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 appropriately positioned and focused so that the mice can be fully visualized throughout the entire Plexiglas enclosure. 3) The enclosure should be thoroughly cleaned between testing pairs to ensure that the current behaviors are not influenced by scents from the prior pair of mice. 4) The bedding material for the enclosure must be sufficient (~1 inch deep) and must be sawdust; mice do not display the inchworming behavior with little/no bedding or when placed on other bedding materials.
While not described here, we have observed inchworming behavior in non-isolated, socially housed BTBR mice placed alone (rather than in pairs) in a similar bedded enclosure. Under these conditions, the BTBR mice still inchworm, but at a lower frequency and duration, thus indicating that the inchworming behavior is a stereotypic behavior possibly induced by social anxiety (data not shown). However, given that studies have failed to reliably demonstrate differences in anxiety between the BTBR and B6 control mice, anxiety is likely a interacting with other factors to induce this behavior12. However, the focus of this methodological article was the standardized modification to the juvenile social interaction protocol because it elicits the behavior rapidly and reliably, with dramatic differences between BTBR and B6 mice. In addition, data are collected from two animals in a single session, thus increasing the information gained from each testing period. In our laboratory, the inchworming behavior has only been confirmed in the age range of P35-P55. Although it is possible that mice younger and older will display this repetitive behavior, failure to identify inchworming at other ages may indicate that the behavior is specific to adolescent and young adult mice.
The data from this experiment indicate clearly that the BTBR mouse strain engages in the repetitive inchworming behavior more rapidly, more frequently and for longer durations than the B6 controls. The inchworming behavior may therefore provide researchers with a new measure of repetitive behaviors in other ASD and disease models. Although it is not yet clear what drives this repetitive digging motion, BTBR mice also display higher levels of self-grooming and social avoidance1,9. As digging is an innate genetically-regulated trait in mice, testing of the inchworming behavior can be applied easily to other strains and has the potential to be a valuable tool for assessing motor stereotypies and compulsive behaviors in other disease models.
One of the hallmark behavioral traits that characterize ASD is repetitive behavior with restricted interests9. In addition to highlighting a valuable motor stereotypy, this experiment has increased the validity of the BTBR mouse model of ASD by demonstrating an additional repetitive behavior similar to the clinical presentation of motor stereotypies in ASD patients. Inchworming is displayed in both the BTBR (ASD) mice and B6 control mice, although the behavior is comparatively rare in B6 mice. The behavior that most closely resembles inchworming is digging behaviors in rodents. Thomas et al.4 suggests that for rodents, digging serves a variety of functions including food storage, predator evasion, and protection from aversive environments. As these functions are not necessary in the laboratory setting, mice who display high levels of digging behavior may be fulfilling a compulsive behavioral need4. Furthermore, Silverman et al.9 suggests that digging behavior should be incorporated into the analysis of the juvenile social interaction test as a measure of non-social activity.
In summary, inchworming is a highly repeated behavior significantly more prevalent in BTBR mice (a model of ASD) compared to B6 mice (control strain). Although the etiology of the behavior is currently unknown, it may be a valuable resource for researchers examining compulsivity and repetition in rodent models of many diseases. The procedural techniques are simple and can be completed with ease in a 24&#160;hr time period. Data analysis is more complex and time-consuming, but video recording the testing procedure allows the researcher to complete the analysis off-line and repetitively to ensure inter-rater consistency and reliability.

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 is significantly shorter for BTBR mice - they typically begin inchworming within the first 10 sec of the start of the recorded session. In contrast, control mice (B6) have a mean latency to inchworm of 4&#160;min&#160;and spend very small portions of their time actually displaying the behavior. These findings are similar to data from prior studies on repetitive digging behaviors in rodents3,6.
Figure 1 depicts the proper experimental set up for testing procedures. Figure 2 shows the average inchworming duration (A), frequency (B) and latency (C) to inchworm for the BTBR mice and the control B6 strain. All parameters were significantly different between the strains. In the experiment reported here, the inchworming test was administered over a 2&#160;day trial period, with alternate pairings in the second day, and the results of each day were compared. No significant differences were found, thus ensuring that the effects were not dependent on either specific mouse pairings or the repetition of the test. Finally, Figure 3 contains still photos of different mice engaged in the two primary components of the inchworming behavior.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/50791/50791fig1highres.jpg" width="500" /> 
Figure 1. Equipment set up for the inchworming experiment. Equipment set up for the inchworming experiment. A 30 cm cube Plexiglas box is set on a table in front of an infrared zoom camera positioned at a 45&#176; angle. The bottom of the enclosure is covered fully with an even layer of at least 1 inch of sawdust.
<img alt="Figure 2" fo:content-width="4in" src="/files/ftp_upload/50791/50791fig2highres.jpg" width="400" /> 
Figure 2. Average inchworming duration, frequency and latency displayed by pairs of BTBR and B6 mice. The BTBR (n = 8) and B6 (n = 8) mice were tested at P35-P40 over a two&#160;day testing period and scored for inchworming behavior. A)&#160;The BTBR strain displayed a significantly longer mean duration of inchworming than the B6 control strain (p&#160;&#60; 0.001). B)&#160;The BTBR mice displayed a significantly higher frequency of inchworming as compared to B6 controls (p&#160;&#60; 0.001). C)&#160;There was a significant difference in BTBR (n=8) and B6 (n=7) latency to dig with BTBR displaying a marked decrease in the time it took to display the behavior compared to B6 controls (p&#160;= 0.001).
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/50791/50791fig3highres.jpg" width="500" /> 
Figure 3. Illustrative representation of different mice in both aspects of the inchworming behavior. The upper panel is an example of mice in the extension phase of the inchworming behavior, whereas the lower panel is an example of mice in the contraction phase of the inchworming behavior. Inchworming is defined as the synchronous inward and synchronous outward movement of the fore and hind limbs of the mouse to displace bedding.

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 ...

inchworming-novel-motor-stereotypy-btbr-t-itpr3tfj-mouse-model

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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.

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

Methods to Explore the Influence of Top-down Visual Processes on Motor Behavior

Kinesthetic awareness is important to successfully navigate the environment. When we interact with our daily surroundings, some aspects of movement are deliberately planned, while others spontaneously occur below conscious awareness. The deliberate component of this dichotomy has been studied extensively in several contexts, while the spontaneous component remains largely under-explored. Moreover, how perceptual processes modulate these movement classes is still unclear. In particular, a currently debated issue is whether the visuomotor system is governed by the spatial percept produced by a visual illusion or whether it is not affected by the illusion and is governed instead by the veridical percept. Bistable percepts such as 3D depth inversion illusions (DIIs) provide an excellent context to study such interactions and balance, particularly when used in combination with reach-to-grasp movements. In this study, a methodology is developed that uses a DII to clarify the role of top-down processes on motor action, particularly exploring how reaches toward a target on a DII are affected in both deliberate and spontaneous movement domains.

It is unclear how top-down signals from the ventral visual stream affect movement. We developed a paradigm to test motor behavior towards a target on a 3D depth inversion illusion. Significant differences are reported in both deliberate, goal-directed movements and automatic actions under illusory and veridical viewing conditions.

Kinesthetic awareness is important to successfully navigate the environment. When we interact with our daily surroundings, some aspects of movement are ...

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

Compensatory Limb Use and Behavioral Assessment of Motor Skill Learning Following Sensorimotor Cortex Injury in a Mouse Model of Ischemic Stroke

Mouse models have become increasingly popular in the field of behavioral neuroscience, and specifically in studies of experimental stroke. As models advance, it is important to develop sensitive behavioral measures specific to the mouse. The present protocol describes a skilled motor task for use in mouse models of stroke. The Pasta Matrix Reaching Task functions as a versatile and sensitive behavioral assay that permits experimenters to collect accurate outcome data and manipulate limb use to mimic human clinical phenomena including compensatory strategies (i.e., learned non-use) and focused rehabilitative training. When combined with neuroanatomical tools, this task also permits researchers to explore the mechanisms that support behavioral recovery of function (or lack thereof) following stroke. The task is both simple and affordable to set up and conduct, offering a variety of training and testing options for numerous research questions concerning functional outcome following injury. Though the task has been applied to mouse models of stroke, it may also be beneficial in studies of functional outcome in other upper extremity injury models.

Mouse models have become increasingly popular in studies of behavioral neuroscience. As models advance, it is important to develop sensitive behavioral measures specific to the mouse. This protocol describes the Pasta Matrix Reaching Task, which is a skilled motor task for use in mouse models of stroke.

Mouse models have become increasingly popular in the field of behavioral neuroscience, and specifically in studies of experimental stroke. As models ...

Testing Sensory and Multisensory Function in Children with Autism Spectrum Disorder

In addition to impairments in social communication and the presence of restricted interests and repetitive behaviors, deficits in sensory processing are now recognized as a core symptom in autism spectrum disorder (ASD). Our ability to perceive and interact with the external world is rooted in sensory processing. For example, listening to a conversation entails processing the auditory cues coming from the speaker (speech content, prosody, syntax) as well as the associated visual information (facial expressions, gestures). Collectively, the &#8220;integration&#8221; of these multisensory (i.e., combined audiovisual) pieces of information results in better comprehension. Such multisensory integration has been shown to be strongly dependent upon the temporal relationship of the paired stimuli. Thus, stimuli that occur in close temporal proximity are highly likely to result in behavioral and perceptual benefits &#8211; gains believed to be reflective of the perceptual system&#39;s judgment of the likelihood that these two stimuli came from the same source. Changes in this temporal integration are expected to strongly alter perceptual processes, and are likely to diminish the ability to accurately perceive and interact with our world. Here, a battery of tasks designed to characterize various aspects of sensory and multisensory temporal processing in children with ASD is described. In addition to its utility in autism, this battery has great potential for characterizing changes in sensory function in other clinical populations, as well as being used to examine changes in these processes across the lifespan.

We describe how to implement a battery of behavioral tasks to examine the processing and integration of sensory stimuli in children with ASD. The goal is to characterize individual differences in temporal processing of simple auditory and visual stimuli and relate these to higher order perceptual skills like speech perception.

In addition to impairments in social communication and the presence of restricted interests and repetitive behaviors, deficits in sensory processing are ...

Paw-Dragging: a Novel, Sensitive Analysis of the Mouse Cylinder Test

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore by rearing and touching the walls of the cylinder with their forelimb paws for postural support. Following ischemic injury to the forelimb sensorimotor cortex, rats rely more heavily on their unaffected forelimb paw for postural support resulting in fewer touches with their affected paw which is termed forelimb asymmetry. In contrast, focal ischemic damage in the mouse brain fails to result in comparable consistent deficits in forelimb asymmetry. While forelimb asymmetry deficits are infrequently observed, mice do demonstrate a novel behaviour post stroke termed &#8220;paw-dragging&#8221;. Paw-dragging is the tendency for a mouse to drag its affected paw along the cylinder wall rather than directly push off from the wall when dismounting from a rear to a four-legged stance. We have previously demonstrated that paw-dragging behaviour is highly sensitive to small cortical ischemic injuries to the forelimb motor cortex. Here we provide a detailed protocol for paw-dragging analysis. We define what a paw-drag is and demonstrate how to quantify paw-dragging behaviour. The cylinder test is a simple and inexpensive test to administer and does not require pre-training or food deprivation strategies. In using paw-dragging analysis with the cylinder test, it fills a niche for predicting cortical ischemic injuries such as photothrombosis and Endothelin-1 (ET-1)-induced ischemia &#8211; two models that are ever-increasing in popularity and produce smaller focal injuries than middle cerebral artery occlusion. Finally, measuring paw-dragging behaviour in the cylinder test will allow studies of functional recovery after cortical injury using a wide cohort of transgenic mouse strains where previous forelimb asymmetry analysis has failed to detect consistent deficits.

Classical forelimb asymmetry analysis of the cylinder test is routinely used to assess behavioural deficits in rats following brain injury or stroke; however, it fails to detect consistent deficits in mice. This study demonstrates that quantifying paw-dragging behaviour is a more sensitive analysis of brain injury in mice.

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore ...

Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the functional status of a motor unit pool in vivo. These measures can provide insight into the normal development and degeneration of the neuromuscular system. These measures have clear translational potential because they are routinely applied in diagnostic and clinical human studies. We present electrophysiological techniques similar to those employed in humans to allow recordings of mouse sciatic nerve function. The CMAP response represents the electrophysiological output from a muscle or group of muscles following supramaximal stimulation of a peripheral nerve. MUNE is an electrophysiological technique that is based on modifications of the CMAP response. MUNE is a calculated value that represents the estimated number of motor neurons or axons (motor control input) supplying the muscle or group of muscles being tested. We present methods for recording CMAP responses from the proximal leg muscles using surface recording electrodes following the stimulation of the sciatic nerve in mice. An incremental MUNE technique is described using submaximal stimuli to determine the average single motor unit potential (SMUP) size. MUNE is calculated by dividing the CMAP amplitude (peak-to-peak) by the SMUP amplitude (peak-to-peak). These electrophysiological techniques allow repeated measures in both neonatal and adult mice in such a manner that facilitates rapid analysis and data collection while reducing the number of animals required for experimental testing. Furthermore, these measures are similar to those recorded in human studies allowing more direct comparisons.

Electrophysiological Motor Unit Number Estimation MUNE Measuring Compound Muscle Action Potential CMAP in Mouse Hindlimb Muscles

We present refined protocols that allow in vivo monitoring of motor unit function in the mouse. Techniques to measure compound muscle action potential (CMAP) and motor unit number estimation (MUNE) in the mouse hind limb muscles innervated by the sciatic nerve are described.

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the ...

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to screen different neurostimulation patterns in hemiparkinsonian (unilateral lesioned) rats. It consists of creating a unilateral Parkinson&#39;s lesion by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle, implanting chronic stimulation electrodes into the subthalamic nucleus and evaluating motor outcomes at the end of 24 hr periods of cable-bound external neurostimulation. The stimulation was conducted with constant current stimulation. The amplitude was set 20% below the individual threshold for side effects. The motor outcome evaluation was done by the assessment of spontaneous paw use in the cylinder test according to Shallert and by the assessment of skilled reaching in the staircase test according to Montoya. This protocol describes in detail the training in the staircase box, the cylinder test, as well as the use of both in hemiparkinsonian rats. The use of both tests is necessary, because the staircase test seems to be more sensitive for fine motor skill impairment and exhibits greater sensitivity to change during neurostimulation. The combination of the unilateral Parkinson model and the two behavioral tests allows the assessment of different stimulation parameters in a standardized way.

Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to ...

Novel Object Exploration as a Potential Assay for Higher Order Repetitive Behaviors in Mice

Restricted, repetitive behaviors (RRBs) are a core feature of autism spectrum disorder (ASD) and disrupt the lives of affected individuals. RRBs are commonly split into lower-order and higher-order components, with lower order RRBs consisting of motor stereotypies and higher order RRBs consisting of perseverative and sequencing behaviors. Higher order RRBs are challenging to model in mice. Current assays for RRBs in mice focus primarily on the lower order components, making basic biomedical research into potential treatments or interventions for higher-order RRBs difficult. Here we describe a new assay, novel object exploration. This assay uses a basic open-field arena with four novel objects placed around the perimeter. The test mouse is allowed to freely explore the arena and the order in which the mouse investigates the novel objects is recorded. From these data, patterned sequences of exploration can be identified, as can the most preferred object for each mouse. The representative data shared here and past results using the novel object exploration assay illustrate that inbred mouse strains do demonstrate different behavior in this assay and that strains with elevated lower order RRBs also show elevated patterned behavior. As such, the novel object exploration assay appears to possess good face validity for higher order RRBs in humans and may be a valuable assay for future studies investigating novel therapeutics for ASD.

Higher order restricted, repetitive behaviors (RRBs) disrupt the lives of affected individuals. These behaviors are challenging to model in rodents, making basic biomedical research into potential treatments or interventions for RRBs difficult. Here we describe novel object exploration as a potential assay for higher order RRBs in mice.

Restricted, repetitive behaviors (RRBs) are a core feature of autism spectrum disorder (ASD) and disrupt the lives of affected individuals.  RRBs are ...

The "Motor" in Implicit Motor Sequence Learning: A Foot-stepping Serial Reaction Time Task

This protocol describes a modified serial reaction time (SRT) task used to study implicit motor sequence learning. Unlike the classic SRT task that involves finger-pressing movements while sitting, the modified SRT task requires participants to step with both feet while maintaining a standing posture. This stepping task necessitates whole body actions that impose postural challenges. The foot-stepping task complements the classic SRT task in several ways. The foot-stepping SRT task is a better proxy for the daily activities that require ongoing postural control, and thus may help us better understand sequence learning in real-life situations. In addition, response time serves as an indicator of sequence learning in the classic SRT task, but it is unclear whether response time, reaction time (RT) representing mental process, or movement time (MT) reflecting the movement itself, is a key player in motor sequence learning. The foot-stepping SRT task allows researchers to disentangle response time into RT and MT, which may clarify how motor planning and movement execution are involved in sequence learning. Lastly, postural control and cognition are interactively related, but little is known about how postural control interacts with learning motor sequences. With a motion capture system, the movement of the whole body (e.g., the center of mass (COM)) can be recorded. Such measures allow us to reveal the dynamic processes underlying discrete responses measured by RT and MT, and may aid in elucidating the relationship between postural control and the explicit and implicit processes involved in sequence learning. Details of the experimental set-up, procedure, and data processing are described. The representative data are adopted from one of our previous studies. Results are related to response time, RT, and MT, as well as the relationship between the anticipatory postural response and the explicit processes involved in implicit motor sequence learning.

We introduce the foot-stepping serial reaction time (SRT) task. This modified SRT task, complementing the classic SRT task that involves only finger-pressing movement, better approximates daily sequenced activities and allows researchers to study the dynamic processes underlying discrete response measures and disentangle the explicit process operating in implicit sequence learning.

This protocol describes a modified serial reaction time (SRT) task used to study implicit motor sequence learning. Unlike the classic SRT task that ...

A Novel Single Animal Motor Function Tracking System Using Simple, Readily Available Software

We have recently demonstrated that implanting intracortical microelectrodes in the motor corteces of rats&#160;results in immediate and lasting motor deficits. Motor impairments were manually quantified through an open field grid test to measure the gross motor function and through a ladder test to measure the fine motor function. Here, we discuss a technique for the automated quantification of the video-recorded tests using our custom Capadona Behavioral Video Analysis System: Grid and Ladder Test, or BVAS. Leveraging simple and readily available coding software (see the Table of Materials), this program allows for the tracking of a single animal on both the open field grid and the ladder tests. In open field grid tracking, the code thresholds the video for intensity, tracks the position of the rat over the 3 min duration of the grid test, and analyzes the path. It then computes and returns measurements for the total distance traveled, the maximum velocity achieved, the number of left- and right-handed turns, and the total number of grid lines crossed by the rat. In ladder tracking, the code again thresholds the video for intensity, tracks the movement of the rat across the ladder, and returns calculated measurements including the time it took the rat to cross the ladder, the number of paw slips occurring below the plane of the ladder rungs, and the incidence of failures due to stagnation or reversals. We envision that the BVAS developed here can be employed for the analysis of motor function in a variety of applications, including many injury or disease models.

The current study aimed to automate the quantification of motor deficits in rats. The initial evaluation model assesses motor loss resulting from an intracortical microelectrode implantation in the motor cortex. We report on the development and use of a tracking algorithm using easily adaptable, simple, and readily available coding software.

We have recently demonstrated that implanting intracortical microelectrodes in the motor corteces of rats&#160;results in immediate and lasting motor ...

Inchworming: A Novel Motor Stereotypy in the BTBR T⁺ Itpr3^tf/J Mouse Model of Autism

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