Simultaneous Measurement of Intracellular Calcium and Membrane Potential in a Mouse Cerebral Endothelium

Take segments of a mouse posterior cerebral artery.

Incubate with an enzyme solution to degrade the tissue matrix, loosening the cells.

Replace the solution with a buffer, then transfer a segment to a recording chamber.

Mechanically dissociate the connective tissue and smooth muscle cell layers, isolating the underlying endothelial tube.

Secure the tube, remove the dissociated layers, and add a physiological buffer.

Place the chamber within a recording system with continuous buffer flow.

Introduce a fluorescent calcium indicator that enters the endothelial cells, then insert a recording electrode into a cell.

Administer a drug that triggers calcium ion influx and induces downstream signaling for calcium release from the endoplasmic reticulum.

The indicator binds to the calcium and, upon excitation, emits fluorescence.

Calcium increase activates calcium-dependent potassium channels, causing potassium outflow and altering the membrane potential.

Simultaneously record changes in intracellular calcium via the fluorescent signal and membrane potential using the electrode.