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的需求，流向活跃脑区的血流量增加，这种现象被称为功能性充血。这种血管反应是血氧水平依赖（BOLD）功能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状况和疾病的标志，其中包括偏头痛、中风和痴呆症的恢复。因此，将神经元活动与血管反应结合起来的精确细胞类型和细胞机制是改善治疗方法和解释功能磁共振成像结果的重要课题。越来越多的证据表明星形胶质细胞通过gq - gpcr介导的Ca2+升高来控制功能性充血。然而，星形细胞gq相关的Ca2+活性与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；技术限制不允许这样做。我们实验室开发的新遗传工具允许在体内选择性刺激或消除星形细胞Gq-GPCR和Ca2+信号，克服这一关键障碍。利用这些基因工具，结合生理视觉刺激

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.