cerebellar granule neurons

Abstract

When the developing brain is exposed to alcohol, neuronal death is a prominent pathologic effect. This loss of neurons may underlie many of the behavioral deficits observed in fetal alcohol syndrome (FAS). Previous studies using whole animals and cultured neurons have demonstrated that the vulnerability of neurons to alcohol-induced death changes with development and can diminish markedly over the course of several days. This study examined the possibility that developmental stage-dependent alcohol resistance depends on a unique signaling pathway involving nitric oxide (NO), the NO-cyclic GMP (cGMP)-cGMP-dependent protein kinase (PKG) pathway. Cerebellar granule neuron (CGN) cultures were established from neonatal rats. The cultures were exposed to alcohol (400 mg/dl) either when they were newly established (1 day in vitro, 1-DIV) or when they were more mature (4 days in vitro, 4-DIV). Viable neurons were quantified 24 h later. Although both 1-DIV and 4-DIV cultures were exposed to alcohol for an identical length of time (24 h), the 1-DIV cultures were much more vulnerable to alcohol-induced neuronal death (22.9% neuronal loss) than the 4-DIV cultures (2.3% neuronal loss). Thus, the cultures have a developmental stage-dependent alcohol resistance. To determine the role of the NO-cGMP-PKG pathway in this developmental stage-dependent alcohol resistance, the CGN cultures at 4-DIV were exposed to agents that either activated or inhibited the pathway, either in the presence or in the absence of alcohol. Inhibition of the pathway at its first step with N G-nitro- l-arginine-methyl ester (NAME) or at its second step with LY83583 converted the 4-DIV cultures from alcohol resistant to alcohol sensitive. Alcohol-induced neuronal losses were as severe in the 4-DIV cultures treated with pathway inhibitors as they were in the 1-DIV cultures. Treatment of the CGN cultures with agents that activate the NO-cGMP-PKG pathway reduced background cell death, and this neurotrophic effect was not inhibited by the presence of alcohol. Furthermore, activation of the NO-cGMP-PKG pathway at sites downstream from sites of pathway inhibition restored alcohol resistance. Thus, the developmental stage-dependent alcohol resistance acquired by CGN cultures depends on a functional NO-cGMP-PKG pathway.