Calcium transients in nNOS neurons underlie distinct phases of the neurovascular response to barrel cortex activation in awake mice

Neuronal nitric oxide (NO) synthase (nNOS), a Ca²⁺ dependent enzyme, is expressed by distinct populations of neocortical neurons. Although neuronal NO is well known to contribute to the blood flow increase evoked by neural activity, the relationships between nNOS neurons activity and vascular responses in the awake state remain unclear. We imaged the barrel cortex in awake, head-fixed mice through a chronically implanted cranial window. The Ca²⁺ indicator GCaMP7f was expressed selectively in nNOS neurons using adenoviral gene transfer in nNOS^cre mice. Air-puffs directed at the contralateral whiskers or spontaneous motion induced Ca²⁺ transients in 30.2 ± 2.2% or 51.6 ± 3.3% of nNOS neurons, respectively, and evoked local arteriolar dilation. The greatest dilatation (14.8 ± 1.1%) occurred when whisking and motion occurred simultaneously. Ca²⁺ transients in individual nNOS neurons and local arteriolar dilation showed various degrees of correlation, which was strongest when the activity of whole nNOS neuron ensemble was examined. We also found that some nNOS neurons became active immediately prior to arteriolar dilation, while others were activated gradually after arteriolar dilatation. Discrete nNOS neuron subsets may contribute either to the initiation or to the maintenance of the vascular response, suggesting a previously unappreciated temporal specificity to the role of NO in neurovascular coupling.