Perforated Patch-Clamp Recordings of Inner Ear Sensory Neuron Cell Bodies

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 pipe