Recording Whole-Cell Currents with GABA Puffing in a Mouse Brain Slice

Begin with a secured prefrontal cortical brain slice in an artificial cerebrospinal fluid, or aCSF, in the recording chamber of an electrophysiology setup.

This aCSF contains a sodium ion channel blocker, which inhibits sodium channels in neurons.

Position a puff micropipette filled with gamma-aminobutyric acid, or GABA, above a postsynaptic neuron.

Place an ionic solution-filled recording micropipette with an electrode near the same neuron.

Apply weak suction to create a tight seal with a small patch of the neuronal membrane.

Then, apply strong suction to disrupt the patched membrane, enabling access to the neuron's interior.

Deliver the GABA, an inhibitory neurotransmitter, which binds with a ligand-gated chloride channel on postsynaptic neurons.

This opens the channel and allows chloride ions to enter the neurons, generating an inhibitory postsynaptic current.

At a constant voltage, record the postsynaptic current. An increase in inhibitory postsynaptic current reflects GABA's role in neuronal activity inhibition.

Transfer a brain slice to the recording chamber using a Pasteur pipette with its fine tip cut to fit the size of the slice. Next, place a platinum slice anchor above the slice to hold it on the platform. After that, fill the recording micropipettes with internal solution, and fill the puff micropipettes with GABA at 10 micromolar, 50 micromolar, and 100 micromolar or aCSF as vehicle control.

To prevent blockage with debris, apply slight positive pressure using a one-milliliter syringe before immersing the recording electrode in the aCSF. Under a microscope, position the recording electrode and puff micropipette so that the tips appear in the center of the monitor. Then, identify a target neuron. Adjust the microscope focus while gradually lowering the puff micropipette, and place it above the recording neuron at an angle of 45 degrees.

Keep the distance between the tip of the puff micropipette and the target neuron in the range of 20 to 40 micrometers. Slowly, and carefully, approach the neuron with the recording electrode and then release the positive pressure. Apply a weak and brief suction through the tubing connected to the electrode holder to create a gigaohm seal. Maintain the voltage at zero millivolts.

After the formation of the gigaohm seal, compensate the fast and slow capacitance manually or automatically. Then, apply a brief and strong suction through the tubing to break into the whole-cell mode. Subsequently, record evoked IPSC currents in voltage-clamp mode.

Deliver single or paired GABA puffs through the puff micropipette controlled by a master eight-voltage step generator. Change the puff duration to obtain the best evoked IPSC currents results and measure the evoked IPSC currents.