Student Research: Kathryn VanDeMark

, Environmental Toxicology (Tox), 2009
Faculty Advisor: Lucio G. Costa

Ethanol Inhibits Muscarinic Receptor-simulated Neuronal Differentiation and Signaling in Pyramidal Hippocampal Neurons


Abstract

Muscarinic receptors have been proposed to play an important role during brain development. This study investigated the effect of Muscarinic receptor activation on prenatal rat hippocampal pyramidal neuron differentiation and the signal transduction pathways involved in this effect. The cholinergic agonist carbachol increased the length of the axon, without affecting the length of minor neuritis. Carbachol-induced axonal growth was also observed in pyramidal neurons from the neocortex, but not in granule neurons from the cerebellum. The effect of carbachol was mediated by the M1 subtype of Muscarinic receptors. The Ca++ chelator BAPTA-AM, two PKC inhibitors (GF 109203X and Ro-32-0432), and the ERK1/2 inhibitors PD98059 and U0126 all blocked carbachol-induced axonal outgrowth. In addition, down-regulation of ERK1/2 with siRNA completely abolished the neuritogenic effect of carbachol. These data suggest an involvement of Ca++, PKC, and ERK1/2 in carbachol-induced axonal growth. Carbachol increased the release of Ca++ from intracellular stores and induced PKC and ERK1/2 activation. Additional experiments showed that PKC, but not Ca++, is involved in carbachol-induced ERK1/2 activation. Altogether, these results show that cholinergic stimulation of prenatal hippocampal pyramidal neurons accelerates axonal growth through the induction of Ca++ mobilization and the activation PKC and especially of ERK1/2.

In utero alcohol exposure can lead to fetal alcohol syndrome which is characterized by facial dysmorphology, growth deficiencies, and central nervous system abnormalities. 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. This study investigated the effect of ethanol on the differentiation of rat hippocampal pyramidal neurons induced by the cholinergic agonist carbachol as a possible mechanism involved in the developmental neurotoxicity of ethanol. Ethanol treatment (20-75 mM) caused an inhibition of carbachol-induced axonal growth, without affecting neuronal viability. Ethanol treatment 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 concentrations used in this study. In conclusion, we have shown that ethanol inhibits carbachol-induced neurite outgrowth by inhibiting PKC and ERK1/2 activation.