This article details the process of using patch-clamp techniques to study inner ear sensory neurons. It outlines the steps for preparing the pipette and establishing a seal with the neuronal membrane to record ionic currents.
Place a culture of inner ear sensory neuron cell bodies in a recording chamber.
Front-fill a patch-clamp pipette with an internal solution.
Backfill with the same solution containing a membrane-perforating antibiotic, allowing it to gradually reach the tip.
Fill two-thirds of the pipette with the antibiotic solution, insert it into a holder containing a recording electrode, and lower it into the chamber.
Visualize under a microscope to approach a neuron.
As the pipette tip contacts the neuron, the neuronal surface forms a dimple, and the resistance increases.
Apply negative pressure to form a tight seal between the pipette and the membrane.
Maintain the holding potential close to the resting membrane potential.
The antibiotic perforates the membrane, allowing ion movement between the cell and electrode to establish a stable ionic environment.
Apply voltage steps to open voltage-gated ion channels, allowing ion influx and generating currents proportional to the ion channel function, which are recorded by the electrode.
Dip the tip of the pipette into the clean solution of the culture dish, to fill it with a clean solution that does not contain amphotericin. Fill the back of the pipette with the internal solution containing amphotericin. Dip the tip of the pipette back into the clean solution of the culture dish while the amphotericin slowly enters the tip from the back of the pipette. Next, finish filling the pipette with the amphotericin solution up to 2/3rds of the pipette.
Lower the pipette into the bath of the recording chamber, and locate the pipette tip in the middle of the field of view. Ensure that the tip is free of air bubbles or other debris. Position the pipette close to the membrane, and land firmly on the center of the cell. Apply negative pressure or suction using a syringe or mouth pipette.
Turn on the holding potential of minus 60 millivolts. The seal resistance should increase until it passes a gigaohm. Once a gigaohm seal forms, release the negative pressure. Amphotericin begins to work. The input resistance slowly decreases, and the current flowing in response to the 5 millivolts voltage step progressively increases as the amphotericin enters the membrane.
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