This article details a method for recording neuronal activity using voltage-sensitive dyes (VSD) in immobilized mouse brain slices. The procedure involves stimulating neurons and measuring changes in membrane potential through fluorescence emission.
Take an immobilized mouse brain slice stained with a voltage-sensitive dye or VSD, fluorescent molecules that integrate into the neuronal membrane and respond to membrane potential changes.
Place the slice in a recording chamber.
Install the ground electrode for a baseline voltage reference.
Next, position the stimulating and recording electrodes on the slice.
Apply an electrical pulse.
The pulse stimulates the neurons, triggering voltage-gated sodium channel opening and sodium ion influx, leading to a membrane potential change.
This induces an action potential, measured by the recording electrode, and alters the VSD's fluorescent properties.
Expose the brain slice to a specific wavelength to excite the VSD and trigger fluorescence emission.
Post-stimulation, the sodium channels close while the voltage-gated potassium channels open, promoting potassium ion efflux.
This changes the membrane potential again and consequently alters the VSD's fluorescence.
Record the fluorescence in real-time to visually represent neuronal activity.
To begin this procedure, turn on the amplifier, computer, and camera system and open the software. Pour aCSF in a 50-milliliter tube, and bubble it with carbogen. Use a peristaltic pump to circulate the aCSF, and adjust the flow rate to approximately one milliliter per minute.
Then, adjust the height of the suction pipette so that the liquid level inside the experiment chamber is always constant. Place the ground electrode in the chamber. Subsequently, fill the glass electrode with a small amount of aCSF, and place it in the electrode holder. Attach the holder to the rod of the manipulator.
Using an amplifier, ensure that the electrode resistance is approximately 1 megaohm. In the recording session, transfer a slice from the moist chamber to an experimental chamber. Press the edge of the ring firmly into the silicone O-ring. Be careful not to break the membrane or the bottom of the experiment chamber.
Under the microscope, place the tip of the stimulating electrode and the field potential recording electrode on the slice. Check the evoked response by delivering a stimulus. Confirm that the field of view covers the right neural circuit in the optical recording system.
Next, adjust the excitation light intensity to approximately 70% to 80% of the maximum capacity of the camera. Using the fluorescent light source, adjust the focus with the acquisition system. Then, start the recording, and analyze the data in an image acquisition software after the acquisition.
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