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Ca 2+动力学提供了星形胶质细胞内信号传导的主要手段之一。意义 星形胶质细胞Ca 2+信号传导包括与神经元的双向通信和神经元的调控。 通过多种机制发挥作用，包括胶质传递和神经递质转运。反映 星形胶质细胞内Ca ~（2+）的作用多种多样，细胞内Ca ~（2+）存在不同的作用机制和空间结构域 身体和外周过程。然而，尽管有大量证据表明星形胶质细胞 Ca 2+在脑功能和行为的细胞机制中，它是否或如何在很大程度上仍然未知 滥用药物影响星形胶质细胞Ca 2+信号传导。这是一个特别突出的问题， 证据表明，奖励回路中的星形胶质细胞在结构和功能上都受到了长期损害。 此外，现有的Ca 2+监测方法的局限性对评估Ca 2+浓度存在重大障碍。 大鼠脑深部结构星形胶质细胞Ca ~（2+）升高与行为相关面对这一挑战，我们 已经开发了两种新型的AAV，其表达可光转化的比率Ca 2+指示剂CaMPARI 2 在星形胶质细胞特异性GfaABC 1D启动子的控制下，允许不可逆地标记Ca 2+激活的 星形胶质细胞一种变体是胞质的，主要在细胞体中表达，定位于主要报告 体细胞Ca 2+升高;相反，Lck融合变体可以报告整个细胞中的Ca 2+升高。 星形胶质细胞和精细的外周过程中。这两种变体都表现出可靠的、星形胶质细胞限制的 在脑桥核、海马和前额叶皮层中表达，我们已经证实， 在活体切片中Ca ~（2+）刺激下的光转化。本提案的目标是验证这些新的 开发工具来量化星形胶质细胞对切片和体内刺激的Ca 2+反应，并优化 未来研究可设计用于评估与药物相关的星形胶质细胞Ca 2+动力学的参数 自我管理和相关行为。目的1优化星形胶质细胞Ca ~（2+）活体监测的条件 切片对ATP和多巴胺受体激动反应。目标2将验证和优化以下条件： 光刺激结合可卡因给药后大鼠体内钙离子监测 建立了体内CaMPARI 2活化的实验设计。这些研究将告诉如何既急性 和慢性可卡因暴露调节星形胶质细胞中的Ca 2+动力学。在这两种情况下， 光转换将通过红色：绿色荧光比率以及使用 定制的抗CaMPARI 2红抗体。结果将允许对星形胶质细胞钙离子的研究应用 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重评估 表达，并询问星形胶质细胞对各种滥用药物的功能反应， 药物滥用的范例。