This method can help answer key questions in the neurophysiology field, such as causal role of theta oscillation in special navigation and associated behaviors. The main advantage of this technique is that it enables a real-time control over the frequency and regularity of hippocampal theta oscillations in behaving mice. Demonstrating the procedure will be Dr.Franziska Bender, a former grad student in my laboratory.
Begin with the fully anesthetized mouse secured in the stereotaxic frame. Drill a hole above the medial septum. Submerge the tip of the injection needle in a droplet of virus on a piece of paraffin film and carefully draw up the virus at about 500 nanoliters per minute while observing the level of the fluid.
To prevent air from entering the injection needle, stop withdrawal before the virus is entirely taken up. Clean the needle with a paper tissue. Check that the virus and oil are separated by an air bubble and mark the level of the virus on the tube.
Position the needle above the craniotomy and slowly insert it into the brain at the first injection point. Inject 450 nanoliters of the virus at a rate of 100 to 150 nanoliters per minute. Following the injection, wait for 10 minutes.
After 10 minutes carefully move the needle up 0.1 millimeters and wait another five minutes. Use a micro stripper to strip 125 micrometers of cladding from a multi-mode optic fiber while the fiber is still attached to the fiber spool. Then, use a diamond knife to cut the fiber to a length of approximately two to three centimeters.
Insert the optic fiber into a zirconia ceramic stick ferrule with an internal diameter of 126 microns. Approximately 0.5 to one millimeter of the fiber should protrude from the convex side of the ferrule. Using a needle, apply one drop of epoxy glue to both ends of the ferrule, but not onto the sides of the ferrule.
After drying use diamond lapping fiber polishing film with three micron grits to polish the convex side of the ferrule. To prepare the tungsten wire array, first glue several 100 micron silica tube guides to the sticky side of a piece of tape. Then use forceps to thread formvar insulated 45 micron tungsten wires through the guide tubes.
Next use a scalpel to strip the insulation from both ends of six enamel insulated fine copper binding wires of approximately five millimeters long. Then strip the insulation from both ends of a grounding wire of approximately two to three centimeters long. Solder each stripped wire to the nanoconnector pins.
Connect each bonding wire to one tungsten wire using one drop of silver conductive paint. After drying, apply a small amount of cement to cover the copper wires. Avoid applying cement to the upper part of the nanoconnector.
After allowing the cement to dry for at least 30 minutes, use blunt stainless steel scissors to cut the tungsten wires at a five to 20 degree angle. Begin by drilling four holes of 0.8 mm diameter in the clean skull of the anesthetized animal. drill two holes rostral to the coronal suture, and two above the cerebellum.
Next, place stainless steel bone screws for grounding and stabilization of the implant. Position the ground screw connected to a copper wire above the cerebellum. Cover the ground screw completely with cement to prevent muscle artifacts during the electrophysiological recordings.
Build a cment ring connecting all screws. Perform a craniotomy above the implantation site, then apply approximately one microliter of sterile saline on the surface of the brain tissue. Use the stereotax to slowly lower the wire array into the craniotomy.
Then apply approximately five microliters of warm liquid wax above the implantation site to protect the brain tissue. Apply cement around the wire array and cover the skull with cement. Apply one drop of flux to the pre soldered ground wire or the pre soldered wire connected to the ground screw, and fuse the wires using a soldering machine.
Lastly, cover the entire ground wire with cement. Check the light output from the patch cord. If the transmission rate of the implanted fiber was 50%ensure that the light output at the tip of the patch cord is 20 milliwatts.
Connect the head stage preamplifier to the implanted connector, and connect the fiber optic patch cord to the implanted hippocampal fiber for optogenetic stimulation. Record the baseline behavior for a duration appropriate for the parameters being measured. Open the software to control the stimulus generator.
Click file, open, and select the protocol file of choice. Click download and start to initiate the light stimulation. Observe control of the theta rhythm by the light pulses.
Hippocampal local field potential during spontaneous theta oscillations is seen here. Note the gamma envelopes, an indicator of physiological theta rhythm. Hippocampal local field potential at seven hertz during optogenetic entrainment is seen here.
Blue stripes indicate the time windows of light application. Note the phase reset by the light pulse indicated here by an arrow. Hippocampal local field potential at 10 hertz reflects optogenetic entrainment.
Note phase-locked gamma envelopes and the phase reversal between stratham orians and stratum radiatum is also maintained during entrainment. Once mastered, this technique can be done in four hours of experiment time, plus six weeks for viral expression. While attempting this procedure, it's essential to remember to ensure efficient viral transduction.
Properly connect optic fiber implant to the patch cord, and obtain good quality of spontaneous local field potential theta oscillations.
