Simultaneous prenatal alcohol and cannabinoid exposure & offspring corticostriatal neurocircuitry

项目摘要 产前酒精暴露（PAE）是发育障碍的最常见原因，但同时 接触其他精神活性物质可能会加剧不良后果。近期偏好 育龄青年同时使用酒精和大麻以及 协同发育损害的临床前证据，支持将SAC作为一种特异性 残疾的发展根源。尽管有迹象表明，PAE和产前大麻素暴露（PCE） 通过大麻素受体1（CNR1）改变后代的内源性大麻素信号，目前尚不清楚是否 CNR1--一种对健康胎儿神经发育至关重要的受体-受损的潜在相关性 关于SAC值得注意的是，PAE损害来自纹状体投射皮质神经元的CNR1调节的突触传递。 在未用药小鼠中，纹状体CNR1调节活性受损导致运动功能障碍、多动和 在PAE和PCE患者中观察到的觅药行为增加、缺陷。因此， 本研究旨在研究SAC对CNR1相关神经回路和暴露于环境中的行为的影响。 后代我们实验室的初步数据表明，调节纹状体的CNR1相关基因网络 胎儿期急性乙醇暴露改变了突触活性，联合SAC暴露增强了生长 单一药物和无药物暴露的神经干细胞缺陷。总的来说，这些文献和数据 告知我们的中心假设，SAC后代将表现出受损的CNR1连接的突触机制， 纹状体对应于增加的运动缺陷和药物寻求行为。 我们将在以下目标中检验这一假设：具体目标1）评估SAC诱导的基因变化 调节皮质纹状体突触活动。我们将使用整合的RNAseq和ATAC-seq来研究一个预- 与CNR1调节的产前皮质纹状体突触可塑性相关的确定的基因网络 暴露的后代具体目的2）评估SAC是否增加行为缺陷和纹状体蛋白 表情我们将进行一系列行为测定，调查运动功能的天然缺陷， 产前暴露后代的乙醇寻求行为。我们将比较行为缺陷和纹状体 使用夹心ELISA的蛋白质表达，定量CNR1和内源性大麻素配体 花生四烯酰胺和2-花生四烯甘油。产前药物暴露将发生在妊娠第12 - 15天， 皮质纹状体神经元的高峰神经发生，并将包括蒸发乙醇吸入和i. p 施用合成大麻素CP-55940。成功完成此提案将确定具体的 SAC（一种与预防相关但研究不足的产前药物）的机制变化 暴露，并为未来的治疗干预目标提供信息。此外，拟议的实验将 提供一个良好的培训环境的框架，包括来自多个赞助商的指导， 合作者，并将随后通知科学严谨的K99/R00赠款申请。

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, the proposed experiments will provide the framework for an excellent training environment, including mentorship from multiple sponsors and collaborators, and will subsequently inform a scientifically rigorous K99/R00 grant application.