Ethanol inhibits muscarinic receptor-induced axonal growth in rat hippocampal neurons

Background: In utero alcohol exposure can lead to fetal alcohol spectrum (FAS) disorders characterized by cognitive and behavioral deficits. In vivo and in vitro studies have shown that ethanol alters neuronal development. One mechanism through which ethanol has been shown to exert its effects is the perturbation of activated signaling cascades. The cholinergic agonist carbachol has been shown to induce axonal outgrowth through intracellular calcium mobilization, protein kinase C (PKC) activation, and ERK1/2 phosphorylation. This study investigated the effect of ethanol on the differentiation of rat hippocampal pyramidal neurons induced by carbachol as a possible mechanism involved in the developmental neurotoxicity of ethanol. Methods: Prenatal rat hippocampal pyramidal neurons were treated with ethanol (50 to 75 mM) in the presence or absence of carbachol for 24 hours. Neurite outgrowth was assessed spectrophotometrically; axonal length was measured in neurons fixed and immunolabeled with the neuron-specific βIII tubulin antibody; cytotoxicity was analyzed using the thiazolyl blue tetrazolium bromide assay. The effect of ethanol on carbachol-stimulated intracellular calcium mobilization was assessed utilizing the fluorescent calcium probe, Fluo-3AM. The PepTag® assay for nonradioactive detection of PKC from Promega was used to measure PKC activity, and ERK1/2 activation was determined by densitometric analysis of Western blots probed for phospo-ERK1/2. Results: Ethanol treatment (50 to 75 mM) caused an inhibition of carbachol-induced axonal growth, without affecting neuronal viability. Neuron treatment for 15 minutes with ethanol did not inhibit the carbachol-stimulated rise in intracellular calcium, while inhibiting PKC activity at the highest tested concentration and ERK1/2 phosphorylation at both the concentrations used in this study. On the other hand, neuron treatment for 24 hours with ethanol significantly inhibited carbachol-induced increase in intracellular calcium. Conclusions: Ethanol inhibited carbachol-induced neurite outgrowth by inhibiting PKC and ERK1/2 activation. These effects may be, in part, responsible for some of the cognitive deficits associated with in utero alcohol exposure.