Daniel E. Bonder, Ph.D. - US grants

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.