Establishing a Whole-Cell Patch Clamp for Electrophysiological Recording from a Flat-Mount Mouse Retina
Trascrizione
In this procedure, filter the internal solution through a syringe filter into the custom backfill filament. Then, insert the filament into a freshly pulled micropipette, and dispense the internal solution near the tip until the solution covers the silver electrode wire for more than 5 millimeters. Afterward, fasten the micropipette onto an electrode holder with a suction pole, through which pressure inside the electrode can be adjusted by pushing or pulling the plunger of a tube-connected 10-milliliter plastic syringe.
Next, locate the pipette under the objective and bring it down to about 100 micrometers above the retina. Under the current follower mode, use DC offset to zero the standing DC voltage signal. Measure the pipette resistance under the current clamp mode by injecting fixed amplitude square wave currents through the pipette while it is in the bath. Neutralize the difference by turning the R access knob and use the reading on it to calculate pipette resistance.
After that, slowly bring the electrode to about 10 micrometers above the retina. Apply positive pressure to the electrode. Then, watch the reflection change to the pipette tip as it approaches the retina.
Quickly but gently force the pipette into the GCL and reduce the positive pressure immediately. Move the pipette toward a labeled neuron and avoid contacting other neurons, blood vessels, and end feet of Muller cells. Apply more positive pressure if needed to prevent electrode clogging.
Next, position the pipette tip near the labeled neuron until a dimple is visible. Then, release the positive pressure and allow the plasma membrane to bounce back onto the pipette tip. Apply 20 to 120 pico amps negative currents to the pipette to help the formation of a giga-ohm seal.
Allowing the cell membrane to bounce back after releasing the positive pressure is important because an excellent seal formed naturally between the membrane and the pipette opening is important to preserve the cell morphology after recording.
If necessary, apply a gentle suction to pull the plasma membrane into the pipette. Wait five minutes after the seal formation to rupture the cell membrane in order to allow the clearance of the spilled internal solution by superfusion. After the membrane has been ruptured, and while in the current clamp mode, switch on the bridge balance and adjust it using the R-axis knob. Record the excitatory and inhibitory postsynaptic currents in the voltage clamp mode by holding the cell at reversal potentials.
