PROJECT SUMMARY/ABSTRACT Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system and mediates diverse functions including sensory and motor processing, as well as learning and memory. The energetic demand of this excitatory activity is met by a localized increase in blood flow. Although this increase in blood flow is important to support the energy demands of neural tissue and represents the basis of the signal monitored with functional magnetic resonance imaging (fMRI), the mechanism(s) that underlie this effect remain unresolved. Unlike other classical neurotransmitters, that are directly recycled into the presynaptic nerve terminal, most glutamate is cleared into astrocytes. This clearance is mediated by two Na+-dependent transporters, called GLT-1 and GLAST (or EAAT2 and EAAT1, respectively). These transporters are almost exclusively expressed by astrocytes and enriched on fine astrocyte processes near synapses and on astrocyte endfeet. We have recently shown that glutamate transporters, Na+/Ca2+ exchangers, and mitochondria are functionally coupled to one another in astrocyte processes. We provide a strong scientific premise for the hypothesis that increases in blood flow upon neuronal activation are due to glutamate transport into astrocytes. In studies proposed in Specific Aim 1, we will use 2-photon imaging combined with pharmacologic and genetic manipulations to test the hypothesis that glutamate transport and Na+/Ca2+ exchange increase calcium in astrocyte endfeet and that this increase in calcium is necessary for stimulus-evoked increases in arteriole diameter in vivo. Normally, excitatory activity causes an increase in blood flow, but under some circumstances, the response becomes inverted. In Specific Aim 2, we will test the hypothesis that preventing mitochondria from docking in astrocyte processes/endfeet results in exaggerated stimulus-evoked calcium signaling in endfeet and inversion of the neurovascular response. After a stroke, decreases in blood flow extend beyond the occluded vessel. In Specific Aim 3, we will test the hypothesis that focal ischemia results in a loss of mitochondria from astrocyte processes, exaggerated stimulus-evoked calcium signaling in endfeet, and inversion of the neurovascular response in the penumbra These studies will define a novel mechanism by which neuronal activity causes an increase in neuronal blood flow, will define a novel mechanism by which this response inverts, and a determine how these phenomena contribute to dysregulated blood flow observed after stroke.