Perforated patch electrophysiology recordings
Enrico Zampese
Abstract
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.
Steps
Prepare patch pipettes
Turn on the Sutter P-1000 puller and enter the desired pull protocol.
Insert a thick-walled borosilicate glass capillary and press pull.
Pipette resistance must be of 3 to 6 megaohms.
Setting up patch rig and environment
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.
Turn on O2/CO2 tank and bubble aCSF solution.
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.
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).
Collect the internal solution with gramicidin in a clean syringe and attach a microfil flexible needle (or alternative electrode filling method).
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).
Turn on water heater and set to desired temperature (~34°C or Room temperature )
Examine slices and patching cells
Transfer brain slice from holding chamber to the recording chamber.
Secure down slice with a harp (slice anchor).
Locate and focus the desired brain region under the 4x objective.
Change the microscope lens to the 60x objective.
Focus on healthy neurons in slices for patching.
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).
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.
Gently place the glass micropipette onto the wire electrode and tighten.
Position the electrode using a micromanipulator.
Under the 60x objective, bring the tip of the glass pipette above the slice.
Apply a positive pressure and maintain it.
Approach the cell diagonally. The positive pressure should create a small dimple on the cell.
Once a dimple is formed, release the positive pressure, and apply a small amount of negative pressure. The resistance should begin to increase rapidly.
As the resistance increases, clamp the cell at your resting potential of interest (typically - 60 mV).
After a giga-ohm seal is formed, monitor series resistance until <100MOhm and stabilized - usually 20-30 minutes).
Start recording.
After the recording
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.
Gently remove electrode.
Remove and discard slice (unless further processing/fixation is needed)
Rinse tubing and microscope chamber.
Save/export files
Shut down the system, dispose of waste according to guidelines, clean work station.
