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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要细胞内 Ca2+ 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 Ca2+ 信号传导包括与神经元的双向通信以及神经元的调控通过不同的机制发挥作用，包括神经胶质细胞传递和神经递质运输。反映了星形胶质细胞 Ca2+ 的作用多种多样，细胞内 Ca2+ 存在不同的机制和空间域身体和外围过程。然而，尽管有大量证据表明星形胶质细胞的基本作用 Ca2+ 在大脑功能和行为的细胞机制中的作用，目前仍不清楚是否或如何滥用药物会影响星形胶质细胞 Ca2+ 信号传导。这是一个特别突出的问题，因为有证据表明，奖赏回路内的星形胶质细胞的结构和功能都长期受损。此外，现有 Ca2+ 监测方法的局限性对评估星形胶质细胞 Ca2+ 升高与大鼠大脑深部结构的行为相关。面对这一挑战，我们开发了两种新型 AAV，可表达光转换、比例 Ca2+ 指示剂 CaMPARI2 在星形胶质细胞特异性 GfaABC1D 启动子的控制下，可对 Ca2+ 激活进行不可逆标记星形胶质细胞。一种变体是细胞质的，主要在细胞体中表达，定位于主要报告体细胞 Ca2+ 升高；相比之下，Lck 融合变体可以报告整个过程中 Ca2+ 的升高。星形胶质细胞和精细外周突起内。两种变体都表现出可靠的、星形胶质细胞限制的在伏隔核、海马和前额皮质中表达，我们已经证实活切片中 Ca2+ 刺激后的光转换。该提案的目标是验证这些新的使用开发了量化星形胶质细胞 Ca2+ 对切片和体内刺激反应的工具，并优化未来研究可以设计的参数来评估与药物相关的星形胶质细胞 Ca2+ 动力学自我管理和相关行为。目标 1 将优化星形胶质细胞 Ca2+ 实时监测的条件切片响应 ATP 和多巴胺受体激动。目标 2 将验证和优化条件大鼠体内光刺激和可卡因给药后的 Ca2+ 监测，使用建立了体内 CaMPARI2 激活的实验设计。这些研究将揭示两者如何急性长期接触可卡因可调节星形胶质细胞中的 Ca2+ 动态。在这两种情况下，CaMPARI2 光转换将通过红：绿荧光比率以及使用免疫组织化学进行评估定制抗 CaMPARI2-red 抗体。结果将允许应用于星形胶质细胞 Ca2+ 的研究跨越广泛的细胞领域，以高分辨率成像并对基因和蛋白质进行多重评估表达，以及询问星形胶质细胞对各种滥用药物和药物的功能反应性药物滥用范式。