The authors would like to acknowledge the Summer Undergraduate Research Program, the Academic Technology User Group, the Center for Digital Learning, and the Science Center at the University of Redlands.
The authors would like to dedicate this paper to the memory of Lou Yango.

The protocol described here was approved by the Institutional Animal Care and Use Committee at the University of Redlands.&#160;The C58, C57, and FVB mice used in these studies were bred at the University of Redlands vivarium from stock originally obtained from the Jackson Laboratory (Bar Harbor, ME).&#160; Sentinels from this vivarium were screened every six months and found to be pathogen free.
1. Equipment and Room Set Up
Note: We used two different arenas for novel object testing: a clear plastic rectangular cage (45 cm x 24 cm x 20 cm) or an opaque circular cage with a base diameter of 41 cm; however, any cage may be used. Pearson et al. used a smaller rectangular cage (20 cm x 30 cm x 20 cm) in their assay. Details from this specific experimental design are included below, but given the novelty of this assay, there is no accepted standards within the field of behavioral phenotyping for any of the variables described.&#160;
<ol>
	<li>Fill the testing arena with approximately &#189; in of corn-cob bedding.</li>
	<li>Select four different novel objects. Select four objects that are approximately the same size, constructed of high density plastic to facilitate cleaning and resist chewing and different from each other in shape and color. Importantly, ensure that test mice are not exposed to these objects until being run in the assay. 
	Note: For example, a pink toy brick, a red monkey, a white tile with blue writing and a standard white die were used here.</li>
	<li>In the rectangular arena, place these objects approximately 3 cm from the corners. In the round arena, place the objects such that they are at equal distances from each other and approximately 10 cm from the sides. Record the placement of each object as a different number, 1-4 (Figure 1). Ensure that the objects are placed in a random or counterbalanced order throughout testing.</li>
	<li>Position a camera directly above the testing arena to record the entire arena during the acclimation and test periods. 
	Note: Having investigators in the same room with the mice can potentially influence activity levels and exploration during testing.</li>
</ol>
2. Novel Object Exploration Test
<ol>
	<li>Test at the beginning of the light cycle in a room illuminated with fluorescent lighting at approximately 100 lux. Ensure that lighting is uniform across the testing arena to standardize appearance while video recording.</li>
	<li>Place a notecard of known dimensions in the bottom of the testing arena and begin video recording.</li>
	<li>Transfer the test mouse into an empty testing arena for 10 min to serve as an acclimation period. Video record the acclimation period.</li>
	<li>After the acclimation period, leave the mouse in the arena, quickly add the four novel objects to the test arena and record the mouse behavior for an additional 10 min.</li>
	<li>Once the full 20 min acclimation/testing period has elapsed, return the test mouse to its home cage and thoroughly clean and dry the novel objects and testing arena with unscented dish soap and water.</li>
</ol>
3. Video Scoring
<ol>
	<li>Complete all behavior scoring using video to facilitate reliability. 
	Note: The behavioral logging software Noldus The Observer was used to perform the steps22 as described here, but a specialized program is not necessary:
	<ol>
		<li>Prior to scoring the first video, set up the project coding scheme in the behavioral logging software by creating a new project or by editing an existing, similar project.
		<ol>
			<li>Within the Project Setup box, set data acquisition to &#39;Offline Observation.&#39; Within the Behavior Coding box, program &#34;Scrabble&#34;, &#34;Digging&#34;, &#34;Rearing&#34;, &#34;Grooming&#34; and &#34;Sniff Object 1, 2, 3 and 4&#34; as &#39;State Events&#39;. Program &#34;Jumping&#34; as a &#39;Point Event&#39;. Note: Definitions for these behaviors are described in detail elsewhere11. 
			Note: State Events have a start and stop time, whereas Point Events simply collect count data. The keystrokes for each discrete behavior are generated by the software and these corresponding keystrokes are programmed into a secondary keyboard (step 3.1.2).</li>
			<li>To program the secondary keyboard, open the keyboard software, click on the appropriate secondary keyboard button displayed on the screen, type in the appropriate keystroke combination, and click OK. Once the secondary keyboard has been programmed, close the software as the program will run in the background of the computer.</li>
		</ol>
		</li>
		<li>Once the project and has been set up, use the behavioral logging software to score the number and duration of rears (defined as both front paws being placed on a wall of the arena), digs (defined as two front paws of the mouse burrowing into the bedding of the arena), self-grooms (defined as the mouse licking any region of their own body and/or the mouse touching any part of the face with their front paws), and jumps (defined as a mouse rearing and then jumping so that all four feet are off the ground simultaneously).
		<ol>
			<li>To score a video, go to File &#62; Open Project and then Observe &#62; Observation &#62; New. The program prompts for a file name. Once named, select the appropriate video media file.</li>
			<li>Begin the scoring by clicking on the Begin Observation button. 
			Note: When scoring mouse repetitive behavior, all State Events require two keystrokes, the first corresponding to the initiation of the behavior and the second corresponding to the end of the behavior. Point Events only require one keystroke.</li>
		</ol>
		</li>
		<li>Score the number of times the mouse sniffed each object. Sniffs are defined as any time a mouse moves its nose within 0.5 cm of an object. Measure sniff duration using behavioral logging software in the same way that repetitive behavior durations were measured (step 3.1.2).
		<ol>
			<li>Every time a mouse sniffs an object, record the corresponding position number, which will lead to a string of numbers by the end of the 10 min testing period (e.g. 1243421&#8230;). Manually record these data. 
			Note: To facilitate efficiency and consistency while scoring videos, the numbers always correspond to a given position, not object.</li>
			<li>If a mouse sniffs an object, looks away, then sniffs the object again, count that number twice.</li>
			<li>Once the full 10 min video has been scored, visualize the data by clicking on Analyze &#62; Behavior Analysis &#62; New. Once the data appear on the screen, export or copy and paste into a separate spreadsheet.</li>
		</ol>
		</li>
		<li>Record the total distance the mouse traveled within the arena during testing. 
		Note: The video tracking software, calibrated to track the test mouse and record the total distance moved in centimeters, was used to perform this step. All videos scored by the video tracking software had a notecard of known dimensions placed in the arena at the start of the video.
		<ol>
			<li>Use the notecard to calibrate each video within the software by setting a calibration line along each end of the notecard and inputting the appropriate length within the software&#39;s calibration screen. Once the lines are drawn, input the known length and width of the notecard that correspond to each line.</li>
			<li>Within Arena Settings, select the entire arena. 
			Note: Separate areas of the arena can be differentiated in the software if, for e.g., mouse movement along the walls vs. through the center was of interest.</li>
			<li>Within Trial Control Settings, select a ten-minute duration. In Detection Settings, choose a dark object on a light background. 
			Note: This would need to be changed if albino mice were being used or if the background was a darker shade.</li>
			<li>Once the settings have been programmed, score the videos. Click Acquisition &#62; Open Acquisition. In the Acquisition Control box, click New Trial and then Start Trial. After ten minutes has elapsed, the program stops and the data can be visualized.</li>
			<li>Click Analyze &#62; Calculate Statistics. Once the data appear on the screen, export or copy and paste into a separate spreadsheet.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Statistical Analyses, Sequencing
<ol>
	<li>Within the string of numbers corresponding to object investigations generated by each mouse, identify the total number of all possible 3 digit combinations without repeat numbers (e.g. 121, 123, 124 but not 112 or 122). 
	Note: A program written in Python programming language was used to identify the number of times each possible sequence appears in the string of numbers. It is not necessary to use an outside program, and this step could be completed many different ways (e.g. using a Find function in Microsoft Word or Excel).</li>
	<li>Record the number of times each sequence occurs and identify the three most often repeated sequences for each mouse. 
	Note: Individual sequences will vary by mouse and the actual sequence is of less interest than the number of times a sequence was repeated (i.e. adherence to a pattern is more important than the pattern itself).</li>
	<li>Because the total number of sequences a mouse repeats will correlate positively to activity level, correct these values by dividing the quantity of most frequent patterns by the total number of patterns for each individual mouse. This will yield a sequence repeat index that is independent of overall activity.</li>
	<li>Compare the number of times each mouse repeats its most common sequences (corrected for activity level) between groups using an appropriate ANOVA, multiple comparison procedure (Dunnett&#39;s test, for e.g.) and post-hoc tests.</li>
</ol>
5. Statistical Analyses, Object Preference
<ol>
	<li>Using the same string of numbers generated above (step 3.1.3.1), identify the novel object preference of each mouse by counting the total number of times each object was investigated, or in other words, counting the total number of 1 sec, 2 sec, 3 sec, and 4 sec in the string of data. Correct for activity level and compare via ANOVA as described above (steps 4.3-4.4). 
	Note: These methods published by our lab22 and described here are based heavily on Pearson et al.17</li>
</ol>

<ol>
	<li>American Psychological Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Diagnostic and Statistical Manual of Mental Disorders. , (2013).</li><li>Baio, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+Autism+Spectrum+Disorder+Among+Children+Aged+8+Years+-+Autism+and+Developmental+Disabilities+Monitoring+Network,+11+Sites,+United+States.">Prevalence of Autism Spectrum Disorder Among Children Aged 8 Years - Autism and Developmental Disabilities Monitoring Network, 11 Sites, United States.</a> Morb. Mortal. Wkly. Rep. 63 (SS02), 1-21 (2010).</li><li>Moy, S. S., 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=Social+approach+and+repetitive+behavior+in+eleven+inbred+mouse+strains.">Social approach and repetitive behavior in eleven inbred mouse strains.</a> Behav. Brain Res. 191 (1), 118-129 (2008).</li><li>Bodfish, J. W., Symons, F. J., Parker, D. E., Lewis, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Varieties+of+repetitive+behavior+in+autism:+comparisons+to+mental+retardation.">Varieties of repetitive behavior in autism: comparisons to mental retardation.</a> J. Autism Dev. Disord. 30 (3), 237-243 (2000).</li><li>Lewis, M. H., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathophysiology+of+restricted+repetitive+behavior.">The pathophysiology of restricted repetitive behavior.</a> J. Neurodev. Disord. 1 (2), 114-132 (2009).</li><li>Lewis, M. H., Bodfish, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repetitive+behavior+disorders+in+autism.">Repetitive behavior disorders in autism.</a> Ment. Retard. Dev. Disabil. Res. Rev. 4, 80-89 (1998).</li><li>Lam, K. S. L., Bodfish, J. W., Piven, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+three+subtypes+of+repetitive+behavior+in+autism+that+differ+in+familiarity+and+association+with+other+symptoms.">Evidence for three subtypes of repetitive behavior in autism that differ in familiarity and association with other symptoms.</a> J. Child Psychol. Psychiatry. 49 (11), 1193-1200 (2008).</li><li>Bishop, S. 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=Subcategories+of+Restricted+and+Repetitive+Behaviors+in+Children+with+Autism+Spectrum+Disorders.">Subcategories of Restricted and Repetitive Behaviors in Children with Autism Spectrum Disorders.</a> J. Autism Dev. Disord. 43 (6), 1287-1297 (2013).</li><li>Lewis, M. H., Tanimura, Y., Lee, L. W., Bodfish, J. W. <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> Behav. Brain Res. 176 (1), 66-74 (2007).</li><li>Korff, S., Stein, D. J., Harvey, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotypic+behaviour+in+the+deer+mouse:+Pharmacological+validation+and+relevance+for+obsessive+compulsive+disorder.">Stereotypic behaviour in the deer mouse: Pharmacological validation and relevance for obsessive compulsive disorder.</a> Prog. Neuropsychopharmacol. Biol. Psychiatry. 32 (2), 348-355 (2008).</li><li>Ryan, B. C., Young, N. B., Crawley, J. N., Bodfish, J. W., Moy, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+deficits,+stereotypy+and+early+emergence+of+repetitive+behavior+in+the+C58/J+inbred+mouse+strain.">Social deficits, stereotypy and early emergence of repetitive behavior in the C58/J inbred mouse strain.</a> Behav. Brain Res. 206 (1), 178-188 (2010).</li><li>Moy, S. S., 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=Mouse+behavioral+tasks+relevant+to+autism:+phenotypes+of+10+inbred+strains.">Mouse behavioral tasks relevant to autism: phenotypes of 10 inbred strains.</a> Behav. Brain Res. 176, 4-20 (2007).</li><li>Moy, S. S., Nadler, J. J., Poe, M. D., Nonneman, R. J., Young, N. B., Koller, B. H., 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=Development+of+a+mouse+test+for+repetitive,+restricted+behaviors:+relevance+to+autism.">Development of a mouse test for repetitive, restricted behaviors: relevance to autism.</a> Behav. Brain Res. 188 (1), 178-194 (2008).</li><li>Moy, S. S., 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=Repetitive+behavior+profile+and+supersensitivity+to+amphetamine+in+the+C58/J+mouse+model+of+autism.">Repetitive behavior profile and supersensitivity to amphetamine in the C58/J mouse model of autism.</a> Behav. Brain Res. 259, 200-214 (2014).</li><li>Scattoni, M. L., Gandhy, S. U., Ricceri, L., 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=Unusual+repertoire+of+vocalizations+in+the+BTBR+T+tf/J+mouse+model+of+autism.">Unusual repertoire of vocalizations in the BTBR T+tf/J mouse model of autism.</a> PLoS ONE. 3 (8), e3067 (2008).</li><li>Muehlmann, A. M., 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=Further+characterization+of+repetitive+behavior+in+C58+mice:+developmental+trajectory+and+effects+of+environmental+enrichment.">Further characterization of repetitive behavior in C58 mice: developmental trajectory and effects of environmental enrichment.</a> Behav. Brain Res. 235, 143-149 (2012).</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+stereotypies+in+the+BTBR+T+tf/J+mouse+model+of+autism.">Motor and cognitive stereotypies in the BTBR T+tf/J mouse model of autism.</a> Genes Brain Behav. 10 (2), 228-235 (2011).</li><li>Belzung, C., Crusio, W. E., Gerlai, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+exploratory+behavior.">Measuring exploratory behavior.</a> Handbook of molecular genetic techniques for brain and behavior research (techniques in the behavioral and neural sciences). , 739-749 (1999).</li><li>Kalueff, A. V., Keisala, T., Minasyan, A., Kuuslahti, M., Tuohimaa, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+stability+of+novelty+exploration+in+mice+exposed+to+different+open+field+tests.">Temporal stability of novelty exploration in mice exposed to different open field tests.</a> Behav. Process. 72, 104-112 (2006).</li><li>Prut, L., Belzung, 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+open+field+as+a+paradigm+to+measure+the+effects+of+drugs+on+anxiety-like+behaviors:+a+review.">The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review.</a> Eur. J. Pharmacol. 46, 3-33 (2003).</li><li>Walsh, R. N., Cumins, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+open-field+test:+a+critical+review.">The open-field test: a critical review.</a> Psychol. Bull. 83 (3), 482-504 (1976).</li><li>Blick, M. G., Puchalski, B. H., Bolanos, V. J., Wolfe, K. M., Green, M. C., Ryan, 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=Novel+object+exploration+in+the+C58/J+mouse+model+of+autistic-like+behavior.">Novel object exploration in the C58/J mouse model of autistic-like behavior.</a> Behav. Brain Res. 282, 54-60 (2015).</li><li>Crawley, J. N., 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=Behavioral+phenotypes+of+inbred+mouse+strains:+implications+and+recommendations+for+molecular+studies.">Behavioral phenotypes of inbred mouse strains: implications and recommendations for molecular studies.</a> Psychopharmacol. 132, 107-124 (1997).</li><li>Logue, S. F., Owen, E. H., Rasmussen, D. L., Wehner, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+locomotor+activity,+acoustic+and+tactile+startle+and+prepulse+inhibition+of+startle+in+inbred+mouse+strains+and+F1+hybrids:+implications+of+genetic+background+for+single+gene+and+quantitative+trait+loci+analyses.">Assessment of locomotor activity, acoustic and tactile startle and prepulse inhibition of startle in inbred mouse strains and F1 hybrids: implications of genetic background for single gene and quantitative trait loci analyses.</a> Neurosci. 80 (4), 1075-1086 (1997).</li><li>Lamprea, M. R., Cardenas, F. P., Setem, J., Morato, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thigmotactic+responses+in+an+open-field.">Thigmotactic responses in an open-field.</a> Braz. J. Med. Biol. Res. 41, 135-140 (2008).</li></ol>

The authors have nothing to disclose.

Here, we present a recently developed assay that may be useful for quantifying mouse behaviors with face validity for higher order repetitive behaviors in humans. Unlike more established assays like the Barnes or T-maze, this novel object exploration assay does not require any mouse training nor is it particularly anxiety provoking. Additionally, novel object exploration does not require any food or social stimuli, allowing for more focus on the behaviors of interest, RRBs, and decreasing the likelihood of confounding va...

Autism spectrum disorder (ASD) is a neurodevelopmental disorder consisting of three core symptoms: social impairment, difficulty communicating through language, and repetitive patterned behaviors1. Since 2000, the number of individuals who have been diagnosed with ASD has increased from 1 in 150 to 1 in 68 in the span of ten years 2. Though the prevalence of the disorder continues to increase, the cause of the disorder is not yet known. There has been a rise in efforts to identify appropriate mouse models for the core and associated symptoms of ASD, as these models could lead to an increased understanding of the underlying symptoms and causes of ASD. There are multiple inbred mouse strains that appear to display behaviors with face validity for the core symptoms of ASD, including repetitive behaviors3.
Restricted, repetitive behaviors (RRBs) are a core symptom of some psychiatric disorders such as ASD. RRBs can increase with the severity of the disorder4, and can drastically disrupt the lifestyle of affected individuals. RRBs are commonly placed into two categories, lower-order repetitive behaviors, which in humans consist of actions such as rocking and hand-flapping; and higher-order repetitive behaviors, which consist of strict adherence to routine and resistance to change5-8.
Lower-order repetitive behaviors have been widely studied in rodents where they manifest as motor stereotypies, which can be easily observed in a laboratory setting9. These behaviors appear to have good face validity for RRBs in humans, and potentially strong construct validity as well10. Testing for the presence of lower order RRBs can be completed through video monitoring of mouse activity to study the bouts and duration of these motor stereotypies11. Higher order repetitive behaviors pose a challenge for basic biomedical research utilizing rodents, as these RRBs are not as easily identified through simple observation. Due to the difficulty in identifying these behaviors, fewer established assays for higher-order repetitive behavior exist. Traditionally, higher-order RRBs have been measured in rodents using a maze paradigm where the test animal is trained to reach competency in escaping. The escape location is then switched and the number of trials required to re-learn the escape location is recorded12. These assays are not ideal as they require a lengthy training period, often induce anxiety, and can result in highly variable results. Hole-board exploration has also been used to quantify higher-order RRBs13,14. This approach does not require extended training sessions, but does rely on food motivation and/or olfactory discrimination. Assays for higher order RRBs that are not anxiogenic or require training would be a nice complement to the existing repertoire of hole-board exploration and maze-based assays currently in use.
The C58/J (C58) inbred mouse strain strongly exemplifies high levels of stereotypic behavior associated with ASD, namely repetitive, purposeless motor stereotypies and elevated levels of self-grooming3,11. Additionally, the C58 mice display RRBs through high levels of rearing, back flipping and scrabbling11,14,16. This strain begins showing these behaviors early in the neonatal period and continues to display them throughout adulthood.&#160; It would be ideal to be able to test for the presence of elevated higher-order RRBs to complement the well-documented lower order RRBs present in this strain as well as other mouse strains.&#160; The novel object exploration assay described here provides the opportunity for researchers to observe lower-order and higher-order RRBs simultaneously, as it gives the ability to measure patterned behaviors as well as repetitive motor stereotypies.
Using novel object exploration as an assay for higher-order repetitive behaviors was developed by Pearson et al.17. This new assessment is an extension of the well-established open field test18-21 with the addition of four novel objects to the arena. Mice were allowed to freely investigate these unfamiliar objects and the number and order of object investigations was tracked. The object investigations were then analyzed for the presence of patterns, with BTBR mice displaying elevated numbers of patterned investigations among the objects. Using this assay, mice can display higher-order repetitive and patterned behaviors while eliminating the need to learn behaviors as well as removing unnecessary stimuli. Novel object exploration induces higher-order RRBs, as it allows the mice to create patterns and form sequences through their natural exploration. Using this assay allows the investigator to quantify the presence of these higher-order RRBs.
Pearson et al. developed this assay and used it to test for the presence of potential higher order repetitive behaviors in the BTBR inbred mouse strain, with intriguing results17. We have recently published a follow-up study looking at the behaviors of the C58, C57BL/6J (C57) and FVB/NJ (FVB) strains, as well as a more detailed investigation into potential confounding variables present in this assay, and possible statistical approaches to analyzing the data generated22.

<table><tbody><tr><td>Standard Polycarbonate Rodent Cage (45 cm x 24 cm x 20 cm)</td><td></td><td></td><td>Multiple cages are desirable to facilitate testing of multiple mice&amp;nbsp;</td></tr><tr><td>Plastic Opaque Circular Testing Arena (41 cm base diameter)</td><td>United States Plastic Corp.</td><td>13931</td><td>Multiple arenas are desirable to facilitate testing of multiple mice&amp;nbsp;</td></tr><tr><td>Standard Corn-Cob Rodent Bedding</td><td></td><td></td><td></td></tr><tr><td>Novel Object - red monkey</td><td>Hasbro, Pawtucket RI</td><td></td><td>from Barrel of Monkeys</td></tr><tr><td>Novel Object - rectangular 2 x 4 LEGO</td><td></td><td></td><td></td></tr><tr><td>Novel Object - tile</td><td>Thinkfun Inc., Alexandria VA</td><td></td><td>from Toot and Otto</td></tr><tr><td>Novel Object - standard white die</td><td></td><td></td><td></td></tr><tr><td>Video Camera</td><td></td><td></td><td></td></tr><tr><td>Behavioral Logging Software - The Observer</td><td>Noldus, Wageningen, The Netherlands</td><td>other programs may be used</td><td></td></tr><tr><td>Video Tracking Software - EthoVision</td><td>Noldus, Wageningen, The Netherlands</td><td>other programs may be used</td><td></td></tr><tr><td>X-Keys input keyboard</td><td>P.I. Engineering, Williamstown MI</td><td>829484</td><td></td></tr><tr><td>MacroWorks II</td><td>P.I. Engineering, Williamstown MI</td><td></td><td></td></tr></tbody></table>

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.

The representative data22 show that female C58/J mice displayed a higher number of sequenced patterns than the other strains in the round arena (Figure 2, panel A), but not in the rectangular arena (Figure 2, panel C). None of the three male strains differed from each other (Figure 2, panels B and D). The representative data show that both male and female C58/J mice display a stronger preference for their most visited object (a...

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

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 ...

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Research

JoVE Journal

Behavior

8.3K Views.  University of Redlands. 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.

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

Marble Burying and Nestlet Shredding as Tests of Repetitive, Compulsive-like Behaviors in Mice

Obsessive-compulsive disorder (OCD) and autism spectrum disorders (ASD) are serious and debilitating psychiatric conditions and each constitutes a significant public health concern, particularly in children. Both of these conditions are highlighted by the repeated expression of meaningless behaviors. Individuals with OCD often show checking, frequent hand washing, and counting. Children with ASDs also engage in repetitive tapping, arm or hand flapping, and rocking. These behaviors can vary widely in intensity and frequency of expression. More intense forms of repetitive behaviors can even result in injury (e.g.&#160;excessive grooming, hand washing, and self-stimulation). These behaviors are therefore very disruptive and make normal social discourse difficult. Treatment options for repetitive behaviors in OCD and ASDs are somewhat limited and there is great interest in developing more effective therapies for each condition. Numerous animal models for evaluating compulsive-like behaviors have been developed over the past three decades. Perhaps the animal models with the greatest validity and ease of use are the marble burying test and the nestlet shredding test. Both tests take advantage of the fact that the target behaviors occur spontaneously in mice. In the marble burying test, 20 marbles are arrayed on the surface of clean bedding. The number of marbles buried in a 30 min session is scored by investigators blind to the treatment or status of the subjects. In the nestlet shredding test, a nestlet comprised of pulped cotton fiber is preweighed and placed on top of cage bedding and the amount of the nestlet remaining intact after a 30 min test session is determined. Presently, we describe protocols for and show movie documentation of marble burying and nestlet shredding. Both tests are easily and accurately scored and each is sensitive to small changes in the expression of compulsive-like behaviors that result from genetic manipulations, disease, or head injury.

Disease, head injury, genetic modifications, and treatment of mice with drugs can have profound effects on behavior. Utilizing well-characterized and validated approaches such as marble burying and nestlet shredding, compulsive-like behaviors can be documented accurately in mice as models of human obsessive-compulsive disorder and autism spectrum disorder.

Obsessive-compulsive disorder (OCD) and autism spectrum disorders (ASD) are serious and debilitating psychiatric conditions and each constitutes a ...

Novel Object Recognition Test for the Investigation of Learning and Memory in Mice

The object recognition test (ORT) is a commonly used behavioral assay for the investigation of various aspects of learning and memory in mice. The ORT is fairly simple and can be completed over 3 days: habituation day, training day, and testing day. During training, the mouse is allowed to explore 2 identical objects. On test day, one of the training objects is replaced with a novel object. Because mice have an innate preference for novelty, if the mouse recognizes the familiar object, it will spend most of its time at the novel object. Due to this innate preference, there is no need for positive or negative reinforcement or long training schedules. Additionally, the ORT can also be modified for numerous applications. The retention interval can be shortened to examine short-term memory, or lengthened to probe long-term memory. Pharmacological intervention can be used at various times prior to training, after training, or prior to recall to investigate different phases of learning (i.e., acquisition, early or late consolidation, or recall). Overall, the ORT is a relatively low-stress, efficient test for memory in mice, and is appropriate for the detection of neuropsychological changes following pharmacological, biological, or genetic manipulations.

The object recognition test (ORT) is a simple and efficient assay for evaluating learning and memory in mice. The methodology is described below.

The object recognition test (ORT) is a commonly used behavioral assay for the investigation of various aspects of learning and memory in mice. The ORT is ...

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often stereotyped actions elicited in response to innately aversive sensory stimuli, but their success requires enough flexibility for adapting to different spatial environments, which can change rapidly. Here, we describe a behavioral assay to evaluate the influence of learned spatial knowledge on defensive behaviors in mice. We have adapted the widely used Barnes maze spatial memory assay to investigate how mice navigate to a shelter during escape responses to innately aversive sensory stimuli in a novel environment, and how they adapt to acute changes in the environment. This new assay is an ethological paradigm that does not require training and exploits the natural exploration patterns and navigation strategies in mice. We propose that the set of protocols described here are a powerful means of studying goal-directed behaviors and stimulus-triggered navigation, which should be of interest to both the fields of instinctive behaviors and spatial memory.

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. The Barnes maze can also assess other mouse behaviors, such as general motivation to escape from the maze platform and exploratory behavior. The Barnes maze can measure whether a genetic mutation or environmental variable can impact the acquisition and retention of spatial memories, as well as provide information about the search strategy employed by the mice. Here we use the Barnes maze to detect a memory deficit in adult mice following a single developmental ethanol exposure event. The newly described Damsel-in-Distress paradigm exposes a male mouse to a female mouse trapped in a chamber in the open center field of the arena. It provides an opportunity for the mouse to socially respond to the trapped female and exhibit prosocial behavior. The Damsel-in-Distress paradigm can also be used to examine mouse behavior in a novel arena and measure locomotor activity. Both the Barnes Maze and the Damsel-in-Distress protocols require minimal financial investment and most aspects of the tests can be constructed from common lab supplies. These flexible and accessible tools can also be used to detect behavioral changes over the course of development.

This protocol measures spatial learning and memory using the Barnes maze. A novel Damsel-in-Distress paradigm is used to assess locomotor activity and prosocial behavior in mice.

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. ...

Novel Object Recognition and Object Location Behavioral Testing in Mice on a Budget

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various physiological or pathological changes. The object location task (OLT) and the novel object recognition task (NORT) are two effective behavioral tasks commonly used to reveal the function and relative health of specific brain regions involved in memory. While both of these tests exploit the inherent preference of mice for the novelty to reveal memory for previously encountered objects, the OLT primarily evaluates spatial learning, which relies heavily on hippocampal activity. The NORT, in contrast, evaluates non-spatial learning of object identity, which relies on multiple brain regions. Both tasks require an open-field-testing arena, objects with equivalent intrinsic value to mice, appropriate environmental cues, and video recording equipment and the software. Commercially available systems, while convenient, can be costly. This manuscript details a simple, cost-effective method for building the arenas and setting up the equipment necessary to perform the OLT and NORT. Furthermore, the manuscript describes an efficient testing protocol that incorporates both OLT and NORT and provides typical methods for data acquisition and analysis, as well as representative results. Successful completion of these tests can provide valuable insight into the memory function of various mouse model systems and appraise the underlying neural regions that support these functions.

Here we provide a protocol which includes comprehensive instructions for the economical establishment of murine object location and novel object recognition behavioral testing, including the design, cost, and construction of required equipment as well as execution of behavioral testing, data collection, and analysis.

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various ...

A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.

A comprehensive behavioral test battery of motor skills, mood&#8212;including social interaction, depression, and anxiety&#8212;and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and ...

Behavioral Assays for Autism Spectrum Disorder in Mouse Models

Strategies for Assessing Autistic-Like Behaviors in Mice

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social communication impairments and restrictive or repetitive behaviors, such as hand flapping or lining up objects. These behavioral patterns can be reliably observed in mouse models with ASD-linked genetic mutations, making them highly useful tools for studying the underlying cellular and molecular mechanisms in ASD. Understanding how genetic changes affect the neurobiology and behaviors observed in ASD will facilitate the development of novel targeted therapeutic compounds to ameliorate core behavioral impairments. Our lab has employed several protocols encompassing well-described training and testing procedures that reflect a wide range of behavioral deficits related to ASD. Here, we detail two assays to study the core features of ASD in mouse models: self-grooming (a measure of repetitive behavior) and the three-chamber social interaction test (a measure of social interaction approach and preference for social novelty).

Rodent models are valuable tools for studying core behaviors related to autism spectrum disorder (ASD). In this article, we expound on two behavioral tests for modeling the core features of ASD in mice: self-grooming, which assesses repetitive behavior, and the three-chamber social interaction test, which documents social impairments.

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

Medicine

A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution of specific genes to brain development and function increases. Relying on mouse models to study the role of specific genetic manipulations is not always feasible since transgenic mouse lines are quite costly and many novel disease-associated genes do not yet have commercially available genetic lines. Additionally, it can take years of development and validation to create a mouse line. In utero electroporation offers a relatively quick and easy method to manipulate gene expression in a cell-type specific manner in vivo that only requires developing a DNA plasmid to achieve a particular genetic manipulation. Bilateral in utero electroporation can be used to target large populations of frontal cortex pyramidal neurons. Combining this gene transfer method with behavioral approaches allows one to study the effects of genetic manipulations on the function of prefrontal cortex networks and the social behavior of juvenile and adult mice.

The role of recently discovered disease-associated genes in the pathogenesis of neuropsychiatric disorders remains obscure. A modified bilateral in utero electroporation technique allows for the gene transfer in large populations of neurons and examination of the causative effects of gene expression changes on social behavior.

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution ...

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

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