Brain slice physiology and optogenetics

Turn on the MultiClamp 700B Amplifier, Axon Digidata 1550B digitizer, PatchStar Micromanipulator, PatchStar Slicescope, computer tower and the associated software.

Turn on O₂/CO₂tank and bubble SIF solution.

Take an aliquot of internal solution, ATP, and GTP from -20°C freezer and thaw On ice.

Once thawed, add ATP and GTP (20µL) to aliquot of internal solution and mix well with a pipette.

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.

Turn on Peri-Star Pro pump and circulate recording solution through chamber.

Adjust and set the rate of Peri-star pump to 3-4mL/min.

Turn on water heater and set to desired temperature (~33-34°C).

Transfer brain slice from incubation beaker 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.

Slowly focus on healthy neurons in slices for patching.

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.

Gently place the glass micropipette onto the wire electrode and tighten.

Apply a positive pressure and maintain it.

Position the electrode using a micromanipulator.

Under the 60x objective, bring the tip of the glass pipette above the slice.

Approach the cell diagonally. The positive pressure should create a small dimple on the cell.

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.

As the resistance increases, clamp the cell at your resting potential of interest (typically -70 mV).

Once gigaseal formed, you can perform fast/slow capacitance compensation.

After a giga-ohm seal is formed, apply a few quick pulses of negative pressure to break into the cell.

Once whole cell configuration is formed, wait for 0h 5m 0s before the start of recording.

Light pulses (1 ms duration) for optogenetic stimulation were delivered using a 478 nm LED through a 60x water immersion objective lens.

Under voltage clamp mode, adjust the intensity of LED to evoke synaptic currents of different amplitude. Repeat 3-5 sweeps per light intensity.

Similar procedures can be performed to evoke optogenetics-induced action potentials under current clamp mode.