Novel cellular markers of drug-mediated calcium signaling in astrocytes

项目摘要 细胞内钙动态是星形胶质细胞内信号传递的主要途径之一。它的意义 星形胶质细胞钙信号转导包括与神经元的双向通讯和神经调控 通过不同的机制发挥作用，包括神经胶质传递和神经递质运输。反映了 星形胶质细胞钙离子的不同作用，细胞内钙离子有不同的机制和空间域 身体和外周突起。然而，尽管有大量证据表明星形胶质细胞的基本作用 钙离子在大脑功能和行为的细胞机制中，是否或如何仍在很大程度上尚不清楚 滥用药物会影响星形胶质细胞的钙信号。这是一个特别突出的问题，因为 有证据表明，奖赏回路中的星形胶质细胞在结构和功能上都存在慢性损害。 此外，现有的钙离子监测方法的局限性给评估钙离子浓度带来了重大障碍。 大鼠脑深部结构中星形胶质细胞钙升高与行为的关系。面对这一挑战，我们 我开发了两种新型的AAVs，它们表达可光转化的比率钙指示剂CaMPARI2 在星形胶质细胞特异性GfaABC1D启动子的控制下，允许对钙激活的不可逆标记 星形胶质细胞。一种变异体是胞浆的，主要在细胞体中表达，定位于主要报告 体细胞钙升高；相反，Lck融合变异体可以报告整个 星形胶质细胞和细小的外周突起内。这两种变种都显示出可靠的、星形胶质细胞限制的 在伏隔核、海马体和前额叶皮质中表达，我们已经证实 活体切片中钙离子刺激下的光转化。本提案目标是验证这些新功能的使用 开发了量化切片和体内星形胶质细胞对刺激的钙反应的工具，并优化了 未来研究可设计用于评估与药物相关的星形胶质细胞钙动力学的参数 自我管理及相关行为。目标1将优化星形胶质细胞活体钙监测条件 切片对三磷酸腺苷和多巴胺受体激动剂均有反应。目标2将验证和优化以下条件 大鼠在体光刺激联合可卡因给药后的钙离子监测 建立了体内激活CaMPARI2的实验设计。这些研究将告诉我们这两种疾病有多严重 慢性可卡因暴露可调节星形胶质细胞的钙动态。在这两种情况下，CAMPARI2 光转化将通过红色：绿色荧光比率以及免疫组织化学方法进行评估 定制抗CAMPARI2-红色抗体。这一结果将为星形胶质细胞钙离子的研究提供应用 在广泛的细胞领域，以高分辨率成像，并对基因和蛋白质进行多路评估 表达，并询问星形胶质细胞在不同的药物滥用和功能反应 药物滥用范例。

Project Summary Intracellular Ca2+ dynamics provide one of the primary means of signaling within astrocytes. The significance of astrocyte Ca2+ signaling includes bidirectional communication with neurons and governance of neural function through diverse mechanisms including gliotransmission and neurotransmitter transport. Reflecting the diverse roles for astrocyte Ca2+, there are distinct mechanisms and spatial domains of Ca2+ within the cell body and peripheral processes. However, despite the substantial evidence for fundamental roles for astrocyte Ca2+ in the cellular mechanisms of brain function and behavior, it remains largely unknown whether or how drugs of abuse affect astrocyte Ca2+ signaling. This is a particularly salient question, given the increasing evidence that astrocytes within the reward circuitry are chronically impaired both in structure and function. Further, limitations of existing methods of Ca2+ monitoring present significant hurdles toward assessment of astrocyte Ca2+ elevations associated with behavior in deep brain structures in rat. Toward that challenge, we have developed two novel AAVs which express the photoconvertible, ratiometric Ca2+ indicator CaMPARI2 under control of the astrocyte-specific GfaABC1D promoter, to allow irreversible marking of Ca2+-activated astrocytes. One variant is cytosolic and expressed primarily in the cell body, localized to primarily report somatic Ca2+ elevations; in contrast, the Lck-fusion variant can report Ca2+ elevations throughout the astrocyte and within the fine peripheral processes. Both variants demonstrate reliable, astrocyte-restricted expression in the nucleus accumbens, hippocampus and prefrontal cortex, and we have confirmed photoconversion upon Ca2+ stimulation in live slice. The goal of this proposal is to validate use of these newly developed tools to quantify astrocyte Ca2+ responses to stimuli in slice and in vivo, and to optimize parameters for which future studies can be designed to assess astroglial Ca2+ dynamics associated with drug self-administration and related behaviors. Aim 1 will optimize conditions for astrocyte Ca2+ monitoring in live slice in response to both ATP and dopamine receptor agonism. Aim 2 will validate and optimize conditions for Ca2+ monitoring following optical stimulation in vivo coupled with cocaine administration in rat, using an established experimental design for CaMPARI2 activation in vivo. These studies will inform how both acute and chronic cocaine exposure regulate Ca2+ dynamics in astrocytes. In both cases, CaMPARI2 photoconversion will be assessed by red:green fluorescence ratios as well as immunohistochemistry using a custom anti-CaMPARI2-red antibody. Results will allow for applications toward investigation of astrocyte Ca2+ across a broad field of cells, imaged at high resolution and with multiplexed assessment of gene and protein expression, and for interrogation of functional responsiveness of astrocytes across varied drugs of abuse and drug abuse paradigms.