Viral Vector Core

酒精滥用和依赖影响了大约8.5%的美国人口，并导致了 巨大的健康和社会成本。斯克里普斯酒精研究中心的首要目标是 研究所（TSRI-ARC）是了解脆弱性的分子和细胞机制， 酒精依赖，重点是应激反应中兴奋性神经传递的失调 大脑区域，如基底外侧杏仁核（BLA）和终纹床核（BNST）。在 此外，将在停药期间检查成年神经发生。病毒载体核心 通过提供经过验证的工具来实现TSRI-ARC Research Components中提出的实验 操纵基因表达局部和标记新生神经元成年大鼠暴露于模型的狂欢 饮酒或酒精依赖。具体目的1是表征七种腺相关病毒的能力， (AAV)BLA和腹内侧前额叶皮层中的神经元能细胞的假型 （vmPFC），其向BLA和BNST发送兴奋性投射。该目标的结果将用于 选择最适合的AAV假型用于生产特定目的2和3中的定制AAV载体。 具体目的2是提供用于BLA中单酰基甘油脂酶表达的局部沉默的AAV载体， vmPFC，并选择性地敲低BLA神经元中促肾上腺皮质激素释放因子受体1型。 神经元具体目标3是提供用于功能性敲低或神经元特异性表达的AAV载体。 BLA中神经元五聚蛋白2的过度表达。具体目的4是提供一种逆转录病毒载体， EGFP。该载体将标记成年大鼠大脑中的新生神经元，并能够表征其特征。 形态学和生理学。对于每个病毒载体，我们建议设计和克隆DNA构建体，获得 来自外部生产设施的高滴度纯化病毒原液，并验证其沉默/过表达 体内效率总而言之，我们预计病毒载体提供的创新分子工具 核心将有助于获得新的见解过度饮酒的神经生物学机制。

Alcohol abuse and dependence affect an estimated 8.5% of the U.S. population and are responsible for substantial health and societal costs. The overarching goal of the Alcohol Research Center at The Scripps Research Institute (TSRI-ARC) is to understand the molecular and cellular mechanisms of vulnerability to alcohol dependence, with a focus on dysregulation of excitatory neurotransmission in stress-responsive brain regions, such as the basolateral amygdala (BLA) and the bed nucleus of the stria terminalis (BNST). In addition, adult neurogenesis will be examined during withdrawal. The Viral Vector Core will be instrumental in the realization of experiments proposed in TSRI-ARC Research Components by providing validated tools to manipulate gene expression locally and to label newborn neurons in adult rats exposed to models of binge drinking or alcohol dependence. Specific Aim 1 is to characterize the ability of seven adeno-associated virus (AAV) pseudotypes to transduce glutamatergic cells in the BLA and the ventromedial prefrontal cortex (vmPFC), which send excitatory projections to the BLA and BNST. Results from this Aim will be used to select the best-suited AAV pseudotype for the production of custom AAV vectors in Specific Aims 2 and 3. Specific Aim 2 is to provide AAV vectors for local silencing of monoacylglycerol lipase expression in BLA and vmPFC, and for knockdown of corticotropin-releasing factor receptor type 1 selectively in BLA glutamatergic neurons. Specific Aim 3 is to provide AAV vectors for functional knockdown or glutamatergic neuron-specific overexpression of neuronal pentraxin 2 in the BLA. Specific Aim 4 is to provide a retroviral vector expressing EGFP. This vector will label newborn neurons in the adult rat brain and enable characterization of their morphology and physiology. For each viral vector, we propose to design and clone DNA constructs, obtain high-titer, purified viral stocks from an outside production facility and validate their silencing/overexpression efficiency in vivo. Altogether, we anticipate that the innovative molecular tools provided by the Viral Vector Core will assist in gaining novel insights into the neurobiological mechanisms of excessive alcohol drinking.