The optical recording method with voltage sensitive dye is thought to be an ideal technology to visualize how the brain works. This method allowed us to record a neuropsychic dynamics stably and reliably over 12 hours, given us a view on psychic activity. We are trying to expand the application to detect circuit changes caused by chemicals on the all stage of development, such as valuable acid application to mothers.
The technique is already well established. Please try to reproduce studies by following the method presented here. To begin this procedure, immerse the decapitated head in ice cold ACSF in a stainless steel surgical tray.
Extract the brain within one minute and place it in a beaker containing chilled ACSF for five minutes. Next, divide the brain section at the midline with a scalpel and place both hemispheres onto a 4%agar block. Then, place the brain block with both hemispheres onto a 4%agar block.
Wipe the excess ACSF from the block with a filter paper. Subsequently, apply superglue to the vibratome platform. Place the agar block on it and wipe the excessive adhesive with a filter paper.
Gently apply a small amount of ice cold ACSF from the top of the brain agar block to help solidify excess superglue and to prevent the glue from covering the brain and disturbing the slicing. Afterward, fix the platform to the vibratome tray and pour the modified ACSF. Set the vibratome to a slow speed and have the blade frequency at its maximum setting.
Then start slicing. Place the slices in the corner of the slice chamber in sequence so that the order of the slices can be easily distinguished. Usually three to five slices can be obtained from one hemisphere.
Next, cut off the brain stem portion using a 30 gauge needle. Using a small tipped paint brush, place the slice on the membrane filter held with a plexiglass ring. Place the ring in a moist recovery chamber and secure the cover to keep the inner pressure high.
When placing the slice onto the membrane filter, adjust the direction and position of the slice within the ring to ensure it is well centered and has a consistent direction. Leave the specimen at 28 degrees Celsius for 30 minutes and then at room temperature for at least 10 to 30 minutes for recovery. To stain the slices, gently apply 100 microliters of the staining solution onto each slice and incubate at 20 minutes for room temperature.
Next, prepare 50 to 100 milliliters of ACSF in a container. Place the ring with the specimen in it to rinse the staining solution. Then, transfer the rinsed slices to another incubation chamber.
Wait more than one hour for recovery before the experiment. 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 one 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 at 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.
This figure shows the representative optical signal upon electrical stimulation of the Schaffer collaterol in area CA1 of a mouse hippocampal slice. These consecutive images show the optical signal before any spacial and temporal filters were applied while these images show the same data after applying a five by five by five cubic filter twice. Due to the high frame rate and high spatial resolution, the application of the filter did not change the signal, but filtered out the noise which can also be observed in the time course recorded in pixels in a single trial.
Here is the comparison of the typical responses in area CA1 of the hippocampal slices between mouse and rat. The small hyperpolarizing response is observed in the distal side of the CA1 after 24 milliseconds in the mouse hippocampal slice, but more massive hyperpolarizing response overtook the depolarizing response in the rat hippocampal slice. Once the slices are stained, they should last for over 12 hours and you can perform other electrophysiological recordings on them.
