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