Abstract Neurovascular coupling (NVC) is the temporal relationship between neural activity and cerebral blood flow (CBF). This neural-evoked hemodynamic response is fundamental to local cerebrovascular homeostasis and is disrupted in cerebrovascular diseases, such as stroke, cerebral amyloid angiopathy, traumatic brain injury, as well as Alzheimer's Disease. The neurons that express neuronal nitric oxide synthase (Nos1) are ideal candidates for the regulation of NVC since nitric oxide (NO) is a very potent vasodilator. Our group has recently developed a Tacr1CreER allele that enables the visualization and manipulation of these neurons. We now have exciting preliminary data supporting the hypothesis that Tacr1 neurons mediate vasodilation. Here, we propose to test this idea through a set of experiments that will: determine the relationship between Tacr1 neurons and blood vessels; examine causality in the regulation of NVC by Tacr1 neurons; and investigate the underlying circuitry. These experiments include correlative studies that will establish whether the structure (place) and function (activity) of Tacr1 neurons positions them to regulate CBF. We will also use optogenetic approaches and laser Doppler flowmetry (LDF) to record CBF in awake behaving mice to test whether Tacr1 neurons necessary and sufficient for vasodilation. Finally, we will use a combination of optogenetic manipulation, GCaMP6f-, and 2P-imaging to elucidate the underlying circuitry of NVC. Overall, our proposal will address a critical gap in knowledge with respect to the specific neural mechanisms that underlie the BOLD signal, which is a widely used, but poorly understood research and clinical tool. Moreover, this insight into NVC is fundamental to our understanding of the pathogenesis of common cerebrovascular diseases and the advancement of pharmacotherapeutics targeting cerebral perfusion.