Novel cellular markers of drug-mediated calcium signaling in astrocytes

星形胶质细胞中药物介导的钙信号传导的新细胞标记物

• 批准号: 10368128
• 负责人: Kathryn Joanna Reissner
• 金额: $ 18.72万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-15 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10368128
• 关键词: ATP Receptors; Acute; Affect; Antibodies; Astrocytes; Basic Science; Behavior; Binding; Brain; Calcium; Calcium Signaling; Cells; Chronic; Cocaine; Code; Communication; Communities; Coupled; Custom; Data; Development; Dopamine Agonists; Dose; Drug Addiction; Drug Exposure; Drug Modelings; Drug abuse; Drug usage; Experimental Designs; Family; Fiber Optics; Fluorescence; Future; Gene Expression; Glare; Glutamates; Goals; Hippocampus (Brain); Illicit Drugs; Image; Immunohistochemistry; Impairment; Investigation; Knowledge; Light; Measures; Mediating; Membrane; Methods; Microscopy; Monitor; Motivation; Neurons; Neurophysiology - biologic function; Neurosciences; Nucleus Accumbens; Optics; Peripheral; Pharmaceutical Preparations; Plasmids; Prefrontal Cortex; Process; Protein Engineering; Public Health; Rattus; Relapse; Reporting; Research; Resolution; Rewards; Rodent; Role; Self Administration; Signal Transduction; Slice; Stimulus; Structure; Substance Use Disorder; Synapses; Time; Ultraviolet Rays; Validation; Variant; Work; addiction; calcium indicator; cellular imaging; cocaine exposure; dependence relapse; design; drug of abuse; in vivo; insight; neuronal cell body; neurotransmitter transport; novel; promoter; protein expression; ratiometric; response; reward circuitry; therapeutic candidate; therapeutic target; tool

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.

项目摘要 细胞内Ca2+动力学提供了星形胶质细胞中信号传导的主要手段之一。意义 星形胶质细胞CA2+信号传导包括与神经元的双向通信和神经治理 通过包括Gliotrassermiss和神经递质转运在内的各种机制的功能。反映 星形胶质细胞CA2+的各种作用，在细胞内有CA2+的不同机制和空间结构域 身体和外围过程。但是，尽管有大量证据表明星形胶质细胞的基本作用 在脑功能和行为的细胞机制中，Ca2+仍然在很大程度上未知 滥用药物会影响星形胶质细胞CA2+信号传导。考虑到不断增加的问题，这是一个特别突出的问题 奖励电路中的星形胶质细胞在结构和功能上长期受损的证据。 此外，CA2+监测现有方法的局限 大鼠深脑结构中与行为相关的星形胶质细胞CA2+升高。面对挑战，我们 已经开发了两个新型的AAV，它们表达了光偏见的比率CA2+指示器CAMPARI2 在星形胶质细胞特异性GFAABC1D启动子的控制下，允许对Ca2+激活的不可逆标记 星形胶质细胞。一种变体是胞质的，主要在细胞体中表达，主要是报告 体细胞Ca2+高程；相比之下，LCK融合变体可以报告整个过程中的Ca2+高程 星形胶质细胞和精细的外围过程。两种变体都表现出可靠的星形胶质细胞限制 在伏隔核，海马和前额叶皮层中的表达，我们已经确认 在活切片中Ca2+刺激上的光转换。该提案的目的是验证使用这些新的 开发了量化星形胶质细胞Ca2+对刺激和体内刺激的响应的工具，并优化 可以设计未来研究以评估与药物相关的星形胶质Ca2+动态的参数 自我管理和相关行为。 AIM 1将优化现场星形胶质细胞CA2+监测的条件 响应于ATP和多巴胺受体激动剂而切片。 AIM 2将验证并优化条件 体内光刺激后的Ca2+监测以及大鼠中可卡因的给药 在体内激活Campari2激活的实验设计。这些研究将告知两者既敏锐 慢性可卡因暴露调节星形胶质细胞中的Ca2+动力学。在这两种情况下，Campari2 光转化将通过红色：绿色荧光比以及免疫组织化学评估 自定义抗碳水化合物2-RED抗体。结果将允许对星形胶质细胞CA2+进行调查的应用 在广阔的细胞领域中，以高分辨率成像，并通过对基因和蛋白质进行多重评估 表达，以及跨各种滥用药物的星形胶质细胞功能反应性的审问 吸毒范式。