Summary Proper brain function depends on neurovascular coupling: neural activity rapidly increases local blood flow to meet moment-to-moment changes in regional brain energy demand 1 . Neurovascular coupling is the basis for functional brain imaging 2 , and its impairment is implicated in neurodegeneration 1 . The underlying molecular and cellular mechanisms of neurovascular coupling remain poorly understood. The conventional view is that neurons or astrocytes release vasodilatory factors that act directly on smooth muscle cells (SMC) to induce arterial dilation and increase local blood flow 1 . Here, using two-photon microscopy to image neural activity and vascular dynamics simultaneously in the barrel cortex of awake mice under whisker stimulation, we found that arteriolar endothelial cells (aECs) play an active role in mediating neurovascular coupling. We found that aECs, unlike other vascular segments of ECs in the CNS, have abundant caveolae. Acute genetic perturbations that eliminated caveolae in aECs, but not in neighboring SMCs, impaired neurovascular coupling. Strikingly, caveolae function in aECs is independent of the eNOS-mediated nitric oxide (NO) pathway. Ablation of both caveolae and eNOS completely abolished neurovascular coupling, whereas each single mutant exhibited partial impairment, revealing that caveolae-mediated pathway in aECs is a major contributor to neurovascular coupling. Our findings indicate that vasodilation is largely due to ECs that actively relay signals from the CNS to SMCs via a caveolae-dependent pathway.

The mouse cerebral cortex contains neurons that express choline acetyltransferase (ChAT) and are a potential local source of acetylcholine. However, the neurotransmitters released by cortical ChAT+ neurons and their synaptic connectivity are unknown. We show that the nearly all cortical ChAT+ neurons in mice are specialized VIP+ interneurons that release GABA strongly onto other inhibitory interneurons and acetylcholine sparsely onto layer 1 interneurons and other VIP+/ChAT+ interneurons. This differential transmission of ACh and GABA based on the postsynaptic target neuron is reflected in VIP+/ChAT+ interneuron pre-synaptic terminals, as quantitative molecular analysis shows that only a subset of these are specialized to release acetylcholine. In addition, we identify a separate, sparse population of non-VIP ChAT+ neurons in the medial prefrontal cortex with a distinct developmental origin that robustly release acetylcholine in layer 1. These results demonstrate both cortex-region heterogeneity in cortical ChAT+ interneurons and target-specific co-release of acetylcholine and GABA.

Highlights d Mouse S1 and S2 encode overlapping information during a tactile working memory task d Recall responses of previous stimuli are more prevalent in S2 and are relayed to S1 d Category information in S1, but not S2, is necessary for task performance d Network properties of S2 allow task information to persist across behavior states