The aim of the following experiment is to activate and study neuronal circuits long term in awake, behaving mice. This method is adapted from the concept originally described in Sparta etal. This is achieved by first assembling a fiber optic couple cord, which connects the optical rotary joint to the fiber optic implant.
Next, the fiber optic is implanted over the brain tissue of interest to create a passage for the light to travel directly to the channel redin expressing neurons. Finally, the light source is connected to the optical rotary joint in order to create a serially connected path from the light source to the brain tissue. Ultimately, light induced firing of channel redsin expressing neurons can be evaluated based on in vivo, electrophysiology, and behavioral studies.
The main advantage of this technique over existing methods like guide cannulas is that it minimizes the damage to the brain tissue caused by repeated insertion and removal of an optical fiber. Though this method can provide insight into neural circuit function, it can also be applied to other systems to better understand the synapse and circuit dependent mechanisms that underlies certain types of neurological disorders. Visual demonstration of this method is critical.
As the implantation steps are difficult to master, each seemingly minor step contributes greatly to the overall stability and reliability of the method. Begin by cutting 35 millimeters of a 125 micron diameter fiber optic with a 100 micron core by positioning a wedge tip carbide scribe perpendicular to the fiber optic and scoring the fiber in a single unidirectional motion followed by pulling straight out. Now, insert an lc ceramic feral with a 127 micron in a diameter bore convex side pointed down into a vice and then insert the fiber optic piece into the feral.
The fiber optic should slide in smoothly and marginally protrude beyond the convex end of the feral. Next apply one drop of freshly prepared heat curable fiber optic epoxy to the flat end of the feral, and then apply a heat gun on the epoxy. The epoxy should fill the feral as it is heated and is cured when it turns black.
After curing, clean any epoxy off the sides of the feral, then using an lc fiber optic polishing disc makes circular rotation patterns to polish the convex end of the feral on aluminum oxide Polishing sheets on a polishing pad on four decreasing grades. Then cut the fiber optic at the flat end to the appropriate length for targeting the region of interest. To test the polished fiber optic, insert the polished end of the implant into the sleeve of the coupler.
Until direct contact with the opposing feral is felt, a bad implant will have a weak focal point near the tip of the fiber optic, whereas a good implant will transmit a smooth concentric circle of light without output. Powers of up to 10 milliwatts store the finished implants in foam until use. Begin this step by measuring and then scoring an off 220 micron diameter fiber optic with a 200 micron core to allow a mouse to move freely but not allow the mouse to chew the fiber.
Then insert the fiber optic into a piece of furation tubing with an inner diameter slightly larger and a length slightly shorter than those of the fiber optic. Now, strip around 25 millimeters from one end of the fiber optic and insert it into the metal end of a multimode FC MM feral assembly. With a 230 micron in a diameter bore until it stops, the fiber optic should stick out through the feral end.
Next, secure the connection with cyanoacrylate at the metal end and cover the connection with a connector Boot polish the feral end with an FC polishing disc as just demonstrated. After stripping the other end of the fiber optic, insert it into an lc ceramic feral with just the convex end distal. Then apply a drop of epoxy to the flat end and heat until cured as just demonstrated.
After polishing the convex end of the feral as just demonstrated, slide an lc feral sleeve over the convex end of the feral until the feral reaches the midpoint of the sleeve. Then place a heat shrink tubing over the ation tubing and sleeve and heat the heat shrink tube into secure and protect the connection. Finally, connect the feral to the laser source and then test it by measuring the light output through the coupler with a spectrophotometer, the light loss between the laser output and measured coupler output should not exceed 30%before beginning the surgery.
Disinfect the surgical area with isop profile alcohol followed by chlorhexidine solution. Next, place the mouse in the stereotaxic rig and secure the head ensuring that the skull is level. Apply ophthalmic ointment to the animal's eyes to prevent dryness and postoperative pain.
Now make an incision through the midline of the scalp to expose the cranium from the eye orbits to the lambda suture. Use serafin clamps to hold back the skin and maintain access to the cranium. Then use a dental pick to etch a checkered pattern throughout the surface of the cranium.
Wash away the resulting debris with sterile saline. After drying thoroughly use a cotton swab to apply 3%hydrogen peroxide to the exposed cranium from around two to three seconds to create micropores. Then wash the skull multiple times with more sterile saline and dry it thoroughly.
After etching, washing and drying the checkerboard pattern into the cranium, again, insert the fiber optic feral implant into the probe holder and then connect it to the stereotaxic arm. Determine the bur hole location with a stereotactic atlas calibrated to bgma and lambda. Then use a rotary dental drill to make a small bur hole craniotomy less than one millimeter in diameter above the region of interest, being careful not to break the dura or damage any tissue.
Using the stereotaxic arm position the implant in place directly above the region of interest, the feral should rest on the surrounding cranial tissue. Now use a sterile toothpick to apply a thin even layer of freshly prepared dental cement across the cranium and onto the lower portion of the implant. Being careful not to come into contact with the skin of the animal, the base layer of dental cement should cover as much surface area on the cranium as possible.
The most difficult aspect of this procedure is applying the dental cement without it touching the mass skin. To ensure success, apply small amounts of cement at a time in thin layers and wait for each layer to completely dry before adding the next one. Apply even layers of the dental cement to form a small mound on top of the cranium and around the implant.
Leave the three to five millimeters of the convex end of the feral clean of cement to allow for a smooth unobstructed connection. Then suture the scalp over the mound of dental cement and around the implant vet bond adhesive can be used for additional binding after suturing. Finally, apply topical analgesics and antibiotics to the suture skin and around the base of the implant, and then place the mouse in a cage for postoperative recovery.
Proper assembly of the fiber optic implant and coupler results in minimal photon loss between the light source and the end of the fiber optic in the region of interest. While polished fiber optics should transmit light in a uniform concentric circle similar to the circle shown here as can be seen in this image with careful implantation and suturing, the implant causes no visible irritation to the mouse and can remain in place for long-term studies without any significant degradation of the fiber optic or to the amount of light transmitted Functional behavioral assays in an awake mouse show a direct response from stimulation through a fiber optic implanted over the motor cortex as evidenced by this in vivo electrophysiology video data showing the activation of targeted neurons through the implant and the behavioral response of the animal during brain stimulation. Following this procedure, other methods like photo stimulation of targeted brain regions can be performed in order to answer additional questions such as what are the patterns of circuit connectivity associated with the target cells?
After watching this video, you should have a good understanding of how to assemble fiber optic implants and coupler cords, and how to implant them over brain tissue expressing optogenetic porters.
