This study was supported in part by an Israel Science Foundation (ISF) grant (297/18). The authors thank M. Bronfeld for establishing the acute rodent model and M. Israelashvili for her comments.

All procedures were approved and supervised by the Institutional Animal Care and Use Committee and adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Bar-Ilan University Guidelines for the Use and Care of Laboratory Animals in Research. This protocol was approved by the National Committee for Experiments in Laboratory Animals at the Ministry of Health.
NOTE: This protocol utilizes female Long-Evans rats (acute and chronic models) and female Sp...

<ol>
	<li>American Psychiatric Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DSM-5.">DSM-5.</a> American Psychiatric Association. , (2013).</li><li>Peterson, B. S., Leckman, J. 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R., Munoz, D. M., Barickman, J., Friedhoff, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+Factors+and+Related+Fluctuation+of+Symptoms+in+Children+and+Adolescents+with+Tourette's+Disorder.">Environmental Factors and Related Fluctuation of Symptoms in Children and Adolescents with Tourette's Disorder.</a> Journal of Child Psychology and Psychiatry. 36 (2), 305-312 (1995).</li><li>Rothenberger, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+and+Tourette+syndrome.">Sleep and Tourette syndrome.</a> Advances in Neurology. 85, 245-259 (2001).</li><li>Conelea, C. a., Woods, D. W., Brandt, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+a+stress+induction+task+on+tic+frequencies+in+youth+with+Tourette+Syndrome.">The impact of a stress induction task on tic frequencies in youth with Tourette Syndrome.</a> Behaviour Research and Therapy. 49 (8), 492-497 (2011).</li><li>Ganos, C., Rothwell, J., Haggard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voluntary+inhibitory+motor+control+over+involuntary+tic+movements.">Voluntary inhibitory motor control over involuntary tic movements.</a> Movement Disorders. 33 (6), 937-946 (2018).</li><li>Yael, D., Vinner, E., Bar-Gad, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+tic+disorders.">Pathophysiology of tic disorders.</a> Movement Disorders. 30 (9), 1171-1178 (2015).</li><li>Kurvits, L., Martino, D., Ganos, C., Eddy, C. 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F., Pittenger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corticostriatal+interactions+in+the+generation+of+tic-like+behaviors+after+local+striatal+disinhibition.">Corticostriatal interactions in the generation of tic-like behaviors after local striatal disinhibition.</a> Experimental Neurology. 265, 122-128 (2015).</li><li>Bronfeld, M., Yael, D., Belelovsky, K., Bar-Gad, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+tics+evoked+by+striatal+disinhibition+in+the+rat.">Motor tics evoked by striatal disinhibition in the rat.</a> Frontiers in Systems Neuroscience. 7, 50 (2013).</li><li>Vinner, E., Israelashvili, M., Bar-Gad, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+striatal+disinhibition+as+a+chronic+animal+model+of+tic+disorders.">Prolonged striatal disinhibition as a chronic animal model of tic disorders.</a> Journal of Neuroscience Methods. 292, 20-29 (2017).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. 6, (2007).</li><li>Flecknell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analgesia+and+Post-Operative+Care.">Analgesia and Post-Operative Care.</a> Laboratory Animal Anaesthesia. , (2016).</li><li>Israelashvili, M., Bar-Gad, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corticostriatal+divergent+function+in+determining+the+temporal+and+spatial+properties+of+motor+tics.">Corticostriatal divergent function in determining the temporal and spatial properties of motor tics.</a> Journal of Neuroscience. 35 (50), 16340-16351 (2015).</li><li>Bronfeld, M., Belelovsky, K., Bar-Gad, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+properties+of+tic-related+neuronal+activity+in+the+cortico-basal+ganglia+loop.">Spatial and temporal properties of tic-related neuronal activity in the cortico-basal ganglia loop.</a> Journal of Neuroscience. 31 (24), 8713-8721 (2011).</li><li>McCairn, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Primary+Role+for+Nucleus+Accumbens+and+Related+Limbic+Network+in+Vocal+Tics.">A Primary Role for Nucleus Accumbens and Related Limbic Network in Vocal Tics.</a> Neuron. 89 (2), 300-307 (2016).</li><li>Rizzo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aripiprazole+Selectively+Reduces+Motor+Tics+in+a+Young+Animal+Model+for+Tourette's+Syndrome+and+Comorbid+Attention+Deficit+and+Hyperactivity+Disorder.">Aripiprazole Selectively Reduces Motor Tics in a Young Animal Model for Tourette's Syndrome and Comorbid Attention Deficit and Hyperactivity Disorder.</a> Frontiers in Neurology. 9, 1-11 (2018).</li><li>Vinner, E., Matzner, A., Belelovsky, K., Bar-gad, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociation+of+tic+expression+from+its+neuronal+encoding+in+the+striatum+during+sleep.">Dissociation of tic expression from its neuronal encoding in the striatum during sleep.</a> bioRxiv. , (2020).</li><li>Webster, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortico-striate+interrelations+in+the+albino+rat.">Cortico-striate interrelations in the albino rat.</a> Journal of Anatomy. 95, 532-544 (1961).</li><li>Ebrahimi, A., Pochet, R., Roger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topographical+organization+of+the+projections+from+physiologically+identified+areas+of+the+motor+cortex+to+the+striatum+in+the+rat.">Topographical organization of the projections from physiologically identified areas of the motor cortex to the striatum in the rat.</a> Neuroscience Research. 14, 39-60 (1992).</li><li>Brown, L. L., Sharp, F. 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The authors have nothing to disclose.

In this manuscript, we detailed the protocols of the acute and chronic models for tic induction in a freely behaving rat. These protocols describe the preparation of all components, the surgery and the experimental process which can be adapted for customization to meet specific research needs. The primary principle underlying these models is the direct local application of bicuculline to the motor areas of the striatum, which is known to play a key role in the pathophysiology of tic disorders10<su...

Tics are the defining symptom of Tourette syndrome (TS) and other tic disorders. Tics are described as sudden, rapid, recurrent movements (motor tics), or vocalizations (vocal tics)1. Tic expression typically fluctuates in its temporal (frequency)2 and spatial (intensity, body location)3 properties over multiple time scales (hours, days, months, and years). These changes are affected by different factors, such as environmental features4,5, behavioral states6,7, and voluntary and temporary suppression8.
Although the neuronal mechanism governing motor tics is still not fully understood, an increasing number of theoretical and experimental studies have provided new evidence as to its nature9. Currently, the pathophysiology of tic generation is thought to involve the cortico-basal ganglia (CBG) loop, and specifically is associated with abnormal inhibition of the striatum, the primary basal ganglia input nucleus10,11,12. Previous studies in rodents and primates have demonstrated that the striatum can be disinhibited by local application of different GABAA antagonists, such as bicuculline and picrotoxin13,14,15,16,17,18. This pharmacological intervention leads to transient motor tic expression in the contralateral side to the injection, thus establishing a robust acute model of tic disorders with face and construct validity. The acute model is simple to induce and makes it possible to study the effects of short-term modulation such as electrical and optical stimulation concurrent with electrophysiological and kinematic recordings before, during and after tic expression. However, the acute model is limited to the short time period following the injection. Based on the acute model, we recently proposed a chronic model of tic generation in rats that utilizes a prolonged, fixed-rate infusion of bicuculline to the striatum via a subcutaneous-implanted mini-osmotic pump19. This model extends the period of tic expression to multiple days/weeks. The constant release of bicuculline over a lengthy period of time allows for the examination of the effects of a variety of factors such as pharmacological treatments and behavioral states on tic expression.
Here, we present protocols for generating the acute and chronic models of tic expression in rats. As a function of the specific research question, the protocols enable the fine-tuning of the parameters including unilateral versus bilateral implantation, the site of the tics (according to the somatotopic organization of the striatum)18 and the angle of the implant-cannula (depending on the location of additional implanted devices). The method used in the chronic model is partially based on commercial products but with critical adjustments to fit the tic model. This article details the adjustments needed to custom tailor these tic models.

<table>
	<tbody>
		<tr>
			<td>Anchor screws</td>
			<td>Micro Fasteners</td>
			<td>SMPPS0002</td>
			<td>#0 x 1/8 - Pan Head Sheet Metal Screws</td>
		</tr>
		<tr>
			<td>Bicuculline methiodide</td>
			<td>Sigma Aldrich</td>
			<td>14343</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate (CA) accelerator</td>
			<td>Zap</td>
			<td>PT29</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate (CA) glue</td>
			<td>BSI</td>
			<td>IC-2000</td>
			<td>This glue was found to be stronger than others</td>
		</tr>
		<tr>
			<td>Dental cement</td>
			<td>Coltene</td>
			<td>H00322</td>
			<td>Hygenic Perm Repair Material Reline Resin Self Cure</td>
		</tr>
		<tr>
			<td>Glue gel</td>
			<td>Loctite</td>
			<td></td>
			<td>Ultra Gel Control</td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>WPI</td>
			<td>501242</td>
			<td>Any hemostat sized approximately 14 cm would be sufficient</td>
		</tr>
		<tr>
			<td>Hypo-tube, extra-thin wall 25G</td>
			<td>Component supply company</td>
			<td>HTX-25X</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypo-tube, regular wall 22G</td>
			<td>Component supply company</td>
			<td>HTX-22R</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypo-tube, regular wall 30G</td>
			<td>Component supply company</td>
			<td>HTX-30R</td>
			<td></td>
		</tr>
		<tr>
			<td>Infusion pump machine</td>
			<td>New Era Pump Systems</td>
			<td>NE-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-osmotic pump</td>
			<td>ALZET</td>
			<td>2001</td>
			<td>1.0&#181;l per hour, 7 days</td>
		</tr>
		<tr>
			<td>PE compatible adhesive</td>
			<td>CEYS</td>
			<td></td>
			<td>Special difficult plastics (suitable for PE)</td>
		</tr>
		<tr>
			<td>PE-10 Catheter Tubing</td>
			<td>ALZET</td>
			<td>PE-10</td>
			<td>ID = 0.28mm, OD = 0.61mm</td>
		</tr>
		<tr>
			<td>Precision glass microsyringe, 10&#181;l</td>
			<td>Hamilton</td>
			<td>80065</td>
			<td>1701 RNR 10&#181;l syr (22s/51/3)</td>
		</tr>
		<tr>
			<td>Tissue adhesive</td>
			<td>3M</td>
			<td>1469Sb</td>
			<td>Vetbond</td>
		</tr>
		<tr>
			<td>Tubing-adapter</td>
			<td>CMA</td>
			<td>3409500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon micro bore tubing, 0.02 inch ID * 0.06 OD</td>
			<td>Component supply company</td>
			<td>TND80-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Wire 0.005-inch</td>
			<td>Component supply company</td>
			<td>GWX-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>Wire 0.013-inch</td>
			<td>Component supply company</td>
			<td>GWX-0130</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating acute and chronic experimental models of motor tic expression in rats

Motor tics are sudden, rapid, recurrent movements that are the key symptoms of Tourette syndrome and other tic disorders. The pathophysiology of tic generation is associated with abnormal inhibition of the basal ganglia, particularly its primary input structure, the striatum. In animal models of both rodents and non-human primates, local application of GABAA antagonists, such as bicuculline and picrotoxin, into the motor parts of the striatum induces local disinhibition resulting in the expression of motor tics.
Here, we present acute and chronic models of motor tics in rats. In the acute model, bicuculline microinjections through a cannula implanted in the dorsal striatum elicit the expression of tics lasting for short time periods of up to an hour. The chronic model is an alternative enabling the extension of tic expression to periods of several days or even weeks, utilizing continuous infusion of bicuculline via a sub-cutaneous mini-osmotic pump.
The models enable the study of the behavioral and neural mechanisms of tic generation throughout the cortico-basal ganglia pathway. The models support the implantation of additional recording and stimulation devices in addition to the injection cannulas, thus allowing for a wide variety of usages such as electrical and optical stimulation and electrophysiological recordings. Each method has different advantages and shortcomings: the acute model enables the comparison of the kinematic properties of movement and the corresponding electrophysiological changes before, during and after tic expression and the effects of short-term modulators on tic expression. This acute model is simple to establish; however, it is limited to a short period of time. The chronic model, while more complex, makes feasible the study of tic dynamics and behavioral effects on tic expression over prolonged periods. Thus, the type of empirical query drives the choice between these two complementary models of tic expression.

Protocols for generating the acute and chronic models for tic induction in rats were presented above. The protocols cover the full preparation for surgery and experiments (Figure 1 for the acute model, Figure 2 for the chronic model). The application of bicuculline into the motor areas of the striatum results in the expression of ongoing motor tics. Tics appear on the contralateral side to the application and are characterized by brief and repetitive muscle cont...

Watch this Scientific Journal Video about Generating Acute and Chronic Experimental Models of Motor Tic Expression in Rats at JoVE.com

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

We present protocols for generating acute and chronic experimental models of tic expression in freely behaving rats. The models are based on striatal cannula implantation and subsequent GABAA antagonist application. The acute model uses transient injections whereas the chronic model utilizes prolonged infusions via a subcutaneous implanted mini-osmotic pump.

Motor tics are sudden, rapid, recurrent movements that are the key symptoms of Tourette syndrome and other tic disorders. The pathophysiology of tic ...

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.

generating-acute-chronic-experimental-models-motor-tic-expression

Research

JoVE Journal

Behavior

7.0K 조회수.  Bar-Ilan University. 이 프로토콜은 일시적 또는 만성 방식으로 자유롭게 행동하는 쥐에서 모터 틱을 유도하는 방법을 배치하여 tic 장애의 독특한 실험 모델을 설명합니다. 이 방법의 주요 장점은 투렛 증후군 및 기타 틱 질환을 연구하는 데 널리 사용되는 틱 발현의 유일한 가능한 모델을 설명한다는 것입니다. 시각적 데모는 맞춤형 장치 준비 및 이식 수술의 복잡한 과정을 명확히 합니다....