Investigating astrocytic Gq-GPCR and Ca2+ signaling in functional hyperemia in vi

描述（由申请人提供）：为了跟上神经元代谢需求的步伐，流向活跃脑区的血流量增加，这种现象称为功能性充血。这种血管反应是血氧水平依赖性（BOLD）功能性MRI的基础，是临床诊断和非侵入性脑研究的重要工具。此外，血管功能障碍是许多使人衰弱的病症和疾病的标志，其中包括偏头痛、中风恢复和痴呆。因此，精确的细胞类型和细胞机制，耦合神经元活动的血管反应是非常感兴趣的主题，以改善治疗和解释功能磁共振成像结果的目的。越来越多的证据表明星形胶质细胞通过Gq-GPCR介导的Ca 2+升高来控制功能性充血。然而，星形胶质细胞Gq-连接的Ca 2+活性和血管对生理刺激的反应之间的直接因果关系尚未在体内得到证实;技术限制还不允许它。我们实验室开发的新的遗传工具允许选择性刺激或消除星形胶质细胞Gq-GPCR和Ca 2+信号在体内，克服这一关键障碍。利用这些遗传工具，结合生理视觉刺激 范例和通过颅窗制备的双光子成像，我将检验星形胶质细胞Gq-GPCR和Ca 2+活性介导体内视皮层功能性充血的假设。本提案的第一个目的是评估星形胶质细胞中选择性激活Gq-GPCR信号级联是否足以诱导皮质血流变化。我将使用腺相关病毒（AAV）载体表达基因工程Gq-GPCR，Gq-DREADD，选择性地在视皮层星形胶质细胞。Gq DREADD不响应内源性配体，而是由血脑屏障渗透性化合物氯氮平-N-氧化物（CNO）激活; CNO不激活内源性受体。腹腔内或皮下注射CNO导致AAV注射动物的星形胶质细胞中选择性的Gq-GPCR信号传导和Ca 2+升高。该系统将用于测试星形胶质细胞Gq-GPCR信号传导的选择性激活是否调节体内皮质血流。第二个目的是解决功能性充血中星形胶质细胞Gq连接的Ca 2+的必要性。为了消除星形胶质细胞Gq连接的Ca 2+信号，我将使用IP 3R 2 KO小鼠系。IP 3R 2 KO星形胶质细胞缺乏对Gq激动剂的可观察到的Ca 2+反应，但神经元功能未改变，并且未观察到行为表型。将通过在急性开颅术中注射指示剂染料或通过表达GCaMP 3（一种遗传编码的指示蛋白）和放置慢性抛光强化薄颅骨（PoRTS）窗来体内监测Ca 2+信号;该系统允许多次成像。这些实验的结果应该澄清星形胶质细胞Gq-GPCR和Ca 2+信号在功能性充血中的作用。所描述的技术也可用于探索可能构成这种生理过程或涉及血管功能障碍的病理状态的其他细胞机制。

DESCRIPTION (provided by applicant): To keep pace with neuronal metabolic demand, blood flow to active brain regions is increased, a phenomenon termed functional hyperemia. This vascular response is the basis of blood oxygen level-dependent (BOLD) functional MRI, an important tool for clinical diagnostics and non-invasive brain research. Additionally, vascular dysfunction is a hallmark of numerous debilitating conditions and diseases, among them migraine, recovery from stroke, and dementia. As such, the precise cell types and cellular mechanisms that couple neuronal activity to vascular responses are topics of great interest for the purpose of improving therapeutics and interpreting fMRI results. There is a growing body of evidence that astrocytes control functional hyperemia by means of Gq-GPCR-mediated Ca2+ elevations. However, a direct, causal link between astrocytic Gq-linked Ca2+ activity and vascular responses to physiological stimulation has yet to be demonstrated in vivo; technical limitations have not permitted it. New genetic tools developed by our lab allow for selective stimulation or elimination of astrocytic Gq-GPCR and Ca2+ signaling in vivo, overcoming this critical barrier. Using these genetic tools, in combination with a physiological visual stimulation paradigm and two-photon imaging through cranial window preparations, I will test the hypothesis that astrocytic Gq-GPCR and Ca2+ activity mediate functional hyperemia in visual cortex in vivo. The first Aim of this proposal will assess if activation of Gq-GPCR signaling cascades selectively in astrocytes is sufficient to induce changes in cortical blood flow. I will ue adeno-associated viral (AAV) vectors to express a genetically-engineered Gq-GPCR, the Gq-DREADD, selectively in visual cortical astrocytes. The Gq DREADD does not respond to endogenous ligands and instead is activated by the blood-brain barrier-permeable compound, Clozapine-N-Oxide (CNO); CNO does not activate endogenous receptors. Injection of CNO intraperitoneally or subcutaneously leads to Gq-GPCR signaling and Ca2+ elevations selectively in astrocytes of AAV-injected animals. This system will be used to test if the selectiv activation of astrocytic Gq-GPCR signaling modulates cortical blood flow in vivo. The second Aim addresses the necessity of astrocytic Gq-linked Ca2+ in functional hyperemia. To eliminate astrocytic Gq- linked Ca2+ signals, I will use the IP3R2 KO mouse line. IP3R2 KO astrocytes lack observable Ca2+ responses to Gq agonists, yet neuronal function is unaltered and no behavioral phenotypes have been observed. Ca2+ signals will be monitored in vivo by indicator dye injection in acute craniotomies or by expression of GCaMP3, a genetically-encoded indicator protein, and placement of chronic Polished, Reinforced Thinned Skull (PoRTS) windows; this system allows for multiple imaging sessions. Results of these experiments should clarify the role that astrocytic Gq-GPCR and Ca2+ signaling play in functional hyperemia. The techniques described can also be used to explore other cellular mechanisms that might underlie this physiological process or pathological states involving vascular dysfunction.