This article describes a method for recording electrical activity from Drosophila photoreceptor cells using a patch clamp technique. The procedure involves creating a high-resistance seal and establishing a whole-cell configuration to measure transient current signals.
Take a Drosophila ommatidium in a recording chamber perfused with an extracellular solution containing calcium ions.
The ommatidium comprises a photoreceptor cell array, which processes visual information.
Assemble a patch pipette comprising an intracellular solution and an electrode connected to an amplifier.
Apply positive pressure to the pipette and advance it perpendicular to the ommatidium's long axis.
The tip forms a dimple on the photoreceptor membrane upon contact.
Calibrate the amplifier parameters for accurate measurements.
Apply negative pressure to form a high-resistance seal.
Then, apply negative pressure pulses to establish a whole-cell configuration.
Set the patched photoreceptor's holding potential to a constant negative value, stabilizing the cell.
Ensure electrical isolation and optimal darkness for accurate measurements.
Apply brief light pulses, triggering signaling pathways, opening the photoreceptor's transient receptor potential channels.
The resulting cation influx causes a transient current signal called a quantum bump, which is recorded.
Choose a suitable ommatidium for the experiment. Select one that meets the following criteria, the outer membrane must be smooth and intact. The long axis must be nearly at a right angle relative to the electrodes' approach from the distal side, and the ommatidium should not be surrounded by any excess tissue. Once selected, center the ommatidium in the field of vision.
Now, fill a pipette with the appropriate intracellular solution for the experiment, and mount the pipette to the electrode holder. Then, blow into the pipette tube to create some positive pressure, and lock the pressure in by closing the tube valve. Next, insert the pipette into the bath chamber, and guide it close to the distal section of the ommatidium, until a small dimple is observed in the membrane, which is due to the positive pressure from the patch pipette.
Now, open the membrane test module in the recording software to apply continuous square voltage pulses of 2 millivolts at a rate of 100 hertz. Next, using the patch clamp amplifier controls, set the junction potential to 0 to set the base of the square pulse to 0 current. Then, open the valve to release the pressure in the pipette, and gently aspirate to create negative pressure. The pipette should then attach to the cell membrane.
Once this occurs, close the valve to lock in the pressure. Now, check the electrode resistance; it should be elevated to between 100 and 150 mega ohms. Then, release the negative pressure in the pipette by opening the valve again. The resistance should now leap up to between 1 and 2 gigaohms, which indicates a seal has been formed between the pipette and the cell.
Next, using the patch clamp amplifier, offset the capacitive currents of the pipette. To make the whole-cell configuration, break the cell membrane using mouth aspiration. Create rapid, short forceful bouts of negative pressure in the electrode. Upon obtaining the whole-cell configuration, a sudden increase in the capacitance will occur.
Then, using the patch clamp amplifier, set the holding potential to the required voltage and offset the capacitive currents in series resistance. Now, close the black front curtain of the Faraday cage and begin the experiment.
繁體中文
