Spatio Temporal INactivation of Gliotransmission

 描述（由申请人提供）：神经科学的一个主要目标是了解大脑中的细胞如何相互交流。星形胶质细胞是一种神经胶质细胞，可以感知突触处的神经元活动，并反过来释放递质（胶质传递）到神经元上以调节突触传递。将开发一种光遗传学方法，该方法使用光来特异性地抑制囊泡释放所需的蛋白质（VAMP 2或VAMP 3）的功能。该工具被称为STING（Gliotransmission的时空失活），将用于确定星形胶质细胞D-丝氨酸在调节NMDAR（N-甲基D-天冬氨酸受体）功能和突触可塑性中的作用。D-丝氨酸作为NMDAR的内源性共激动剂，对于诱导长时程增强是至关重要的，长时程增强是一种突触强化形式，被认为是学习和记忆的细胞基础。我们假设，当大脑中的D-丝氨酸水平在清醒时达到峰值时，突触可塑性增强。我们将使用STING来检测星形胶质细胞中D-丝氨酸的释放，以确定对突触的影响。STING是通过将miniSOG（一种光敏单线态氧发生器）融合到囊泡释放的关键介体来实现的。功能丧失是通过用480 nm光触发miniSOG释放单线态氧来实现的，单线态氧氧化氨基酸残基并使蛋白质功能失活。我们将验证miniSOG-VAMP 2/3在腺相关病毒（AAV）转导后在脑中表达。我们将在海马切片中进行电生理学，并评估STING如何影响神经胶质递质D-丝氨酸的释放。鉴于D-丝氨酸水平以警戒状态（清醒与睡眠）依赖性方式在一天中振荡，我们将在清醒期间和睡眠期间使用STING来检查D-丝氨酸对突触可塑性的贡献。目标1：神经胶质传递的时空失活（STING）：确定miniSOG-VAMP 2/3构建体的细胞和亚细胞定位。目的2：证明miniSOG-VAMP 2/3在神经元或星形胶质细胞中的失活影响突触传递。目标3：确定星形胶质细胞对突触可塑性的贡献使用STING，我们将获得对D-丝氨酸对突触可塑性的星形胶质细胞贡献的有价值的见解，这与精神分裂症具有主要的翻译相关性，其中存在NMDAR功能减退。改变星形胶质细胞胶质传递可能是调节NMDAR的可行途径，这是这种神经精神疾病的主要治疗靶点。实现光控功能丧失将为神经回路和行为中的蛋白质功能分析提供前所未有的控制，并可广泛应用于生物医学科学的许多领域。完成拟议的研究将准备申请人作为一个成功的职业生涯在该领域的神经胶质生物学的独立研究人员。

 DESCRIPTION (provided by applicant): A major goal of neuroscience is to understand how cells in the brain communicate with each other. Astrocytes, a type of glial cell, can perceive neuronal activity at synapses and in return release transmitters (gliotransmission) onto neurons to modulate synaptic transmission. An optogenetic approach will be developed that uses light to specifically inactivate the function of a protein required for vesicular release, either VAMP2 or VAMP3. This tool is called STING (Spatio-Temporal INactivation of Gliotransmission) and will be used to determine the role of astrocytic D-serine in modulating NMDAR (N-methyl D-aspartate receptor) function and synaptic plasticity. D-serine, which acts as the endogenous co-agonist of the NMDAR, is critical for inducing long-term potentiation, a form of synaptic strengthening thought to be the cellular basis of learning and memory. We hypothesize that as D-serine levels in the brain peak during wakefulness, synaptic plasticity is heightened. We will use STING to inactivate the release of D-serine from astrocytes to determine the effects on synapses. STING is achieved by fusing miniSOG, a light-sensitive singlet oxygen generator to a critical mediator of vesicle release. Loss of function is achieved by triggering miniSOG with 480nm light to release singlet oxygen which oxidizes amino acid residues and inactivates protein function. We will validate that miniSOG-VAMP2/3 is expressed in the brain following adeno-associated virus (AAV) transduction. We will perform electrophysiology in hippocampal slices, and assess how STING affects the release of the gliotransmitter D-serine. Given that D- serine levels oscillate throughout the day in a vigilant-state (wake versus sleep)-dependent manner, we will examine the contribution of D-serine to synaptic plasticity using STING during wakefulness and during sleep. Aim 1: Spatio-Temporal INactivation of Gliotransmission (STING): Determining the cellular and subcellular localization of the miniSOG-VAMP2/3 construct. Aim 2: Demonstrate that inactivation of miniSOG-VAMP2/3 in either neurons or astrocytes affects synaptic transmission. Aim 3: Determine the astrocytic contribution to synaptic plasticity Using STING, we will gain valuable insight into the astrocytic contribution of D-serine to synaptic plasticity, which has major translational relevance to schizophrenia, where there is a hypofunction of NMDARs. Altering astrocytic gliotransmission may be a viable pathway for modulating NMDARs, which are a major therapeutic target for this neuropsychiatric disease. Achieving light-controlled loss of function will provide unprecedented control in the analysis of protein function in neural circuits and behavior, and can be broadly applied across many fields in biomedical sciences. Completion of the proposed studies will prepare the applicant for a successful career as an independent researcher in the field glial biology.