Mechanisms underlying diverse effects of low-dose embryonic ethanol on development and function of hypocretin/orexin neurons

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PROJECT SUMMARY Alcohol use disorder (AUD) is a chronic psychiatric disorder with severe societal consequences that impacts over 15 million adults in the USA and 76 million worldwide. Ethanol exposure early in life, particularly in utero, strikingly alters fetal brain development and increases the risk for AUD. Even exposure to low concentrations of ethanol can produce the most prevalent, milder forms of fetal alcohol spectrum disorder. Our recent studies in the rat demonstrate that prenatal exposure to low levels of ethanol, in addition to increasing ethanol consumption and anxiety in the offspring, stimulates neurogenesis with negligible cytotoxic effects. Interestingly, prenatal ethanol stimulates the density of neurons expressing hypocretin/orexin (Hcrt), an orexigenic neuropeptide located almost exclusively in the hypothalamus that has a major role in promoting AUD-related behaviors. These compelling effects observed in our rodent model led us to seek a simpler vertebrate model to investigate in real- time, at cellular and anatomical levels, the mechanisms involved in ethanol’s effects on embryonic development of Hcrt neurons. The zebrafish (ZF) is a perfect vertebrate model system for our studies, due to its optical accessibility and external development, its small size and low cost, and its comparable CNS that develops early and rapidly alongside a relatively sophisticated behavioral repertoire. With our recent publications showing a conservation of ethanol’s effects across species, we established in our laboratory a variety of techniques particularly suited for comprehensive studies of the ZF brain, including time-lapse live imaging, optogenetics, calcium imaging, and in vivo targeted laser ablation, and obtained preliminary results revealing robust and unexpected changes in Hcrt neurons and neuroimmune systems that may lead to the formation of specific asymmetric Hcrt subpopulations. Based on these new findings, we propose to test the following hypothesis: Embryonic exposure to ethanol at low doses has diverse effects on the development of Hcrt neurons, which are mediated by dynamic changes in local inflammatory chemokine systems and contribute to the formation of asymmetric, abnormally-located Hcrt subpopulations that exhibit altered neural activity and are causally related to disturbances in ethanol consumption and associated behaviors. In 3 specific aims, we plan in ZF embryonically exposed to low-dose ethanol: 1) to thoroughly characterize Hcrt neuronal development along with behavior, under normal conditions and after ethanol exposure, and directly test the behavioral functions of these neurons; 2) to precisely determine, at a single-cell level, if Hcrt neurons in specific subpopulations are unique in their birth date, site of origin, migratory path, and signaling activity, and if they are causally related to behavioral disturbances; and 3) to test the possibility that effects of embryonic ethanol on Hcrt neuronal development and behavior are mediated by local neuroimmune systems, specifically CXCL12a/CXCR4b/CXCR7b and CCL2/CCR2. This research aims to elucidate in depth the dynamic and diverse neuropeptide, neuroimmune and behavioral changes caused by low levels of in utero ethanol exposure that increase the risk for AUD.