Functional consequences of cocaine self-administration on astrocytes

项目总结/摘要 越来越多的证据已经确定，神经核星形胶质细胞是药物滥用的突出目标。为 例如，许多研究表明，大鼠自我给药多种药物类别导致受损的 谷氨酸稳态，以及减少的结构特征和突触共定位的核 星形胶质细胞。这项提议的初步数据显示，雄性大鼠海马星形胶质细胞表现出 在10天的长- 进入（6h/d）可卡因自我给药。这些发现表明星形胶质细胞的生理功能丧失 作为神经回路的调节器。然而，驱动这些观察结果的机制尚不清楚， 对神经功能的影响我们假设星形胶质细胞病理学强调了神经系统的 适应和增加的措施，观察到长期禁欲的渴望（也称为 孵育）。因此，本提案的目标是确定可卡因自身与 给药和脑桥核星形胶质细胞功能障碍的发生，以及 星形胶质细胞功能障碍、突触传递和行为之间的联系。目标1将评估操作性 可卡因自身给药对诱发的Ca2+反应的影响， 并将确定星形胶质细胞Ca2+对可卡因的功能意义 寻求行为。目的2将研究可卡因自我管理如何影响星形胶质细胞的能力， 负性调节D1和D2受体阳性中型棘神经元的兴奋性突触传递。 最后，目标3将建立多巴胺和G蛋白介导的星形胶质细胞信号传导在病理性星形胶质细胞损伤中的作用。 可卡因对大鼠星形胶质细胞结构的影响。对于每个目标，我们将根据以下因素评估终点： 穿越禁欲的时间这些目标将通过利用多模式 方法，跨越细胞生理学电路分析和动物行为，结合大鼠可卡因自我， 施用荧光标记的星形胶质细胞的高分辨率成像，Ca2 +-Ca2+的急性切片成像， 使用光可转换Ca2+指示剂（astro-CaMPARI2）标记的星形胶质细胞，星形胶质细胞DREADD刺激， 使用星形胶质细胞表达的PMCA 2 Ca 2+泵，全细胞膜片钳， 电生理学，使用Drd1a-Cre和Drd2-Cre转基因大鼠的神经元亚型特异性分析，以及 腺苷生物传感器GRABAdo的荧光成像。总的来说，这些研究将确定 可卡因自我给药戒断后星形胶质细胞功能障碍与神经生理学之间的关系， 行为

Project Summary/Abstract Accumulating evidence has identified nucleus accumbens astrocytes as salient targets of drug abuse. For example, numerous studies indicate that rat self-administration of multiple drug classes results in impaired glutamate homeostasis, as well as decreased structural features and synaptic colocalization of nucleus accumbens astrocytes. Preliminary data for this proposal reveal that male rat accumbens astrocytes exhibit striking (~40%) reductions in surface area, volume, and synaptic colocalization 45 days after 10 days of long- access (6h/d) cocaine self-administration. These findings suggest loss of physiological function of astrocytes as regulators of neural circuits. However, the mechanism(s) driving these observations are unknown, as are the consequences to neural function. We hypothesize that astrocyte pathology underscores the neural adaptations and increased measures of craving observed across prolonged abstinence (also known as incubation). Accordingly, the goal of this proposal is to define the relationship between cocaine self- administration and the onset of astrocyte dysfunction in the nucleus accumbens, as well as the relationship between astrocyte dysfunction, synaptic transmission, and behavior. Aim 1 will assess the effects of operant cocaine self-administration on evoked Ca2+ responses in accumbens astrocytes as a function of both self- administration and abstinence, and will determine the functional significance of astrocyte Ca2+ to cocaine seeking behaviors. Aim 2 will examine how cocaine self-administration affects the ability of astrocytes to negatively regulate excitatory synaptic transmission in D1 and D2 receptor-positive medium spiny neurons. Lastly, Aim 3 will establish the role of dopamine and G protein-mediated astrocyte signaling in the pathological effects of cocaine on accumbens astrocyte structure. For each aim, we will evaluate endpoints as a function of time across abstinence. These objectives will collectively be accomplished by leveraging a multimodal approach that spans cellular physiology to circuit analysis and animal behavior, combining rat cocaine self- administration with high-resolution imaging of fluorescently-labeled astrocytes, acute slice imaging of Ca2+- labeled astrocytes using a photoconvertible Ca2+ indicator (astro-CaMPARI2), astrocyte DREADD stimulation, astrocyte Ca2+ depletion using an astrocyte-expressed PMCA2 Ca2+ pump, whole cell patch-clamp electrophysiology, neuronal subtype-specific analyses using Drd1a-Cre and Drd2-Cre transgenic rats, and fluorescent imaging of an adenosine biosensor GRABAdo. Collectively, these studies will define the relationship between astrocyte dysfunction across abstinence from cocaine self-administration with neural physiology and behavior.