Investigating cytoskeletal dynamics in astrocyte structure and cocaine seeking behavior

摘要 可卡因滥用在整个美国都是一个重大的公共卫生问题，因为 自2013年以来，美国与可卡因有关的死亡人数几乎增加了两倍。尽管迫切需要 干预，缺乏FDA批准的治疗可卡因使用障碍的方法。因此，有相当大的 需要对导致复发脆弱性的机制进行调查。最近的进展表明， 可卡因诱导的星形胶质细胞结构适应可能导致复发易感性。星形胶质细胞是 大脑中最丰富的神经胶质细胞，调节各种关键功能，包括突触传递和 可塑性。赖斯纳实验室的研究表明，伏隔核中的星形胶质细胞显著 在可卡因自我给药和灭绝后结构受损。此外，来自我们的初步数据 实验室表明，大鼠长时间给药(LGA，6h/天)后长期戒断(45d) 导致星形胶质细胞体积、表面积和突触共定位显著减少约40%。然而， 推动这些观察的机制尚不清楚。我假设星形胶质细胞的下调 细胞骨架动力学是这些效应的主要贡献者。因此，这项提案将审查如何 长期应用可卡因后星形胶质细胞的细胞骨架动力学改变 禁欲，以及细胞骨架蛋白的操纵如何影响可卡因的寻找。值得注意的是， 肌动蛋白-细胞骨架连接蛋白Ezrin的磷酸化 星形胶质细胞，被急性可卡因暴露下调。因此，这一提议的首要假设是 可卡因和戒断引起的星形胶质细胞结构缺陷是星形胶质细胞改变的结果 细胞骨架动力学，这进一步促进了寻求可卡因的行为，这种行为在下列情况下加剧 长期禁欲。为了验证这一假设，在目标1中，我将测量可卡因自我给药的效果 以及对细胞骨架蛋白的戒断，包括Ezrin，我将使用星形胶质细胞特异的AAV来操纵 Ezrin的表达，以检测其在星形胶质细胞结构中的作用，并进一步在可卡因寻找行为中的作用。在目标2中，我 将使用RiboTag AAV，结合RNAseq以无偏见的方式分离星形胶质细胞特异的mRNAs 测量可卡因和戒断诱导的星形胶质细胞转录组变化的方法可能 助长了寻求可卡因的行为。我将具体分析结果以测量相对表达谱 这些星形细胞mRNAs中的细胞骨架蛋白。这些结果将使我们更好地理解 星形胶质细胞结构功能障碍如何积极促进可卡因滥用和复发。

ABSTRACT Cocaine abuse presents a significant public health concern across the United States, as the number of cocaine-related deaths in the United States has almost tripled since 2013. Despite an urgent need for intervention, an FDA-approved treatment for Cocaine Use Disorder is lacking. Hence, there is a considerable need for investigations into the mechanisms that drive relapse vulnerability. Recent advances indicate that cocaine-induced structural adaptations in astrocytes may contribute to relapse vulnerability. Astrocytes are the most abundant glial cell in the brain and regulate varied critical functions, including synaptic transmission and plasticity. Research in the Reissner lab has revealed that astrocytes in the nucleus accumbens are significantly structurally impaired following cocaine self-administration and extinction. In addition, preliminary data from our lab indicate that rat long-access (LgA, 6h/day) self-administration followed by prolonged abstinence (45d) leads to a significant ~40% decrease in astrocyte volume, surface area, and synaptic colocalization. However, the mechanisms driving these observations are unknown. I hypothesize that downregulation of astrocyte cytoskeletal dynamics is a major contributor to these effects. Accordingly, this proposal will examine how astrocyte cytoskeletal dynamics are altered following chronic cocaine self-administration and prolonged abstinence, and how manipulation of cytoskeletal proteins can influence cocaine seeking. Of note, phosphorylation of ezrin, an actin-cytoskeleton linker protein that is abundantly and preferentially expressed in astrocytes, is downregulated by acute cocaine exposure. Thus, the overarching hypothesis of this proposal is that cocaine and abstinence-induced astrocyte structural deficits result from alterations in astrocyte cytoskeletal dynamics, which further contribute to cocaine-seeking behaviors that are exacerbated following long-term abstinence. To test this hypothesis, in Aim 1 I will measure the effects of cocaine self-administration and abstinence on cytoskeletal proteins, including ezrin, and I will use an astrocyte-specific AAV to manipulate ezrin expression, to examine its role astrocyte structure and further in cocaine-seeking behaviors. In Aim 2, I will use a RiboTag AAV, to isolate astrocyte-specific mRNAs in conjunction with RNAseq as an unbiased approach to measure cocaine and abstinence-induced changes in the astrocyte transcriptome that may contribute to cocaine-seeking behavior. I will specifically analyze results to measure relative expression profiles of cytoskeletal proteins among these astrocyte mRNAs. These results will provide a greater understanding of how dysfunctions in astrocyte structure actively contribute to cocaine abuse and relapse.