Generating Acute and Chronic Experimental Models of Motor Tic Expression in Rats

This protocol describes a unique experimental model of tic disorders by laying out the method for inducing motor tics in a freely-behaving rat, either in a transient or chronic manner. The main advantage of this method is that it describes the only available model of tic expression, which is widely used to study Tourette syndrome and other tic disorders. Visual demonstration clarifies the complex process of custom-made device preparation and implantation surgery.

Demonstrating the procedure will be Esther Vinner, a graduate student from my lab, and Katya Belelovsky, the lab manager. To begin, prepare an implant cannula by cutting a 25 gauge stainless steel hypotube and a 0.013 inch dummy removable internal wire. Insert this dummy wire into the implant cannula until it reaches the end and bend the excess wire.

Then cut a 70 centimeter flexible polymer microbore tube to prepare the injector. Prepare an injection cannula by cutting a 30 gauge stainless steel hypotube with a rotary tool. Insert three millimeters of this injection cannula into a flexible tube, and glue the joint between them to obtain an injector.

When performing the surgery, slide the implant cannular onto the cannula holder and lower it up to the implantation target in the rat's brain. Attach the implanted cannula to the skull of the rat by applying dental cement. Insert the prepared dummy into this implant cannula and cover all the implants by applying dental cement to the rest of the skull area.

To perform the microinjection, attach the injector to the precision glass microsyringe filled with bicuculline and configure the settings to a rate of 0.35 microliters per minute and a total volume of 0.35 microliters. Place the rat in the experimental cage. Remove the dummy and insert the injector into the implanted cannula through the end.

Start the infusion pump machine and keep track of the tic initiation and termination times using a stopwatch. After one minute of injection, remove the injector and slowly reinsert the dummy. Prepare a cannula guide by cutting a 12 millimeter 25 gauge stainless steel hypotube and an infusion cannula by cutting a 30 gauge stainless steel hypotube.

Insert a 0.005 inch diameter wire into the infusion cannula and bend it into L shape in the intended location. Next, prepare flexible catheter tubing by cutting an eight centimeter polyethylene PE10 tube. Remove the inner wire from the infusion cannula, glue the cannula guide on the three millimeter overlap near the bent part of the infusion cannula using cyanoacrylate glue and accelerator.

Then insert the horizontal part of the infusion cannula onto the catheter tubing. Eject the translucent cap of the pump flow moderator. Immerse the tubing adapter in 70%alcohol and attach it to the short cannula part of the flow moderator until it touches the white flange.

Insert the flexible catheter tubing into the tubing adapter and hold its long cannula part using a clip stand while gluing all the connections. Wrap the mini osmotic pump with a paper wipe and fix it vertically with the opening facing upwards using a clip holder stand. Fill the pump with aCSF using a syringe with a 27 gauge blunt needle, taking care to avoid air entry.

Fill the long cannula part of infusion tube with aCSF using a syringe with a 27 gauge blunt needle. And insert it into the pump. Place the pump in a saline beaker for at least four to six hours at 37 degrees Celsius.

Create a subcutaneous pocket in the rat's back by alternatively opening and closing an alcohol-sterilized hemostat under the skin through the mid scapular line. Remove the pump from the water bath and place it on the rats back covered with a paper wipe. Slide that cannula guide of the infusion tube on the cannula holder.

Hold the pump with the hemostat and gently insert it into the subcutaneous pocket. Implant the infusion cannula in the target and glue it to the skull using gel glue. Fix the infusion cannula, ensure that the catheter is kink-free to allow for neck movement and cover all the implants with dental cement.

To perform pump replacement surgery, start injecting bicuculline into the pump using a syringe with a 27 gauge blunt needle. Continue to inject bicuculline while removing the syringe to prevent air from entering. Insert the flow moderator inside the pump and place the pump in a saline beaker for at least four to six hours in a 37 degrees Celsius water bath.

Make an incision on the skin above the implanted pump. Wash the pocket with room temperature aCSF and dry it with gauze pads. Detach the aCSF-filled pump from the flow moderator using a hemostat and discard it.

Similarly, detach and discard the flow moderator from the bicuculline-filled pump. Gently attach the bicuculline-filled pump to the implanted flow moderator and glue the incision line with a tissue adhesive. In the acute model, tics start to appear several minutes after the bicuculline microinjection and last for dozens of minutes then eventually decay and cease.

In the chronic model, tics typically start to appear on the first day following the bicuculline-filled pump implantation, which fluctuates during the day and is most clearly observable during the quiet waking state. Motor tics have a stereotypic kinematic signature that can be detected in the accelerometer and gyroscope signals. Similarly, tic timing can also be assessed using the local fields potential signal throughout the cortico-basal ganglia pathway because of the appearance of large amplitude LFP transient spikes.

These models enabled the study of tic expression following different behavioral, environmental and pharmacological interventions for prolonged periods of time.