Significance: Neurovascular coupling (NVC) is the process that increases cerebral blood flow in response to neuronal activity. NVC is orchestrated by signaling between neurons, glia, and vascular cells. Elucidating the mechanisms underlying NVC at different vascular segments and in different brain regions is imperative for understanding of brain function and mechanisms of dysfunction.

Aim: Our goal is to describe a protocol for concurrently monitoring stimulation-evoked neuronal activity and resultant vascular responses in acute brain slices.

Approach: We describe a step-by-step protocol that allows the study of endogenous NVC mechanisms engaged by neuronal activity in a controlled, reduced preparation.

Results: This ex vivo NVC assay allows researchers to disentangle the mechanisms regulating the contractile responses of different vascular segments in response to neuronal firing independent of flow and pressure mediated effects from connected vessels. It also enables easy pharmacological manipulations in a simplified, reduced system and can be combined with Ca^2  + imaging or broader electrophysiology techniques to obtain multimodal data during NVC.

Conclusions: The ex vivo NVC assay will facilitate investigations of cellular and molecular mechanisms that give rise to NVC and should serve as a valuable complement to in vivo imaging methods.