Establishing a Whole-Cell Patch Clamp for Electrophysiological Recording from a Flat-Mount Mouse Retina

Place a flat-mount mouse retina in a physiological solution within a recording chamber.

Using a microscope, position a recording pipette above the retina. The pipette contains an electrode and is filled with an internal solution.

Apply positive pressure to prevent pipette clogging, insert it into the retina, and then reduce the pressure to avoid tissue damage.

Advance the pipette toward the starburst amacrine cells, or SACs, which are synaptically linked to other neurons.

Approach a cell until a dimple forms on its membrane. Release the pressure, allowing the membrane to adhere to the tip.

Use a negative current to draw a membrane patch into the pipette.

Apply suction to rupture the membrane, establishing continuity between the electrode and the cell cytoplasm, a technique called whole-cell patch-clamp.

To record synaptic currents, apply a voltage to keep the membrane potential of the SAC constant.

The neurotransmitters released by synaptically linked neurons trigger ion flow through SAC synaptic receptors, measured by the electrode.

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.