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

描述(申请人提供)：为了跟上神经元代谢需求，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)功能磁共振成像的基础，是临床诊断和无创脑研究的重要工具。此外，血管功能障碍是许多令人衰弱的疾病和疾病的标志，其中包括偏头痛、中风恢复和痴呆症。因此，准确的细胞类型和将神经元活动与血管反应相结合的细胞机制对于改进治疗和解释功能磁共振结果是非常感兴趣的话题。越来越多的证据表明，星形胶质细胞通过GQ-GPCR介导的钙升高来控制功能性充血。然而，星形胶质细胞GQ相关的钙离子活性和血管对生理刺激的反应之间的直接因果联系尚未在体内得到证实；技术限制还不允许这样做。我们实验室开发的新基因工具可以在体内选择性地刺激或消除星形细胞GQ-GPCR和钙信号，克服这一关键障碍。使用这些基因工具，结合生理视觉刺激 范式和双光子成像通过颅窗准备，我将在活体内验证星形胶质细胞GQ-GPCR和钙离子活性介导视觉皮质功能性充血的假设。这项建议的第一个目的是评估星形胶质细胞中选择性激活GQ-GPCR信号是否足以引起皮质血流量的变化。我将使用腺相关病毒(AAV)载体在视觉皮质星形胶质细胞中选择性地表达基因工程GQ-GPCRGQ-DREADD。GQ DREADD对内源性配体没有反应，而是被血脑屏障通透性化合物氯氮平-N-氧化物(CNO)激活；CNO不激活内源性受体。腹膜或皮下注射CNO可选择性地诱导AAV注射动物星形胶质细胞Gq-GPCR信号转导和钙离子升高。该系统将被用来测试星形胶质细胞GQ-GPCR信号的选择性激活是否在体内调节皮质血流。第二个目的是探讨功能性充血时星形胶质细胞GQ连接的钙离子的必要性。为了消除星形细胞GQ连接的钙信号，我将使用IP3R2 KO鼠系。IP3R2 KO星形胶质细胞对GQ激动剂缺乏明显的钙反应，但神经元功能没有改变，也没有观察到行为表型。通过在急性开颅手术中注入指示剂染料，或通过表达GCaMP3(一种遗传编码的指示蛋白)，以及放置长期抛光的、加固的薄颅骨(PORTS)窗口，将在体内监测Ca2+信号；该系统允许进行多次成像会话。这些实验的结果应该阐明星形胶质细胞GQ-GPCR和钙信号在功能性充血中所起的作用。所描述的技术也可以用来探索其他细胞机制，这些机制可能是这种生理过程或涉及血管功能障碍的病理状态的基础。

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.