VII. SCJ version nucleated patch in organotypic slices

Procedure

1. All procedures are the same as those used for whole-cell patch recording.

2. Prepare low-impedance recording pipettes (2–4 MΩ).

3. Stabilize the setup if the table is shaking. (Removing vibration is very important.)

4. Move a slice to the recording chamber.

5. Select a neuron suitable for nucleated patch recording.

   • Thicker slices contain many cells suitable for nucleated patch recordings because they have multiple cell layers.
     Select a neuron located on the surface of the slice.
     This is important because sealing can be disrupted when pulling out the cell body if the neuron is located too deep.

   • Sometimes organotypic slices have dura matter covering the surface.
     This can make the procedure difficult or impossible.
     Gently tease and remove the dura matter locally using recording pipettes
     (move slightly up/down or left/right; do not touch neurons)
     to expose the target neuron.

6. Form a giga-seal and establish whole-cell configuration.

   • Do not attempt a nucleated patch if a giga-seal cannot be achieved.

   • Lower leak current is better for nucleated patch recording.

   • Whole-cell capacitance: 15–20 pF

   • Series resistance: ~10 MΩ

7. Retract the recording pipette 2–3 min after establishing whole-cell configuration.

   • Take sufficient time when retracting the pipette (2–3 min).

   • Monitor the leak waveform on the oscilloscope.

   • Continue retracting the pipette until the leak current reaches ~0 pA.

In the case of outside-out patch recordings, use higher-impedance pipettes (4–6 MΩ).