Studying the Excitability of Fluorescent Neurons using a Whole-Cell Patch Clamp

Take an immobilized transfected mouse coronal slice in a recording chamber perfused with aCSF.

The slice contains a sparse pyramidal neuron population expressing a target enzyme and a fluorescent protein.

Assemble a recording pipette comprising an intracellular solution and an electrode connected to an amplifier for measuring neuronal signals.

Microscopically identify the target tissue area and locate a fluorescent neuron.

Apply positive pressure to the pipette to prevent clogging and advance it to the target neuron.

The positive pressure causes a slight indentation on the neuronal membrane upon contact.

Release the pressure, forming a tight seal between the membrane and the tip.

Set the holding potential at a constant negative value to stabilize the neuron.

Apply brief negative pressure to rupture the membrane, connecting the cytoplasm to the pipette interior and establishing a whole-cell configuration.

Switch to current-clamp mode and apply positive current pulses, generating action potentials indicative of neuronal excitability.

Transfer a slice to the recording chamber using a Pasteur pipette or a small brush. Hold down the slice with a harp, and perfuse it with ACSF at a rate of 2 milliliters per minute. To patch a GFP-positive neuron, locate the area of interest through the microscope at 10x. Then, find a GFP-positive cell using the 60x objective.

Next, fill the recording electrode with intracellular solution. Subsequently, place a glass pipette in the pipette holder. Afterward, place the pipette tip in the bath and focus on the tip. Once the pipette is in the bath, apply positive pressure through the back pressure control system.

Approach the cell of interest under visual guidance while maintaining back pressure in the pipette. Upon the appearance of a small dimple on the cell surface, release the pressure. At this point, a tight seal with a resistance larger than 1 gigaohm may be formed. Otherwise, apply a light negative pressure to facilitate it.

While the seal is being formed, bring the holding voltage-clamp to negative 60 millivolts. Once the gigaohm seal is formed, apply a pulse of suction to rupture the cell membrane and break into the whole-cell mode. Once it is in whole-cell mode, switch from voltage-clamp to current-clamp mode and start recording.