The authors are grateful for the technical and logistical assistance and expertise provided by Rose Tobias, Younghee Ahn, and David N. Ruskin. The work described here was funded by the Alberta Children&#39;s Hospital Foundation and the Alberta Children&#39;s Hospital Research Institute.

All procedures were carried out in accordance with the Canadian Animal Care Committee and approved by the University of Calgary Animal Care Committee. 
1. Animal Preparation
<ol>
	<li>Isolate mice overnight (12 hr) prior to testing. To do this, place all of the BTBR and B6 mice (P35-P40) needed for experimental analysis in cages by themselves, the day before testing.</li>
	<li>Place the isolated mice in the behavioral analysis room 30 min prior to testing so that animals will habituate to the novel environment.</li>
</ol>
2. Equipment Preparation
<ol>
	<li>Place the 30 x 30 cm Plexiglas box with a removable lid in the testing room. Place the box on a table and position the video camera to a 45&#176; angle from the bottom; this location of the camera will permit visualization of the entire floor of the enclosure and the mouse. Use an infrared backlight to help minimize glare from the wall of the box.</li>
	<li>Use a video camera that possesses a high zoom function with infrared capabilities to optimize filming in a low lux environment. Focus the camera on the enclosure enlarging the image so that the mice are visualized throughout the entire box.</li>
	<li>Cover the entire floor of the box with sawdust bedding (should be approximately 1 inch deep).</li>
</ol>
3 Testing Procedure
<ol>
	<li>Place an intra-strain pair of mice (BTBR - BTBR or B6-B6) within the testing enclosure.</li>
	<li>Turn the lights off in the testing room.</li>
	<li>Start the video camera to record and exit the room. This will prevent experimenter influence on the behavior of the mice. Record the activity for 11 min.</li>
	<li>Once the recording is complete, re-enter the testing room and return the mice to their original home cages.</li>
	<li>Dispose of the sawdust bedding, wash the entire testing box with 70% ethanol, and place new sawdust bedding in the bottom of the testing enclosure.</li>
	<li>Repeat the entire procedure for the rest of the intra-stain pairs and return the mice to their original home cages.</li>
</ol>
Note: To verify the reliability of the inchworming data, the procedure may be repeated on subsequent days with different BTBR-BTBR and B6-B6 pair combinations. This will ensure that the observed behavior is not dependent upon the specific partner in the enclosure, but the inchworming individual.
4. Data Analysis
<ol>
	<li>Inchworming is defined as the synchronous inward and outward movement of the animal&#39;s fore- and hind-feet, with a minimum of one co-occurring inward and then subsequent outward movement that effectively displaces the bedding. Manually watch and score each video twice (once for each mouse) to ascertain the total duration, frequency, and latency to inchworm in the 10 min video. Manual scoring of inchworming duration, frequency and latency should be based on the following definitions: 
	 
	Inchworming Duration: Is defined as the total time each mouse spends engaged in the inchworming behavior. To score this aspect of inchworming, use a timer that can be started and stopped. Start timing when the mouse begins to inchworm and stop the timer when the mouse stops inchworming. When and if the mouse begins to inchworm again, re-start and re-stop the timer. Repeat the starting and stopping of the timer for the duration of the 10 min observation period each time the mouse inchworms. This will generate an inchworming duration score that is between 0:00 and 10:00 min. 
	 
	Inchworming Frequency: Is defined as the number of times the mouse engages in the inchworming behavior in the 10 min observation period. A single count is awarded each time the mouse starts and stops inchworming. The inchworming frequency score will be a number between 0 and &#8734;. 
	 
	Latency to Inchworm: Is defined as the time that passes prior to the first onset of inchworming. Start the timer when the video starts, this will be time 0:00, and stop the timer when the mouse inchworms for the first time. If the mouse does not display the inchworming behavior the latency to inchworm will be 10:00. When analyzing latency to inchworm, a score between 0:01 and 10:00 min will be obtained. Mice that fail to inchworm for the duration of the testing period should be removed from the sample population to ensure that the results are pertinent to a population of mice that display the inchworming behavior.</li>
	 
	<li>Calculate the total frequency and duration of inchworming for each mouse in the video. Calculate the time required for the first mouse in the pair to inchworm for the latency to inchworm for that specific pair of mice. These parameters are based on prior methodological work related to repetitive digging behaviors in mice6.</li>
	<li>Calculate the average duration, frequency, and latency to dig for each experimental group in the analysis (i.e. BTBR vs. B6). Use an ANOVA or a t-test to compare data between strains/experimental groups. 
	 
	Note: When the procedure is completed on a second day, use a repeated measures ANOVA to ensure that there were no subsequent trial effects or pairing effects.</li>
</ol>

<ol>
	<li>McFarlane, H. G., Kusek, G. K., Yan, M., Phoenix, J. L., Bolivar, V. J., Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autism-like+behavioral+phenotypes+in+BTBR+T+tf/J+mice.">Autism-like behavioral phenotypes in BTBR T+tf/J mice.</a> Genes, Brain, and Behavior. 7, 152-163 (2008).</li><li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+mouse+behavioral+tasks+relevant+to+autistic-like+behaviors.">Designing mouse behavioral tasks relevant to autistic-like behaviors.</a> Mental Retardation and Developmental Disabilities Research Reviews. 10 (4), 248-258 (2004).</li><li>Deacon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digging+and+marble+burying+in+mice:+simple+methods+for+in+vivo+identification+of+biological+impacts.">Digging and marble burying in mice: simple methods for in vivo identification of biological impacts.</a> Nature Protocols. 1 (1), 122-124 (2006).</li><li>Thomas, A., Burant, A., Bui, N., Graham, D., Yuva-Paylor, L. A., Paylor, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marble+burying+reflects+a+repetitive+and+perseverative+behavior+more+than+novelty-induced+anxiety.">Marble burying reflects a repetitive and perseverative behavior more than novelty-induced anxiety.</a> Psychopharmacology. 204, 361-373 (2009).</li><li>Dudek, B. C., Adams, N., Boice, R., Abbott, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+influences+on+digging+behaviours+in+mice+(Mus+musculus)+in+laboratory+and+seminatural+settings.">Genetic influences on digging behaviours in mice (Mus musculus) in laboratory and seminatural settings.</a> Journal of Comparative Psychology. 97 (3), 249-259 (1983).</li><li>Webster, D., Williams, M. H., Owens, R., Geiger, V., Dewsbury, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digging+behavior+in+12+taxa+of+muriod+rodents.">Digging behavior in 12 taxa of muriod rodents.</a> Animal Learning and Behavior. 9 (2), 173-177 (1981).</li><li>Pobbe, R. H., Pearson, B. L., Defensor, E., Bolivar, V. J., Blanchard, D. C., Blanchard, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+social+behaviours+of+C57BL/6J+versus+BTBR+inbred+mouse+strains+in+the+visible+burrow+system.">Expression of social behaviours of C57BL/6J versus BTBR inbred mouse strains in the visible burrow system.</a> Behavioural Brain Research. 214, 443-449 (2010).</li><li>Lewis, M. H., Tanimura, Y., Lee, L., Bodfish, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+restricted+repetitive+behavior+in+autism.">Animal models of restricted repetitive behavior in autism.</a> Behavioural Brain Research. 176, 66-74 (2007).</li><li>Silverman, J. L., Yang, M., Lord, C., Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+phenotyping+assays+for+mouse+models+of+autism.">Behavioural phenotyping assays for mouse models of autism.</a> Nature Reviews. 11, 490-502 (2010).</li><li>Deacon, R., Rawlins, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+lesions,+species-typical+behaviours+and+anxiety+in+mice.">Hippocampal lesions, species-typical behaviours and anxiety in mice.</a> Behavioural Brain Research. 156, 241-249 (2005).</li><li>Pearson, B. L., 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=Motor+and+cognitive+sterotypies+in+the+BTBR+T+tf/J+mouse+model+of+autism.">Motor and cognitive sterotypies in the BTBR T+tf/J mouse model of autism.</a> Genes, Brain, and Behavior. 10, 228-235 (2011).</li><li>Pobbe, R. H., Defensor, E., Pearson, B. L., Bolivar, V. J., Blanchard, D. C., Blanchard, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+and+social+anxiety+in+the+BTBR+T++tf/J+mouse+strain.">General and social anxiety in the BTBR T+ tf/J mouse strain.</a> Behavioural Brain Research. 216 (1), 446-451 (2011).</li></ol>

The authors have nothing to disclose.

Inchworming is a novel measure of repetitive behavior that can be used to study many disorders that exhibit stereotypies such as ASD. There are 4 steps that are of critical importance to generating reliable inchworming data from the modified juvenile social interaction protocol: 1) the extended isolation period is essential to the inchworming test and should not be removed from the protocol or shortened. This requirement is specific to testing of inchworming in pairs described here. 2) The video camera needs to be approp...

Although the pathophysiology of autism spectrum disorders (ASD) remains unclear, diagnosis depends upon the presence of three broad symptom categories: abnormal social reciprocity, impairments in communication, and repetitive/stereotypic behaviors1. Most rodent models are unable to simultaneously reproduce all three symptoms in a single animal. Thus, models that can reliably simulate some or all aspects of the disorder hold great value for the development of efficacious preventative and therapeutic strategies. One model that does display all three aspects of ASD is the BTBR mouse1,2.
In combination with ultrasonic vocalizations and social approach assays, the juvenile social interaction test is commonly used to characterize mouse models of pediatric ASD; these tests measure decreased and abnormal social behaviors which represent core features of ASD. In addition to the juvenile social interaction test, many other tests have been used to measure stereotypic behaviors in animal models of ASD. For example, the marble burying test, which examines the tendency of an animal to displace bedding material using the snout and forepaws in effort to cover an object, is thought to measure compulsive stereotypic behaviors that occur in response to aversive stimuli3,4. Accordingly, digging tests have been implemented to quantify this repetitive behavior in ASD animal models and different species of rodents3,5-7. Digging behaviors are defined as obvious directed actions of the snout or paws to displace bedding materials and have been hypothesized to be a sensitive parameter of repetitive motor behavior4.
Here, we report a novel type of digging, which we have termed &#34;inchworming,&#34; in the BTBR strain of mice. The characterization of this novel behavior may provide a robust measure of a repetitive motor behavior that may be used for further validation of rodent models such as ASD, Huntington&#39; Disease or obsessive compulsive behavior8,9. We define &#34;inchworming&#34; as at least one synchronous movement of the fore- and hind-limbs inwards followed by a synchronous movement outwards which leads to the displacement of bedding material. This differs from typical digging behavior in many ways. First, the inchworming behavior is usually observed as a combination of episodes rather than a single isolated event. In contrast, digging may occur as single coordinated movement of either the hind or fore paws3. In addition, inchworming movements tend to occur horizontally across the enclosure floor as opposed to the stationary positions that are characteristic of digging behaviors. Finally, the inchworming behavior described below occurs frequently in the BTBR inbred strain of mice but is rarely identified in the B6 control strain, whereas typical digging behaviors are common in B6 and BTBR strains of mice10. The inchworming test procedures outlined in this study represent modifications of a standard juvenile social interaction protocol1.
Given that marble burying, digging, and juvenile play have established value for measuring abnormal behavior in mouse models of ASD, the inchworming behavior complements these previously defined measurements of stereotypic behaviors. In particular, the inchworming behavior provides a useful measure of lower order motor stereotypies that are commonly identified in ASD8,11. In addition, this new inchworming procedure may provide researchers with an additional valuable tool for the analysis of repetitive motor behaviors relevant to developmental disorders such as ASD.

<table border="1">
 <tbody>
 <tr>
 <td>Material Name </td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>30 cm X 30 cm Plexiglas box with lid</td>
 <td></td>
 <td></td>
 <td>Can be constructed </td>
 </tr>
 <tr>
 <td>IR Camera </td>
 <td>Survshop (Calgary, Alberta)</td>
 <td>Sony CCD Camera DP955V- 30' Infrared Armor Dome </td>
 <td>Find a camera with high-resolution and zoom capabilities</td>
 </tr>
 <tr>
 <td>DVR</td>
 <td>Supercircuits (Austin, Texas)</td>
 <td>BLACK Enterprise-Class 4-Channel H.264 security DVR with DVD Burner</td>
 <td>High data capacity</td>
 </tr>
 <tr>
 <td>Sawdust</td>
 <td></td>
 <td></td>
 <td>Same as bedding material </td>
 </tr>
 </tbody>
 </table>

inchworming: a novel motor stereotypy in the btbr t+ itpr3tf/j mouse model of autism

Autism Spectrum Disorder (ASD) is a behaviorally defined neurodevelopmental disorder characterized by decreased reciprocal social interaction, abnormal communication, and repetitive behaviors with restricted interest. As diagnosis is based on clinical criteria, any potentially relevant rodent models of this heterogeneous disorder should ideally recapitulate these diverse behavioral traits. The BTBR T+ Itpr3tf/J (BTBR) mouse is an established animal model of ASD, displaying repetitive behaviors such as increased grooming, as well as cognitive inflexibility. With respect to social interaction and interest, the juvenile play test has been employed in multiple rodent models of ASD. Here, we show that when BTBR mice are tested in a juvenile social interaction enclosure containing sawdust bedding, they display a repetitive synchronous digging motion. This repetitive motor behavior, referred to as &#34;inchworming,&#34; was named because of the stereotypic nature of the movements exhibited by the mice while moving horizontally across the floor. Inchworming mice must use their fore- and hind-limbs in synchrony to displace the bedding, performing a minimum of one inward and one outward motion. Although both BTBR and C56BL/6J (B6) mice exhibit this behavior, BTBR mice demonstrate a significantly higher duration and frequency of inchworming and a decreased latency to initiate inchworming when placed in a bedded enclosure. We conclude that this newly described behavior provides a measure of a repetitive motor stereotypy that can be easily measured in animal models of ASD.

The modified version of the juvenile play test described here can be used to observe and analyze the novel repetitive digging behavior termed inchworming. Inchworming is exhibited to a far greater extent by BTBR mice than B6 controls. In a typical 10&#160;min&#160;session, BTBR mice will display the distinct inchworming behavior (synchronous inward and outward movement of the paws in an effort to displace the sawdust bedding) for approximately 5 min, or half of the observation period. Furthermore, the latency to inchworm...

Watch this Scientific Journal Video about Inchworming: A Novel Motor Stereotypy in the BTBR T+ Itpr3tf/J Mouse Model of Autism at JoVE.com

Inchworming: A Novel Motor Stereotypy in the BTBR T+ Itpr3tf/J Mouse Model of Autism

Inchworming is a highly repetitive synchronous digging motion displayed by BTBR T+ Itpr3tf/J (BTBR) mice when placed in a testing cage with sufficient sawdust bedding. The procedure is a modification of the juvenile social interaction protocol and is used here to assess repetitive motor stereotypies relevant to Autism Spectrum Disorder.

Inchworming: A Novel Motor Stereotypy in the BTBR T+ Itpr3tf/J Mouse Model of Autism

Autism Spectrum Disorder (ASD) is a behaviorally defined neurodevelopmental disorder characterized by decreased reciprocal social interaction, abnormal ...

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11.5K Views.  University of Calgary Faculty of Medicine. Inchworming is a highly repetitive synchronous digging motion displayed by BTBR T+ Itpr3tf/J (BTBR) mice when placed in a testing cage with sufficient sawdust bedding. The procedure is a modification of the juvenile social interaction protocol and is used here to assess repetitive motor stereotypies relevant to Autism Spectrum Disorder.