This article details a protocol for performing targeted patch-clamp recordings from fluorescent protein-expressing neurons in brain slices. The method involves precise alignment and insertion of a multichannel probe into the tissue to obtain electrical recordings.
Start by perfusing an aerated artificial cerebrospinal fluid or aCSF over a mesh grid in a perfusion chamber.
Transfer a brain slice containing fluorescent protein-expressing neurons and anchor it with a platinum-wired device.
Rotate the mesh grid for alignment.
Lower the multichannel probe toward the slice.
Adjust the filter cube for fluorescent protein visualization.
Rotate the slice to align the probe.
Adjust the probe height below the tissue surface.
Now, perfuse the aCSF and focus on the fluorescently labeled neurons.
Using a high-power objective, focus on the tissue and adjust the excitation light.
Use an appropriate light filter to observe fluorescence in neurons.
Adjust the objective lens to create space for the patch pipette and apply positive pressure.
Next, position the pipette near the neuron to form a membrane dimple.
Finally, apply weak suction to create a membrane seal, followed by strong suction to break the membrane and obtain electrical recordings from the targeted neuron.
To place the multi-channel probe in the ex vivo brain tissue slide, perfuse bubbled experimental ACSF at three to six milliliters per minute, and transfer the brain slice containing the area of interest onto the mesh grid in the microscope perfusion chamber. Anchor the brain slice with a platinum harp. Rotate the mesh grid so that the line of electrode contacts on the distal end of the multichannel probe is approximately perpendicular to the PL surface.
Under broad-field illumination and fine control of the micromanipulator, lower the multichannel probe toward the surface of the slice. Engage the appropriate filter cube for visualization of the fluorescent reporter protein expressed in axon terminals of cortical afferents.
If necessary, rotate the slice to more precisely align the probe with the PL surface. Position the probe just above the plane of the slice, 200 micrometers short of the final target position along the x-axis, leaving at least one channel outside of the area of tissue being recorded. Slowly, insert the probe into the slice along its longitudinal axis.
To minimize damage to the tissue, only advance the probe to the extent that the sharp tips are just visible below the tissue surface. Switch experimental ACSF source to bagged control solution, and identify the fluorescently labeled cell for targeted patch-clamp recording. Restrict the aperture to the smallest diameter, and engage the high-power water-immersion objective, taking care to avoid contact between the multichannel probe and objective lens. Bring the tissue into focus.
Center the light over an area of tissue adjacent to, but not overlapping the multi-channel probe. Engage the appropriate filter set to allow imaging of cells expressing the Cre-dependent fluorescent marker. Raise the objective lens to create ample space for lowering a patch pipette.
Load a patch pipette with internal solution and mounted on the electrode holder. Use a one-milliliter syringe to apply positive pressure corresponding to approximately 0.1 milliliter air. Lower the patch pipette into the solution, bringing the tip into focus under visual guidance, and obtain whole-cell recording from the targeted cell.
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