Organotypic hippocampal slice cultures – electrophysiology and random-access two-photon voltage imaging

Solutions

Artificial cerebrospinal fluid (ACSF)

The ACSF is continuously bubbled with 95% O₂ and 5% CO₂ and has a final pH = 7.4 and osmolarity of 300 mOsm.

Intracellular solution

Titrate this solution with KOH to obtain a final pH of 7.2 and adjust the osmolarity to 295 mOsm.

Whole-cell recordings

1.     Place organotypic brain slice under the microscope. During recordings, slices are continuously perfused with oxygenated artificial cerebrospinal fluid. Experiments can be performed at room temperature or the ACSF can be warmed with a heater before reaching the recording chamber.

2.     Find a neuron expressing the voltage-indicator using the multiphoton microscope. A general guideline in organotypic slices is to target cells located at a depth of 20 to 70 µm from the surface of the slice. Neurons targeted for recordings had ovoid shapes, recognizable neuronal features such as dendrites and axons, and were not excessively bright.

3.     Pull glass capillaries to make recording electrodes (WPI inc., TW150F-4)

4.     Fill recording electrode with patch solution and position on the micromanipulator.

5.     Inject positive pressure (~equivalent to 0.5 ml if using a 5 ml syringe)

6.     Position the pipette in the recording batch

7.     Lower the pipette above the neuron of interest, without penetrating the tissue.

8.     Approach the neuron of interest using slow manipulator speed.

9.     Touch the neuron of interest. As soon as a deflection is seen on the membrane and that the pipette resistance increases ~0.2 MΩ, release the pressure.

10.  Apply slight but constant negative pressure. A gigaseal will form, and the resistance should reach > 1 GΩ.

            11.Apply slight but constant negative pressure until the whole-cell configuration is obtained.

Two-photon voltage imaging

Imaging is performed with a custom-built random-access two-photon microscope (Otsu et al., 2008; Chamberland et al., 2014).

1.     Obtain a whole-cell recording from a neuron expressing the voltage indicator.

2.     Set the wavelength of the laser to 920 nm, the peak excitation wavelength for ASAP voltage indicators. In our hands, wavelengths of 900 – 920 nm yield high-quality traces.

3.     Identify the neuronal membrane, based on the adjacent green ASAP and red Alexa-594 fluorescence.

4.     Position the optical recording sites on the neuronal membrane. In our hands, an exposition time of 50 μs per pixel yield satisfactory results. Position recording sites on adjacent neurites to record from multiple neuronal compartments.

5.     The number of recording sites limits the recording speed. To record AP-evoked optical transients, it is advisable to obtain a final scan speed no lower than 1 kHz.

6.     Switch the recording mode to current-clamp. Evoked action potentials by brief current injections of 0.5 – 2 nA for 2 – 4 ms.

7.     Synchronize the electrophysiological equipment with the microscope.

8.     Trigger the firing of action potentials while accumulating optical data.

9.     Average sequential sweeps to increase the signal-to-noise ratio.