Fetal alcohol syndrome (FAS) is an important cause of mental retardation. Exposure of the developing brain to alcohol can induce the death of neurons, which contributes strongly to learning deficits and neurological problems associated with FAS. Understanding the factors that influence neuronal vulnerability to alcohol induced loss in the developing brain is of considerable significance. We hypothesize that as neurons mature, they acquire a neuroprotective, signaling pathway that protects them against alcohol toxicity. We have discovered a neuroprotective pathway, which is mediated by nitric oxide (NO). This proposal will elucidate the function and molecular mechanisms of the NO-signaling pathway in protecting the developing brain against alcohol-induced neuronal loss. Our experiments will utilize a mouse strain carrying a null mutation for neuronal nitric oxide synthase (nNOS-/-), thus these animals lack NO-mediated neuroprotection. Specific Aim 1 includes morphological experiments examining the importance of NO-mediated neuroprotection on neuronal survival. Stereological methods will measure alcohol-induced neuronal losses in the hippocampus and cerebellum of mice that express or lack the protective pathway. Alcohol exposure will occur at a variety of ages in order to determine the impact of NO-mediated neuroprotection at different stages of brain development. Specific Aim 2 utilizes behavioral testing (eyeblink conditioning, Morris Water Maze) to determine whether the enhanced alcohol-induced neuronal losses in nNOS -/- mice are linked to greater functional deficits. Alcohol-induced deficits on these tests will be compared in nNOS / and nNOS+/+ mice and linked with neuronal losses. Specific Aim 3 includes molecular studies to determine the mechanism by which NO-signaling provides neuroprotection against alcohol toxicity. Experiments will determine whether nNOS is developmentally regulated in vivo and link alcohol vulnerability to expression levels and activity of nNOS. "Rescue" experiments in which the nNOS gene will be transfected into pathway-deficient neurons in culture derived from nNOS -/- mice will determine whether neuroprotection against alcohol toxicity can be restored. Immunohistochemistry and quantitative RT-PCR will explore the role of cAMP-responsive element binding protein (CREB) as a downstream effector molecule for the NO-signaling and elucidate CREB's role in regulating apoptotic proteins, which may underlie this neuroprotection.