Animal models of Fetal Alcohol Syndrome (FAS) show that there is a significant impairment in the early development of the serotonin (5-HT) system; there is a long-lasting reduction in 5-HT neurons, decreased 5-HT, and fewer reuptake sites. Our recent in vitro studies suggest that ethanol causes this damage by inducing programmed cell death (apoptosis) in these neurons. Chronic in utero ethanol exposure also retards the development of the astrocytes and other glial cells that provide essential trophic factors, such as S100B, to nearby 5-HT neurons. Maternal treatment with a 5-HT1A agonist prevents most of the damage to developing 5-HT neurons and to the S100B-immunopositive astrocytes proximal to these neurons. 5-HT1A agonists also stimulate the release of S100B from astrocytes. Importantly, recent studies from this and other laboratories show that 5-HT1A agonists prevent damage to neurons by reducing apoptosis and that S100B might contribute to these neuroprotective effects. Despite the frequency of CNS damage that is caused by FAS and alcohol related neurodevelopmental disorder (ARND) and the tremendous public health costs associated with FAS and ARND, there is no therapeutic treatment to prevent CNS damage. This grant proposal will investigate the hypothesis that a 5-HT1A agonist that is used clinically as an anxiolytic in humans exerts neuroprotective effects against ethanol-induced apoptosis in developing 5-HT and other fetal rhombencephalic neurons, and that these neuroprotective effects are mediated by the activation of specific prosurvival pathways. It will also investigate the cellular and molecular mechanisms associated with the neuroprotective effects of S100B against ethanol-induced apoptosis. In addition, these studies will identify specific cellular and molecular changes that contribute to the neurotoxic effects of ethanol on developing CNS neurons. Using in vitro experiments, we will determine the cellular and molecular mechanisms that are responsible for the anti-apoptotic and pro-survival effects of 5-HT1A agonists on ethanol-treated developing rhombencephalic neurons and 5-HT neurons. We will perform a systematic investigation of two pro-survival pathways that have been linked to the activation of the 5-HT1A receptors: the phosphatidylinositol-3 kinase pathway and the mitogen-activated protein kinase kinase (MAPKK/MEK) pathway. The potential involvement of S100B in the protective effects of a 5-HT1A agonist will also be evaluated. In vivo studies will be performed to confirm that a 5-HT1Aagonist acts through similar neuroprotective mechanism(s) in the intact animal. Analyses will include western blot analyses, real-time quantitative RT-PCR, fluorescence and light microscopy, immunohistochemistry, and enzyme assays.
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