TITLE: Ex vivo electrophysiology
AUTHORS: Beatriz E Nielsen

[DESCRIPTION]
This protocol describes the steps to perform whole-cell electrophysiology recordings in acute brain slices.

[STEPS]
SECTION: Set up
1. Rig set-up.
SECTION: Set up
1.3. Set 1X ACSF (add drugs needed for particular experiments) in a jug or bottle and bubble it with O2/CO2. 
SECTION: Set up
1.2. Open the N2 tank connected to the rig anti-vibration table.
SECTION: Set up
1.1. Turn on required devices (computer, amplifier, manipulators, light sources, video camara controls, etc.) 
SECTION: Set up
2. Recording electrodes or patch pipettes:
SECTION: Set up
2.1. Pull patch pipettes (1.5 – 2 MΩ) (World Precision Instruments) using an electrode puller (Narishige, PC-10).
SECTION: Set up
2.2. Thaw an aliquot of the appropriate internal solution (stored at -80 °C (see 'Solutions' section below) (add ATP, GTP and Phosphocreatine if solution does not already have it). Fill a syringe with filter with the internal solution and keep on ice by the rig.

SECTION: Set up
3. Electrical stimulation electrodes:


SECTION: Set up
3.1. Pull electrodes (World Precision Instruments) using a puller (Narishige, PC-10).
SECTION: Set up
3.2. Fill electrodes with 1X ACSF using a syringe with filter. 
SECTION: Set up
1.4. Place the intake line into the ACSF container and allow circulation. Wait until the fluid has entered the recording chamber, then turn on the in-line heater (Warner Instruments) and set it to desired temperature 32-34 °C.

SECTION: Whole-Cell recordings
4. Transfer brain slice from incubation vial to the recording chamber and secure down the slice using a harp.

(The protocol for obtaining acute brain slices is linked below).
 

SECTION: Whole-Cell recordings
5. Locate and focus the desired region of the brain under the 4x objective.

SECTION: Whole-Cell recordings
6. Change the microscope lens to the 40x or 60x objective focus on healthy neurons selected for patching.


    
SECTION: Whole-Cell recordings
7. Fill a pipette tip with internal solution. Remove any air bubbles by gently flicking the glass pipette.

SECTION: Whole-Cell recordings
8. Place the pipette onto the wire electrode on the holder and tighten.
SECTION: Whole-Cell recordings
9. Apply a positive pressure and maintain it by quickly closing the stopcock. 
SECTION: Whole-Cell recordings
10. Position the electrode using a micromanipulator.
SECTION: Whole-Cell recordings
11. Under the 40x-60x immersion objective, bring the tip of the pipette above the slice.
SECTION: Whole-Cell recordings
12. Approach the cell until the positive pressure create a small dimple.
SECTION: Whole-Cell recordings
13. Zero the pipette offset, release the positive pressure, and apply a small amount of negative pressure. The resistance should start to increase rapidly until a giga-Ohm seal is formed. 
SECTION: Whole-Cell recordings
14. Clamp the cell at your resting potential of interest (typically -60 mV).
SECTION: Whole-Cell recordings
15. Apply a few quick pulses of negative pressure to break into the cell and reach the whole-cell configuration.
SECTION: Whole-Cell recordings
16. Wait 5 min to allow the internal solution to dialyze the cell before start your recordings. Cells were discarded if series resistance was ≥15 MΩ.
Reordings can be made in voltage-clamp or current-clamp modes and the acquisition protocol will differ depending on the experiment.

SECTION: Solutions
18. Internal solutions:

K-Gluconate + 10 mM BAPTA
 
+ 1 mg/mL, 0.1 mg/mL and 1.5 mg/mL
pH=7.35,  275 mOsm

K-Gluconate + 0.1 mM EGTA
 
+ 1 mg/mL, 0.1 mg/mL and 1.5 mg/mL
pH=7.35,  275 mOsm

SECTION: Solutions
19. External solutions:
1X ACSF 

For 1L: 
 900 mL + 100 mL  + 1.8 g  + 2 g 

10X ACSF stock
 
 

 
 
SECTION: Whole-Cell recordings
17. Once finished the recording of a particular cell, set the holding potential to 0 mV and remove the electrode. Discard the electrode in sharps container.
SECTION: Whole-Cell recordings
16.2. Optogenetic stimulation: deliver light pulses using using a 478 nm LED. Select the appropriate number of pulses, intensity and duration of light stimulation for the experiment.
SECTION: Whole-Cell recordings
16.1. Electrical stimulation: position the monopolar glass stimulating electrode filled with ACSF consistently 200 µm away from the recorded cell. Select the appropriate number of pulses, intensity and duration of electrical stimulation for the experiment.