PROJECT SUMMARY Prenatal alcohol exposure (PAE) is the most common cause of developmental disorders, though concurrent exposure to other psychoactive substances may exacerbate adverse outcomes. Recent preference for simultaneous use of alcohol and cannabis (SAC) among young adults of child-bearing age combined with early preclinical evidence for synergistic developmental harm, support a premise for investigating SAC as a specific developmental source of disability. Despite indications that both PAE and prenatal cannabinoid exposure (PCE) alter endocannabinoid signaling through cannabinoid receptor 1 (CNR1) in offspring, it is yet unknown whether impairments to CNR1 – a receptor essential to healthy fetal neurodevelopment – underly impairments associated with SAC. Notably, PAE impairs CNR1-regulated synaptic transmission from striatal-projecting cortical neurons. In drug-naïve mice, impaired striatal CNR1-regulated activity contributes to motor dysfunction, hyperactivity and increased drug-seeking behaviors, deficits observed in humans with PAE and PCE. Therefore, the objective of this proposal is to investigate the impact of SAC on CNR1-associated neural circuits and behaviors in exposed offspring. Preliminary data from our lab have shown that a CNR1-associated gene network that regulates striatal synaptic activity is changed by acute fetal ethanol exposure, and combined SAC exposure augments growth deficits in neural stem cells from single-drug and drug-free exposures. Collectively, the literature and these data inform our central hypothesis that SAC offspring will exhibit impaired CNR1-linked synaptic mechanisms within the striatum corresponding with increased motor deficits and drug-seeking behaviors. We will test this hypothesis in the following aims: Specific Aim 1) To assess SAC-induced changes in genes that regulate corticostriatal synaptic activity. We will use integrative RNAseq and ATAC-seq to investigate a pre- determined gene network associated with CNR1-regulated corticostriatal synaptic plasticity in prenatally exposed offspring. Specific Aim 2) To assess whether SAC augments behavioral deficits and striatal protein expression. We will perform a battery of behavioral assays investigating native deficits in motor function and ethanol-seeking behaviors in prenatally exposed offspring. We will compare behavioral deficits with striatal protein expression using sandwich ELISAs, quantifying CNR1 and endogenous cannabinoid ligands anandamide and 2-arachidonylglycerol. Prenatal drug exposure will occur from gestational days 12-15, a period of peak neurogenesis for corticostriatal neurons, and will incorporate vaporized ethanol inhalation and i.p administration of synthetic cannabinoid CP-55940. Successful completion of this proposal will identify specific mechanistic changes underlying SAC, a translationally-relevant but under-investigated form of prenatal drug exposure, and inform future targets for therapeutic intervention. Furthermore, t...