The overall goal of this procedure is to provide a novel head-fixation technique using dental acrylic which is widely available in laboratories performing implantation surgery in rodents. This method can answer key questions in the brain and behavior research field. The main advantages of this technique is that it uses no metal head-posts or plates, so it can be used for laboratories that can not easily fabricate CNC machined head-posts.
Using CAD software design a rectangular fixation bar that is 123 by 35 by 6 millimeters, with a centered rectangular port that measures 16 by 20 millimeters This port is used to access the brain so its size should be modified as needed. Add slopes or indentations to serve as guide rails along the borders of the port. On either side of the fixation-bar prepare plates to attach via elongated anchoring holes.
The holes should pass a 6 millimeter screw, also in front of the port add a three millimeter diameter hole to fit a locking hook. Next, design the walls and floor of a 135 by 305 by 76 millimeter tall cube. The larger side walls are used to support the bar.
They need 6 millimeter holes to serve this purpose. Once all the parts have been machined from acrylic, assemble the box and bar using cyanoacrylade. Then attach the bar to the supporting walls of the box using 6 millimeter screws.
Lastly adjust the slope of the attachment to properly fit a rat's head by adjusting the screw's position. For mice use the same design with the smaller dimension bar, the same anchoring plates and a smaller box. Begin by preparing the bar.
First, wrap the port with paraffin film. Then, coat the border of the paraffin with a thin layer of acrylic resin. Next, disinfect the frame with a thorough cleaning using 70%ethanol.
After anesthetizing the rat confirm its anesthetized state using a toe-pinch. As needed provide half-doses of the drug cocktail to maintain the anesthesia. After fixing the rat on the stereotaxic frame, apply ophthalmic ointment and shave the hair from the skull.
Then provide a subcutaneous injection of 2%lidocaine at the incision site. Now it is time to incise the scalp along the mid-line and completely scrape off the Periosteum. Follow with several saline rinses to expose a clean region of the skull.
Next, make burr holes for the screws near the rim of the skull. Implant the screws perpendicularly to the skull. There should be no fluids leaking from the implanted screws.
Rinse around the implant sites and air dry the preparation. Next, coat most of the skull's surface with a thin layer of dental adhesive. But leave the bregma exposed for coordinate navigation.
Perform other electrode or hyperdrive implantations. Then secure the implants with acrylic resin on the dental cement layer. In doing this, leave enough space on the mounting resin so that the bar can be adhered to the skull.
Now, align the head-fixation bar about 5 millimeters above the skull. Apply some more resin between the skull and the resins on the bar. After the resins have hardened remove the ear bar and pull the mold and mounter out of the fixation-bar port.
To finish the procedure, inject the animal with antibiotics and an analgesic. Then, transfer it to a recovery cage and house it individually once it has regained sternal recumbency. Allow the animal to recover for at least a week before habituating it to head-fixation.
On the first day of habituation handle the animals frequently in short sessions. On the second day introduce the fixation-box. Let the animals explore the environment freely.
On several occasions during the exploratory period briefly grab a hold of the head-mounter for a second or two. On the third day introduce the experience of head-fixation. Put the animal in the box and direct the head to the port on the head-fixation bar.
Then grab the head-mounter and put it into the fixation bar. Bind the head with the locking hook and a rubber band. Then, let the animal stand for five minutes without touching it, which will only cause the animal to struggle more.
Excretion and defecation are expected and will gradually reduce with more exposure to the fixation. After five minutes remove the rubber band and eject the mounting bar. Then return the animal to its home cage and give it cereal or a sunflower seed as a reward.
After half an hour repeat the process. Fixate the animalfive times on this first day of fixation. On day four, increase the number of exposures to fixation to seven.
Moving forward, gradually extend the fixation period from five minutes to ten minutes, to 20 minutes, to half an hour and finally up to 50 minutes. If at any point an animal stops accepting the reward do not further increase the fixation time, as this is a sign of high stress. During the habituation process weigh the animals daily.
If an animal's body weight decreases during the process it should not be used for the study. In an experiment with eight rats, bilateral stimulating electrodes were positioned into the medial forebrain bundle. In the M1 forelimb area rats were equipped with microdrives and tungsten wire electrodes.
Once habituated the rats performed 40 to 60 minutes of behavioral tasks during head-fixation. All the molded head-mounters stayed attached to the animals for all 40 days of the experiment. In the experiment the animals were taught to pull levers for stimulation rewards.
In an experiment with mice implanted with four electrodes the head-mounter stayed attached for 80 days. The mice experienced 20 to 40 minutes of daily head-fixation over those 80 days. After watching this video you should have a good understanding of how to design a head-fixation bar and how to help treat the fixed animals.
With practice implanting the acrylic head-mounter can be accomplished within minutes. Later, the head-fixation is quick and easy since it requires no screws or clamps. Don't forget that working with dental acrylic resin can lead to skin irritation.
And precautions such as wearing a mask and gloves and working in a well ventilated area should always be taken while performing this procedure.
