Loss of hemodynamic functional connectivity during high arousal does not reflect neuronal uncoupling

Ascending neuromodulatory projections from deep brain nuclei generate internal brain states that differentially engage specific neuronal cell types. Because neurovascular coupling is cell-type specific and neuromodulatory transmitters have vasoactive properties, we hypothesized that the impulse response function (IRF) linking spontaneous neuronal activity with hemodynamics would depend on brain state. To test this hypothesis, we used mesoscopic optical imaging to measure (1) release of neuromodulatory transmitters norepinephrine (NE) or acetylcholine (ACh), (2) Ca2+ activity of local cortical neurons, and (3) changes in hemoglobin concentration and oxygenation across the dorsal surface of cerebral cortex during spontaneous neuronal activity in awake mice. Fluctuations in total hemoglobin (HbT), reflective of dilation dynamics, were well predicted by a weighted sum of positive Ca2+ and negative NE contributions, while ACh signals were largely redundant with Ca2+. The hemodynamic IRF varied in time and depended on the arousal (pupil dilation, whisking) which was captured by NE but not ACh release. In every case, we obtained a good fit for the IRF using a weighted sum of two alpha functions with the coefficients derived from the Ca2+/NE contributions to HbT. During high arousal, the dynamic nature of the IRF resulted in the loss of hemodynamic coherence between cortical regions (known as “functional connectivity” in BOLD fMRI studies) despite coherent behavior of the underlying neuronal Ca2+activity. We conclude that dynamics of the hemodynamic IRF challenge the metric of functional connectivity because the loss of hemodynamic coherence can be falsely interpreted as “functional uncoupling” of the underlying neuronal activity.