VII. SCJ version nucleated patch in organotypic slices

Procedure

1. All procedures are same with the way to make whole cell patch.

2. Prepare the low impedance recording pipettes (2-4 MOhm).

3. Fix it if the table is shaking (Removing vibration is very important).

4. Move a slice to the recording chamber.

5. Select a neuron to be good for nucleated patch.

- Thicker slices have many cell that are so good for making nucleated patches, because they have much layers of cells. Select the neuron located on the surface of slice. This is important because when you pull out the cell body, sealing can be broken if the cell is located deeply.

- Sometimes organotypic slices have the dura matter covering the surface of slice. It makes the procedure more difficult or impossible. Tease and remove the matter locally with recording pipettes (Move it slightly up and down or right and left. Don’t touch neurons) and expose the neuron that you want to record.

6. Make a giga seal and make a whole cell.

- Don’t try to make a nucleated patch if you cannot get a giga sealing

- Less leak current, better for a nucleated patch

- Whole cell cap. 15~20 pF, Series resistance ~ 10

7. Pull out the recording pipette 2 - 3 min after making a whole cell.

- Spend enough time to pull out the pipette (2 – 3 min).

- Check the leak wave in the oscilloscope monitor.

- Pull out the pipette until the leak current becomes ~ 0 pA.

* In the case of outside out patch, use the higher impedance pipettes (6-4 MOhm).