Jun 06, 2023
Brain slice physiology and optogenetics
- Hong-Yuan Chu1,2
- 1Van Andel Institute;
- 2ASAP
Protocol Citation: Hong-Yuan Chu 2023. Brain slice physiology and optogenetics. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly7m2rlx9/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 01, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82723
Keywords: Brain slice physiology, Examine slices and patching cells, Optogenetics, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-020572
Abstract
This protocol describes Chu Lab standard external solutions for electrophysiology.
Attachments
726-1837.docx
25KB
Materials
Solutions to prepare:
Electrophysiology solutions are prepared as 10× stock solutions (below) and diluted for use on the day of the experiment. Bicarbonate is added to all working solutions.
| A | B | C | D | |
| Bicarbonate | ||||
| mM | MW | 10×, g/L | ||
| NaHCO3 | 26 | 84.01 | 21.8426 | |
| A | B | C | D | |
| Synthetic Interstitial Fluid (SIF) (recording solution) | ||||
| mM | MW | 10×, g/L | ||
| NaCl | 126 | 58.44 | 73.6344 | |
| KCl | 3 | 74.55 | 2.2365 | |
| NaH2PO4.H2O | 1.25 | 137.99 | 1.7249 | |
| CaCl2.2H2O | 1.6 | 147.02 | 2.3523 | |
| MgSO4.7H2O | 1.5 | 246.48 | 3.6972 | |
| D-Glucose | 10 | 180.16 | 18.0160 | |
Brain slice physiology: Setting up patch rig and environment
Brain slice physiology: Setting up patch rig and environment
Turn on the MultiClamp 700B Amplifier, Axon Digidata 1550B digitizer, PatchStar Micromanipulator, PatchStar Slicescope, computer tower and the associated software.
Note
Note: amplifier and digitizer must be turned on prior to opening software.
Turn on O2/CO2 tank 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 (20 µL ) 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 3-4 mL /min.
Turn on water heater and set to desired temperature (~33-34 °C ).
Brain slice physiology: Examine slices and patching cells
Brain slice physiology: Examine slices and patching cells
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.
Note
- Change slowly to ensure there is enough clearance and the lens does not get damaged.
- If there is not enough clearance, move back to the 4x and raise the objective up, and then try again.
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 00:05:00 before the start of recording.
5m
Brain slice physiology: Optogenetics
Brain slice physiology: Optogenetics
Light pulses (1 ms duration) for optogenetic stimulation were delivered using a 478 nm LED through a 60x water immersion objective lens.
Note
Note: depending on the experimental design, synaptic blockers may be needed to isolate glutamatergic or GABAergic currents. Similarly, TTX and 4-AP can be applied to isolate monosynaptic responses from microcircuits with intensive local excitatory connections.
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.