Novel tools for spatiotemporal modulation of astrocytes in neuronal circuits

星形胶质细胞是大脑中的一类主要的非神经细胞，它与突触处的神经元相互作用。 而电路水平仍然知之甚少。而在体内，双光子显微镜揭示了时空 细胞内钙瞬变的不同星形细胞特征，操纵基因的工具的稀缺 星形胶质细胞在体内具有时空精确度的组成和生理活性受到限制 它们对神经元的生理影响的研究主要是相关研究。在这里，我们建议 开发三种相互独立的新工具，针对星形胶质细胞的三个关键功能：基因 表达、细胞内信号转导和谷氨酸摄取。在目标1中，我们将开发CRISPR/CAS9- 基于平台同时选择性地敲除星形胶质细胞中的多个基因。当代小鼠星形细胞 基因消融研究依赖于少量的Cre-loxP重组酶转基因株系，它们只针对一个 单基因，往往缺乏时间和空间控制。我们建议创造一种新的星形胶质细胞特异性， 时间可诱导的CRISPR/Cas9条件转基因小鼠模型，具有创新的病毒平台 使用单个病毒多gRNA、Cys4介导的通用靶向系统消融多个基因 (MRCUTS)。我们将在培养的星形胶质细胞中应用这个系统来靶向ITPR2和Adra1a/b基因(目标1a)。 验证该工具并将其有效性与当前的Cre-loxP方法(目标1b)进行比较，并探索新的功能角色 应用MRCUTS同时消融两种亚型去甲肾上腺素受体对觉醒中星形胶质细胞的影响 (议程1a/b)(目标1c)。在目标2中，我们将开发一种光遗传激活G蛋白信号的方法 星形胶质细胞中的级联反应。目前调节星形胶质细胞信号的方法，如DREADDS，缺乏时间性 精确度。我们将开发和表征光遗传激活的G蛋白受体(opto-xr)的用途。 在星形胶质细胞中探索生理相关时间尺度上的星形胶质细胞信号转导，首先是体外(目标2a)， 然后在体内使用双光子显微镜测量星形胶质细胞的钙动力学(Aim 2b)，随后 用OPTO-XR研究星形胶质细胞G蛋白信号转导对神经元生理学的影响 结合星形胶质细胞-神经元双钙显像(Aim 2c)。在目标3中，我们将开发一种体内方法 光基因干扰星形胶质细胞对谷氨酸的摄取。筛选ChR2的突变，并将四个 突变，导致光门离子通道，ChromeQ，它在 钙和质子电导，同时增加钠电流。我们将记录急性脑中的星形胶质细胞 切片以参数化光基因激活的钠电流并确定对两个星形胶质细胞的影响 转运蛋白电流和附近神经元(目标3a)，研究通过chromeQ干扰谷氨酸摄取如何影响 星形胶质细胞内钙动力学和神经元反应特性(Aim 3b)，并探讨 ChromeQ关于神经元生理学和运动学习(目标3c)。这里提出的工具将使更深层次的 了解星形胶质细胞-神经元在正常脑功能中的串扰及其在脑部疾病中的干扰。

Astrocytes are a major class of non-neuronal cells in the brain whose crosstalk with neurons at the synaptic and circuit levels remains poorly understood. While in vivo two-photon microscopy has revealed spatiotemporally diverse astrocytic signatures of intracellular Ca2+ transients, the scarcity of tools that manipulate the genetic makeup and physiological activity of astrocytes with spatial and temporal precision in vivo has restricted investigation of their physiological impact on neurons to predominantly correlational studies. Here, we propose developing three novel and mutually independent tools that target three crucial functions of astrocytes: gene expression, intracellular signal transduction, and glutamate uptake. In Aim 1, we will develop a CRISPR/Cas9- based platform to simultaneously knockout multiple genes selectively in astrocytes. Current mouse astrocytic gene ablation studies rely on a small number of Cre-LoxP recombinase transgenic lines, which target only a single gene and often lack temporal and spatial control. We propose creating a novel astrocyte-specific, temporally inducible, CRISPR/Cas9 conditional transgenic mouse model with an innovative viral platform for ablating multiple genes using a single virus Multi-gRNA, Cys4-mediated, Universal Targeting System (MRCUTS). We will apply this system in cultured astrocytes to target the Itpr2 and Adra1a/b genes (aim 1a), validate the tool and compare its efficacy to current Cre-LoxP methods (aim 1b), and probe a new functional role of astrocytes in arousal by using MRCUTS to simultaneously ablate two subtypes of noradrenergic receptors (Adra1a/b) (aim 1c). In Aim 2, we will develop a method for optogenetically activating G-protein signaling cascades in astrocytes. Current methods for modulating astrocyte signaling, such as DREADDs, lack temporal precision. We will develop and characterize the use of optogenetically activated G-protein receptors (opto-XR) in astrocytes to probe astrocyte signal transduction on physiologically-relevant timescales, first in vitro (aim 2a), then in vivo using 2-photon microscopy to measure astrocyte calcium dynamics (aim 2b), and subsequently explore the effects of astrocytic G-protein signal transduction on neuronal physiology using opto-XR in conjunction with astrocyte-neuron dual-calcium imaging (aim 2c). In Aim 3, we will develop an in vivo method for optogenetically disrupting glutamate uptake by astrocytes. Screening for mutations in ChR2, and combining four mutations, results in a light-gated ion channel, ChromeQ that possesses order-of-magnitude reductions in calcium and proton conductance while increasing sodium currents. We will record from astrocytes in acute brain slices to parameterize optogenetically activated sodium currents and determine effects on both astrocyte transporter currents and nearby neurons (aim 3a), examine how disrupting glutamate uptake via chromeQ affects astrocyte calcium dynamics and neuronal response properties in vivo (aim 3b), and explore the effects of ChromeQ on neuronal physiology and motor learning (aim 3c). The tools proposed here will enable a deeper understanding of astrocyte-neuron crosstalk in normal brain function and its disruption in brain disorders.