TITLE: Brain slice physiology and optogenetics
AUTHORS: Hong-Yuan Chu

[DESCRIPTION]
This protocol describes Chu Lab standard external solutions for electrophysiology.

[STEPS]
SECTION: Brain slice physiology: Setting up patch rig and environment
1. Turn on the MultiClamp 700B Amplifier, Axon Digidata 1550B digitizer, PatchStar Micromanipulator, PatchStar Slicescope, computer tower and the associated software. 
 


SECTION: Brain slice physiology: Setting up patch rig and environment
2. Turn on O2/CO2 tank and bubble SIF solution.
SECTION: Brain slice physiology: Setting up patch rig and environment
3. Take an aliquot of internal solution, ATP, and GTP from -20 °C freezer and thaw on ice.

SECTION: Brain slice physiology: Setting up patch rig and environment
4. Once thawed, add ATP and GTP (20 µL) to aliquot of internal solution and mix well with a pipette.

SECTION: Brain slice physiology: Setting up patch rig and environment
5. Fill syringe with internal solution, place a filter on the end of the syringe, and place a MicroFil Pipette Filler on the end of the filter.
SECTION: Brain slice physiology: Setting up patch rig and environment
6. Turn on Peri-Star Pro pump and circulate recording solution through chamber.
SECTION: Brain slice physiology: Setting up patch rig and environment
7. Adjust and set the rate of Peri-star pump to 3-4 mL/min.

SECTION: Brain slice physiology: Setting up patch rig and environment
8. Turn on water heater and set to desired temperature (~33-34 °C).

SECTION: Brain slice physiology: Examine slices and patching cells
9. Transfer brain slice from incubation beaker to the recording chamber.
SECTION: Brain slice physiology: Examine slices and patching cells
10. Secure down slice with a harp (slice anchor).
SECTION: Brain slice physiology: Examine slices and patching cells
11. Locate and focus the desired brain region under the 4x objective.
SECTION: Brain slice physiology: Examine slices and patching cells
12. Change the microscope lens to the 60x objective.
 

SECTION: Brain slice physiology: Examine slices and patching cells
13. Slowly focus on healthy neurons in slices for patching.
SECTION: Brain slice physiology: Examine slices and patching cells
14. Fill a glass micropipette one-third full of internal solution (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: Brain slice physiology: Examine slices and patching cells
15. Gently place the glass micropipette onto the wire electrode and tighten.
SECTION: Brain slice physiology: Examine slices and patching cells
16. Apply a positive pressure and maintain it.
SECTION: Brain slice physiology: Examine slices and patching cells
17. Position the electrode using a micromanipulator.
SECTION: Brain slice physiology: Examine slices and patching cells
18. Under the 60x objective, bring the tip of the glass pipette above the slice.
SECTION: Brain slice physiology: Examine slices and patching cells
19. Approach the cell diagonally. The positive pressure should create a small dimple on the cell.
SECTION: Brain slice physiology: Examine slices and patching cells
20. 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: Brain slice physiology: Examine slices and patching cells
21. As the resistance increases, clamp the cell at your resting potential of interest (typically -70 mV).
SECTION: Brain slice physiology: Examine slices and patching cells
22. Once gigaseal formed, you can perform fast/slow capacitance compensation.
SECTION: Brain slice physiology: Examine slices and patching cells
23. After a giga-ohm seal is formed, apply a few quick pulses of negative pressure to break into the cell.
SECTION: Brain slice physiology: Examine slices and patching cells
24. Once whole cell configuration is formed, wait for 5 min before the start of recording.

SECTION: Brain slice physiology: Optogenetics
25. Light pulses (1 ms duration) for optogenetic stimulation were delivered using a 478 nm LED through a 60x water immersion objective lens. 
 

SECTION: Brain slice physiology: Optogenetics
26. Under voltage clamp mode, adjust the intensity of LED to evoke synaptic currents of different amplitude. Repeat 3-5 sweeps per light intensity.
SECTION: Brain slice physiology: Optogenetics
27. Similar procedures can be performed to evoke optogenetics-induced action potentials under current clamp mode.