TITLE: Ex vivo mouse brain patch clamp recordings and Fura-2 imaging
AUTHORS: taylor.panczyk

[DESCRIPTION]
In this protocol we detail the steps to perform ex-vivo brain slices electrophysiology and Fura-2 recordings. For both types of recordings, the brain slices with the desired nucleus must be placed into the recording chamber and patch clamp high resistance seal must be performed. For cell attached configuration recording, the sual must be kept unbroken. For whole cell configuration, quick pulses of negative pressure to break into the cell. For Tura-2 recordings, a whole cell patch clamp must be performed to allow the Fura-2 to enter the cell.

[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 2.5 to 5 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
6. Take an aliquot of internal solution from the -20° fridge.

SECTION: Setting up patch rig and environment
7. Fill syringe with internal solution (or aCSF for cell attached recordings), place a filter on the end of the syringe, and place a MicroFil Pipette Filler on the end of the filter.
SECTION: Setting up patch rig and environment
8. Turn on the pump and circulate recording aCSF solution through chamber.

SECTION: Setting up patch rig and environment
9. Adjust and set the rate of the pump to 3-4 mL/min.

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

SECTION: Examine slices and patching cells
11. Transfer brain slice from incubation beaker 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. Slowly focus on healthy neurons in slices for patching.
SECTION: Examine slices and patching cells
16. For whole cell configuration, fill a glass micropipette one-third full of internal solution. For cell attached configuration fill it with aCSF. Ensure there is no residual internal solution on exterior of glass micropipette, as this may introduce salts into the micromanipulator and add additional noise to recordings. Remove any air bubbles by gently flicking the glass micropipette.
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, zero the pipette voltage, 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, leave it this way to record in cell attached configuration or apply a few quick pulses of negative pressure to break into the cell to record in whole cell configuration.
SECTION: Examine slices and patching cells
25. Start recording.
SECTION: Fura 2 imaging
26. Transfer slices to a 2PLSM and turn on the 780-nm wavelength laser.
SECTION: Fura 2 imaging
27. Add Fura-2 (100 uM) and Alexa Fluor 594 hydrazide (25 uM) to the internal solution.
SECTION: Fura 2 imaging
28. Fill the patch pipettes with the internal solution.
SECTION: Fura 2 imaging
29. Perform a whole cell patch clamp recording as previously explained.
SECTION: Fura 2 imaging
30. Wait 10 minutes until the cell is completely filled with the internal solution containing Fura-2.
SECTION: Fura 2 imaging
31. Choose a dendrite from the patched neuron 20-100 um far from soma.
SECTION: Fura 2 imaging
32. Draw a strait line ROI over the chosen dendrite.
SECTION: Fura 2 imaging
33. Start line scan acquisitions with 0.195-m pixels and 12-s dwell time.