Compelling evidence suggests that ethanol exposure early in life, even at low levels, disturbs the development of neurons in the brain and increases the risk for developing alcohol use disorder (AUD). Low-dose maternal ethanol (ME) consumption in both zebrafish (ZF) and rats has revealed conserved neurobehavioral effects across species, with ME consumption significantly increasing in the offspring the expression of the hypothalamic orexigenic peptide hypocretin/orexin (Hcrt), a potent stimulator of consummatory behavior, and the consumption of ethanol and anxiety-related behaviors. While there is evidence linking neuroimmune signaling to AUD and showing ethanol in rats to stimulate various inflammatory chemokines and cytokines in glial cells, there are few studies examining neuroimmune factors within neurons and investigating how they coexpress with neuropeptides and affect the development of these neurons and the behaviors they control. Focusing on hypothalamic Hcrt neurons and neuroimmune systems that exist within these neurons, this proposal will use a dual-species approach to test the following hypothesis: Maternal ethanol consumption disturbs specific neuroimmune transcripts in Hcrt neurons of the offspring which, in turn, stimulate the development of these neurons and contribute to an increased propensity for greater ethanol consumption and preference in the offspring.
In Aim 1, transgenic Hcrt:EGFP ZF will be used, an advantageous vertebrate due to its external development, genetic tractability, optical transparency and small size, to first evaluate ME?s effects on the well- studied chemokine CXCL12a and its receptor CXCR4b within Hcrt neurons of the offspring. Then, the Hcrt transcriptome will be sequenced using RNA-seq, and bioinformatic analyses will be performed to identify the top differentially expressed neuroimmune gene induced by ethanol exposure. This analysis will be followed by CRISPR/Cas9 gene editing, which will be used to determine in ZF offspring the functional role of the most strongly affected candidate gene as well as CXCL12a and CXCR4b in altering the development of Hcrt neurons, to be examined using quantitative live imaging, and also in stimulating ethanol consumption and related behaviors.
In Aim 2, the ZF findings will be translated directly to the rat model, first by confirming results obtained in the ZF and then by knocking down through injection into rat embryo brain of AAV delivered shRNA for the target neuroimmune gene identified in Aim 1. To determine the functional role of this target gene in mediating the effects of ME on neuronal development and behavior, Hcrt neurons will be examined in adolescent rat offspring using iDISCO brain clearing, and ethanol consumption will be measured using the intermittent access two-bottle choice paradigm. With this research being in line with multiple objectives of the NIAAA strategic plan, the proposed studies which will provide extensive training with different techniques should also produce novel insights into how maternal consumption of low ethanol levels acts through the neuroimmune system to affect the development of Hcrt neurons in the offspring and contribute to their increased ethanol intake later in life.
Human and animal studies show that the risk for developing alcohol use disorder is increased by maternal consumption of alcohol during pregnancy, even at low doses, and that this risk is attributed to disturbances in the development of brain systems that control behavior. While ethanol is known to stimulate a neuropeptide hypocretin/orexin and particular neuroimmune factors in the hypothalamus, little is known about how the neuroimmune system interacts within neuropeptide neurons to control their development and ultimately affect behavior of the offspring. The proposed studies will evaluate specific neuroimmune transcripts stimulated within hypocretin/orexin neurons by low levels of maternal ethanol consumption and directly test their functional role in promoting an increased propensity for greater ethanol consumption in the offspring, thus generating new information and potential strategies to alleviate the effects caused in offspring by maternal alcohol consumption during pregnancy.
