TITLE: Perforated patch electrophysiology recordings
AUTHORS: enrico.zampese

[DESCRIPTION]
In this protocol we detail the steps to perform ex-vivo brain slices perforated patch electrophysiology recordings.
The protocol is originally meant to record from striatal MSNs, but can be adapted to other cell types.

[BEFORE_START]
Please refer to existing protocols to obtain brain slices for electrophysiology experiments.

[GUIDELINES]
Please adhere to institutional guidelines.

[STEPS]
SECTION: Prepare patch pipettes
1. Turn on the Sutter P-1000 puller and enter the desired pull protocol.

SECTION: Prepare patch pipettes
2. Insert a thick-walled borosilicate glass capillary and press pull.
SECTION: Prepare patch pipettes
3. Pipette resistance must be of 3 to 6 megaohms.
SECTION: Setting up patch rig and environment
4. Turn on the MultiClamp 700B Amplifier, Axon Digidata 1550B digitizer, micromanipulator, computer tower and the associated software. Note: amplifier and digitizer must be turned on prior to opening software.
SECTION: Setting up patch rig and environment
5. Turn on O2/CO2 tank and bubble aCSF solution.
SECTION: Setting up patch rig and environment
8. Collect the internal solution with gramicidin in a clean syringe and attach a microfil flexible needle (or alternative electrode filling method).

SECTION: Setting up patch rig and environment
9. Start circuling experiemental aCSF through chamber, either using a gravity flow system or a peristaltic pump (a gravity flow system is recommended because of the reduced electrical noise).

SECTION: Setting up patch rig and environment
10. Turn on water heater and set to desired temperature (~34 °C or Room temperature )

SECTION: Examine slices and patching cells
11. Transfer brain slice from holding chamber to the recording chamber.
SECTION: Examine slices and patching cells
12. Secure down slice with a harp (slice anchor).
SECTION: Examine slices and patching cells
13. Locate and focus the desired brain region under the 4x objective.

SECTION: Examine slices and patching cells
14. Change the microscope lens to the 60x objective.
SECTION: Examine slices and patching cells
15. Focus on healthy neurons in slices for patching.
SECTION: Examine slices and patching cells
16. Front-fill the patch electrode with a small volume of gramicidin-free internal solution (possibly filling only the tip of the pipette - smaller volumes will result in faster achievement of perforated patch configuration, larger volumes will allow more time to patch cell, since once gramicidin at the tip of the electrode will compromise the ability to obtain a seal).
SECTION: Examine slices and patching cells
17. Gently place the glass micropipette onto the wire electrode and tighten.
SECTION: Examine slices and patching cells
18. Position the electrode using a micromanipulator.
SECTION: Examine slices and patching cells
19. Under the 60x objective, bring the tip of the glass pipette above the slice.
SECTION: Examine slices and patching cells
20. Apply a positive pressure and maintain it.
SECTION: Examine slices and patching cells
21. Approach the cell diagonally. The positive pressure should create a small dimple on the cell.
SECTION: Examine slices and patching cells
22. Once a dimple is formed, release the positive pressure, and apply a small amount of negative pressure. The resistance should begin to increase rapidly.
SECTION: Examine slices and patching cells
23. As the resistance increases, clamp the cell at your resting potential of interest (typically - 60 mV).
SECTION: Examine slices and patching cells
24. After a giga-ohm seal is formed, monitor series resistance until <100MOhm and stabilized - usually 20-30 minutes).
SECTION: Examine slices and patching cells
25. Start recording.
SECTION: Setting up patch rig and environment
6. Filter the internal solution containing Alexa dye in a clean eppendorf tube. Save a small amount of internal solution with Alexa dye in a separate tube or in the syringe.
SECTION: Setting up patch rig and environment
7. Add appropriate amount of gramycidin solution to the desired final concentration (e.g. 10 microM, but the protocol might be optimized based on specific needs).
SECTION: Examine slices and patching cells
16.1. Gently back fill the electrode with internal solution containing gramicidin. Avoid formation of bubbles, but move carefully to avoid mixing the two solutions too quickly.
SECTION: After the recording
26. If Alexa dye was added to the internal solution, a fluorescent light source can be used to verify that the Alexa signal is not detected inside the patch cell.
SECTION: After the recording
27. Gently remove electrode.
SECTION: After the recording
28. Remove and discard slice (unless further processing/fixation is needed)
SECTION: After the recording
29. Rinse tubing and microscope chamber.
SECTION: After the recording
30. Save/export files
SECTION: After the recording
31. Shut down the system, dispose of waste according to guidelines, clean work station.