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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Whole-cell patch-clamp recordings
- Transfer the first tissue section to the recording chamber submerged in a continuous flow of Krebs solution. Mount the section using light weights and submerge the objective.
- Identify the striatal region in the microscope and search for GFP-positive (reprogrammed) neurons. Select a neuron that is extensive in morphology and not covered by fiber bundles or blood vessels.
- Prepare borosilicate glass pipettes (3–7 MΩ) for patching and fill with the following intracellular solution (in mM): 122.5 potassium gluconate, 12.5 KCl, 0.2 EGTA, 10 HEPES, 2 MgATP, 0.3 Na3GTP and 8 NaCl, adjusted to pH 7.3 with KOH.
- Attach the glass pipette to the recording electrode and dip it into the solution. Double-check the electrode's resistance. Then, slowly approach the cell with the pipette, keeping a slight positive pressure on the electrode to avoid plugging the tip.
CAUTION: Be careful to keep track of your cell while descending the electrode and not to bleach the fluorescence in the cell (i.e., turn off the fluorescence lamp when you do not need it).
- Rinse the surrounding tissue carefully using positive pressure of the electrode and approach the cell with the electrode. Locate the electrode right on top of the cell and descend until the electrode touched the membrane. Make a Giga-Ω seal between the electrode and cell membrane, and with negative pressure pulses, rupture the membrane to create a whole-cell patch.
CAUTION: Reprogrammed neurons are sensitive. Be careful when patching and do not put too much negative pressure when reaching a Giga-Ω seal or opening the cell membrane. Also, patching onto older animals requires practice and patience as their connective tissue is thicker, and it is harder to visualize the neurons.
- Check the resting membrane potentials immediately after breaking in in current-clamp mode and write them down for analysis.
- In the current clamp, maintain the cell between -60 mV to -80 mV and inject 500 ms currents from -20 pA to +90 pA, with 10 pA increments to induce action potentials.