Light-Evoked Postsynaptic Responses in Neurons of Mouse Retinal Slices
Transcrição
For patch-clamp recordings, prime the perfusion tubes with Ames' medium and allow all the bubbles to pass from the tubing. After making recording pipettes with a glass puller, backfill the tip with pipette solution. Next, use a micropipette filler to fill about one-third of the pipette. Once filled, store each pipette in a moist pipette box.
Next, turn on the equipment for patch-clamp recordings, including the computer, amplifier, CCD camera, and microscope. Working in dark conditions place a retinal slice preparation onto the microscope stage with the aid of an infrared viewer. After it is immobilized, begin continuous perfusion.
Set the perfusion temperature to 33 to 37 degrees Celsius. View the slice surface with a CCD camera. Focus on the target location where the target cell types reside. Select a healthy-looking cell for patch-clamp recording. Once a healthy cell is identified, place a recording pipette in a pipette holder, and advance the pipette to the slice preparation.
When it is close to the slice preparation, find the tip of the pipette with a microscope. Once the pipette tip is visible under the microscope, move the tip down towards the target cell. Next, set the amplifier, and adjust the pipette at picoamp 5 volts per nanoamp. Start two continuous electric pulse of approximately 5 millivolts and approximately 10 hertz and check the pipette resistance.
An ideal resistance is between 5 and 12 megaohms for most retinal neurons. When ready, use a mouthpiece or syringe to blow out the internal solution, until the tip of the pipette is on the surface of the target cell. When the positive pressure makes a small dimple on the cell surface, advance the tip slightly and stop blowing out.
If the pipette resistance is continuously increasing, leave it and monitor the resistance until it reaches greater than 1 gigaohm. If the resistance does not spontaneously increase, gently apply negative pressure until it becomes a gigaseal. After the gigaseal is achieved, change the holding potential to negative 70 millivolts.
Then, intermittently apply negative pressure to rupture the membrane inside the pipette tip. When the whole-cell configuration is made, the pipette resistance can be between 500 megaohms and one gigaohm, and the capacitive current is observable. Record the I-V relationship from negative 80 to 40 millivolts. Different types of voltage-gated channels will be activated depending on the cell type. Lastly, record light-evoked synaptic currents or voltages.
