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.

In this procedure, prepare the trituration apparatus using a microscope, a camera, and an aluminum stage holding a chamber and micromanipulators. Secure a microsyringe with a pump controller adjacent to the stage and the specimen. Next, completely backfill a trituration pipette with mineral oil, and secure it over the microsyringe piston.

Then, using the microsyringe with pump controller, withdraw about 130 nanoliters of dissociation solution into the pipette while ensuring the absence of air bubbles. Subsequently, place intact arterial segments into one milliliter of dissociation solution with required concentrations of enzymes in a 10-milliliter glass tube.

Incubate at 34 degrees Celsius for 10 to 12 minutes for partial digestion. Following digestion, replace the enzyme solution with 5 milliliters of fresh dissociation solution. Using a 1-milliliter pipette, transfer one segment into the chamber containing dissociation solution at room temperature.

Next, place the pipette into the dissociation solution in the chamber, and position it close to one end of the digested vessel. Set a rate within the range of 2 to 5 nanoliters per second on the pump controller for gentle trituration. While viewing through 100 times to 200 times magnification, withdraw and eject the arterial segment to dissociate smooth muscle cells while producing an endothelial tube.

If necessary, carefully use fine-tipped forceps to separate dissociated adventitia and internal elastic lamina from the endothelial tube. Confirm that all smooth muscle cells are dissociated and that only endothelial cells remain as intact tube.

Using micromanipulators, secure each end of the endothelial tube on the glass coverslip of the superfusion chamber using borosilicate glass pinning pipettes. Wash out the dissociated adventitia and smooth muscle cells from the chamber, and replace the dissociation solution with two millimolar calcium chloride PSS.

Transfer the mobile platform secured to endothelial tube onto the microscope of superfusion and experimental rig. Then, use six clean 50-milliliter reservoirs for continuous delivery of PSS and respective drug solutions during the experiment.

Use the in-line flow control valve to manually set the flow rate as consistent as laminar flow while matching flow feed to vacuum suction. Deliver PSS to the chamber for the superfusion of the endothelial tube for at least five minutes before recording the background data and dye loading.

To measure the membrane potential simultaneously with intracellular calcium concentration, pull a sharp electrode and backfill it with two molar potassium chloride to record from cells loaded with Fura-2 dye.

To study the intercellular coupling via dye transfer, backfill the microelectrode with 0.1% propidium iodide dissolved in two molar potassium chloride. Next, place the electrode over a silver wire coated with chloride in the pipette holder attached to an electrometer head stage secured with a micromanipulator.

Use the micromanipulator to briefly position the tip of the electrode into the flowing PSS in the chamber while viewing through the four times objective. Subsequently, set the resting membrane potential to zero as consistent with grounded bath potential.

If desired, use audible baseline monitors linked to electrometers to associate sound pitch with potential recordings. Afterward, increase magnification to 400 times using a 40 times objective, and position the tip of electrode just over a cell of the endothelial tube.

Adjust the photometric window using the photometry software to focus on about 50 to 80 endothelial cells. Then, gently place the electrode into one of the cells of the endothelial tube using the micromanipulator, and wait at least two minutes for the resting membrane potential to stabilize.

Once resting membrane potential is stable at its expected value, turn on the photomultiplier tube on the fluorescence interface in the absence of light and begin acquisition of the intracellular calcium concentration by exciting Fura-2, alternately, at 340 and 380 nanometers while collecting fluorescence emission at 510 nanometers.

Once simultaneous measurements of membrane potential and intracellular calcium concentration are established, allow about five minutes for the super-fusion of endothelial tube with PSS at a constant laminar flow rate before the application of drugs.

Apply the drug prepared in PSS to the superfusion chamber at a constant flow rate. During drug treatment, measure the membrane potential in the F340/F380 ratio simultaneously. Once the experiment is done, withdraw the electrode from the cell. Then, stop respective recordings of membrane potential and intracellular calcium concentration and save the files for data analysis.