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 rectangu...

<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>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Standard Polycarbonate Rodent Cage (45 x 24 x 20 cm)</td>
			<td></td>
			<td></td>
			<td>Multiple cages are desirable to facilitate testing of multiple mice&#160;</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&#160;</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 2x4 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 colspan="2">Noldus, Wageningen, The Netherlands</td>
			<td>other programs may be used</td>
		</tr>
		<tr>
			<td>Video Tracking Software - EthoVision</td>
			<td colspan="2">Noldus, Wageningen, The Netherlands</td>
			<td>other programs may be used</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...

Watch this Scientific Journal Video about Novel Object Exploration as a Potential Assay for Higher Order Repetitive Behaviors in Mice at JoVE.com

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 ...

The overall goal of this behavioral assay is to identify the presence of higher order repetitive behaviors in mice. This assay can help validate potential mouse models and autistic like behavior, such as the inbred C 58 J mouse strain. The main advantages of this technique is that it does not require any mouse training and it is not particularly anxiety provoking.Begin by filling the testing arena. A clear plastic rectangular cage with a half inch of corn cob bedding. Select four novel objects that are different in shape and color.For example, a pink toy brick, a red monkey, a white tile with blue writing, and a standard white die. Ensure that these objects are approximately the same size and constructed with high density plastic to facilitate cleaning and resist chewing. In the rectangular arena, place these objects three centimeters from the corners in a random and counter-balanced order throughout testing.Record the placement of each object as a different number from one to four. Finally, position a camera directly above the testing arenas so that the entire arena is recorded during the acclimation and rest periods. Begin testing 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. Then, place a note card of known dimensions in the bottom of the testing arena for later use as a calibration standard. And push the record button on the video camera.Next, transfer a test mouse into the empty testing arena for 10 minutes to serve as an acclimation period and continue recording. After the acclimation period, leave the mouse in the arena and quickly add the four novel objects to the arena. Record mouse behavior for an additional 10 minutes during this testing period.Finally, after the testing period has completed, return the test mouse to its home cage. Clean and dry the novel objects as well as the testing arena with unscented dish soap and water. Begin by setting up the project coding scheme in the behavioral logging software.Within the project setup box, set data acquisition to offline, offline observation. Within the behavior coding box, program scrabble, digging, rearing, grooming, and sniff object one, two, three, and four as state events. Program jumping as a point event.To program the secondary keyboard, open the keyboard software, click on the secondary keyboard button displayed on the screen, type in the appropriate keystroke combination, and click okay. Once the secondary keyboard has been programmed, close the software and let the program run in the background of the computer. After the project has been set up in the software, prepare to score the number and duration of each of the following repetitive behaviors.Rears, digs, self-grooms, and jumps. To score a video, click file, open project, observe, observation, and new. Choose a name for the new file and then select the video media file to score.Begin scoring the repetitive behaviors by clicking on the begin observation button. Use two keystrokes for all state events. One to signify the initiation and the other for the end of the behavior.And use only one keystroke for point events. Next, use the software to score the number of times and length of duration the mouse sniffed each object, which is defined as any time a mouse moves its nose within 0.5 centimeters of an object. Each time a mouse sniffs an object, record the corresponding position number of the object and manually record the string of position numbers until the end of the 10 minute testing period.Count a position twice if a mouse sniffs an object, looks away, then sniffs the object again. After the 10 minute testing video has been scored for sniffs, visualize the data by clicking on analyze, behavior analysis, and new. Then, once the data appear on the screen, export into a separate spreadsheet.Next, record the total distance the mouse traveled within the arena during testing. To do this, use the note card to calibrate each video by setting a calibration line along each end of the note card and inputting the appropriate length within the software's calibration screen. After the lines are drawn, input the known length and width of the note card that correspond to each line.Next, within arena settings, select the entire arena. Within trail control settings, select a 10 minute duration. And in detection settings, choose a dark object on a light background.Once these settings have been programmed, score the videos by clicking acquisition and open acquisition. In the acquisition control box, click new trial. And then start trials.After 10 minutes have elapsed, stop the program and observe the data. Finally, click analyze and calculate statistics. After the data appear on the screen, export into a separate spreadsheet.The percentage of the number of times the three most common three pattern sequences were repeated were observed across three different inbred mouse strains. The C 58 females displayed a strong adherence to their preferred patterns in the round arena, but the not the rectangular arena. While male mice did not differ from each other in either arena.Further more, male and female C 58 mice in the round arena showed a stronger preference for their most preferred object than the other two strains. While no differences in object preference were shown in the rectangular arena. Once mastered, this technique can be completed in under one hour, with 15 minutes needed for data acquisition and 20 minutes needed for behavioral scoring.While attempting this procedure, it is important to remember that mice are very sensitive to changes in their surroundings. Please ensure that the testing environment remains consistent throughout the experiment. Following this procedure, other methods like the Barnes Maze or Team Maze can be used to further validate the presence of higher order repetitive behaviors.After watching this video, you should have a good understanding of how to use novel object exploration to identify the presence of higher order repetitive behaviors in mice.

novel-object-exploration-as-potential-assay-for-higher-order

Research

JoVE Journal

Behavior

8.2K vistas.  University of Redlands. The overall goal of this behavioral assay is to identify the presence of higher order repetitive behaviors in mice. This assay can help validate potential mouse models and autistic like behavior, such as the inbred C 58 J mouse strain. The main advantages of this technique is that it does not require any mouse training and it is not particularly anxiety provoking.Begin by filling the testing arena. A clear plastic rectangular cage with a half inch of corn cob bedding. Select four novel...

Video: 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...

marble burying and nestlet shredding as tests of repetitive, compulsive-like behaviors in mice

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

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...

novel object recognition and object location behavioral testing in mice on a budget

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

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...

novel object recognition test for the investigation of learning and memory in mice

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

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

strategies for assessing autistic-like behaviors in mice

Author Spotlight: Optimizing Behavioral Protocols and Investigating Molecular Mechanisms in Autism Spectrum Disorder

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...

Strategies for Assessing Autistic-Like Behaviors in Mice

repetitive tumor challenge assay: an in vitro culture assay to evaluate chimeric antigen receptor (car) t cells for repetitive tumor killing potential

In this video, we demonstrate the repetitive tumor challenge assay to evaluate the CAR T cells&#8217; repetitive tumor killing potential during high tumor cell loads. Further, the CAR T cells can be analyzed using flow cytometry to determine the different subpopulations of CAR T cells in the...

Repetitive Tumor Challenge Assay: An In Vitro Culture Assay to Evaluate Chimeric Antigen Receptor (CAR) T Cells for Repetitive Tumor Killing Potential

Resuspend the glioblastoma cells to a 1.6 times 10 to the fifth tumor cells per milliliter of fresh co-culture medium concentration and dilute T cells harvested from a CAR T cell culture to the appropriate percentage of CAR positive T cells per milliliter of co-culture medium...

the generation of higher-order laguerre-gauss optical beams for high-precision interferometry

The Generation of Higher-order Laguerre-Gauss Optical Beams for High-precision Interferometry

Large laser-interferometers are being constructed to create a new type of astronomy based on gravitational waves. Their sensitivities, as for many other high-precision experiments, are approaching fundamental noise limits such as the atomic vibration of their components. We are pioneering technologies to overcome these limits using novel laser beam...

modified fear conditioning for inducing flight behaviors in mice

Author Spotlight: Exploring Neural Correlates of Defensive Behaviors in Fear Learning and Extinction

To study flight behaviors in a fearful context, we introduce a modified fear conditioning protocol. This protocol ensures that mice consistently exhibit flight behaviors during cue presentation in the fear...

Modified Fear Conditioning for Inducing Flight Behaviors in Mice

covalent labeling with diethylpyrocarbonate for studying protein higher-order structure by mass spectrometry

Covalent Labeling with Diethylpyrocarbonate for Studying Protein Higher-Order Structure by Mass Spectrometry

The experimental procedures for performing diethylpyrocarbonate-based covalent labeling with mass spectrometric detection are described. Diethylpyrocarbonate is simply mixed with the protein or protein complex of interest, leading to the modification of solvent accessible amino acid residues. The modified residues can be identified after proteolytic digestion and liquid chromatography/mass spectrometry...

a repetitive concussive head injury model in mice

A Repetitive Concussive Head Injury Model in Mice

Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient...

laser-free hydroxyl radical protein footprinting to perform higher order structural analysis of proteins

Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins

This protocol presents a method to use inline radical dosimetry and a plasma light source to perform flash oxidation protein footprinting. This method replaces the hazardous UV laser to simplify and improve the reproducibility of fast photochemical oxidation of protein...

novel assay for cold nociception in drosophila larvae

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold...

Novel Assay for Cold Nociception in Drosophila Larvae

modeling highly repetitive low-level blast exposure in mice

Author Spotlight: Deciphering the Long-Term Effects of Low-Level Blast Exposures in Mice

Presented here are methods for producing repeated low-intensity blast exposures using...

Modeling Highly Repetitive Low-level Blast Exposure in Mice

assessment of spontaneous alternation, novel object recognition and limb clasping in transgenic mouse models of amyloid-&#946; and tau neuropathology

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

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...

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

asymmetric walkway: a novel behavioral assay for studying asymmetric locomotion

Asymmetric Walkway: A Novel Behavioral Assay for Studying Asymmetric Locomotion

Here, we present a protocol to quantify precise stepping in rodents. Cortical and the spinal central pattern generator signals are required for precise foot-placement during obstructed locomotion. We report here the novel constrained walking task that directly examines precise stepping...

observational fear as a model of affective empathy in mice

Author Spotlight: Evaluating Empathy-Like Traits in Rodents to Study the Generation of Emotional Contagion by Brain

The observational fear paradigm assesses vicarious freezing in rodents as a model for affective empathy. This procedure entails exposing observer rodents to conspecific demonstrators receiving aversive foot shocks to elicit empathic freezing responses. By employing observational fear assays, researchers can investigate the neural mechanisms underlying affective...

Observational Fear as a Model of Affective Empathy in Mice

a novel surgical technique as a foundation for in vivo partial liver engineering in rat

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

a within-subject experimental design using an object location task in rats

A Within-Subject Experimental Design using an Object Location Task in Rats

This protocol provides detailed steps for an object location task with four repetitions using the same cohort of rats. Weak and strong encoding can produce short- and long-term memories. The flexibility of the protocol with repetition can be beneficial for studies involving surgical operations by saving time and...

limited bedding and nesting as a model for early-life adversity in mice

Author Spotlight: Exploring Microglial Interactions with Stress-Response Circuitry Using the Limited Bedding and Nesting Model

This protocol describes an animal model for studying how early-life adversity, provoked by an impoverished environment and unpredictable maternal care during the early postnatal period, affects brain development and the future risk of mental...

Limited Bedding and Nesting as a Model for Early-Life Adversity 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 ...

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 ...

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for quantifying locomotor deficits in rodents is to measure minute disturbances of unconstrained gait overground (e.g., manual BBB score or automated CatWalk). However, cortical inputs are not required for the generation of basic locomotion produced by the spinal central pattern generator (CPG). Thus, unconstrained walking tasks test locomotor deficits due to motor cortical impairment only indirectly. In this study, we propose a novel, precise foot-placement locomotor task that evaluates cortical inputs to the spinal CPG. An instrumented peg-way was used to impose symmetrical and asymmetrical locomotor tasks mimicking lateralized movement deficits. We demonstrate that shifts from equidistant inter-stride lengths of 20% produce changes in the forelimb stance phase characteristics during locomotion with preferred stride length. Furthermore, we propose that the asymmetric walkway allows for measurements of behavioral outcomes produced by cortical control signals. These measures are relevant for the assessment of impairment after cortical damage.

Here, we present a protocol to quantify precise stepping in rodents. Cortical and the spinal central pattern generator signals are required for precise foot-placement during obstructed locomotion. We report here the novel constrained walking task that directly examines precise stepping behavior.

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for ...

Neuronavigation-guided Repetitive Transcranial Magnetic Stimulation for Aphasia

Repetitive transcranial magnetic stimulation (rTMS) is widely used for several neurological conditions, as it has gained acknowledgement for its potential therapeutic effects. Brain excitability is non-invasively modulated by rTMS, and rTMS to the language areas has proved its potential effects on treatment of aphasia. In our protocol, we aim to artificially induce virtual aphasia in healthy subjects by inhibiting Brodmann area 44 and 45 using neuronavigational TMS (nTMS), and F3 of the International 10-20 EEG system for conventional TMS (cTMS). To measure the degree of aphasia, changes in reaction time to a picture naming task pre- and post-stimulation are measured and compare the delay in reaction time between nTMS and cTMS. Accuracy of the two TMS stimulation methods is compared by averaging the Talairach coordinates of the target and the actual stimulation. Consistency of stimulation is demonstrated by the error range from the target. The purpose of this study is to demonstrate use of nTMS and to describe the benefits and limitations of the nTMS compared to those of cTMS.

This study is designed to test the hypothesis that neuronavigational system-guided transcranial magnetic stimulation has higher accuracy for targeting the intended target as demonstrated by eliciting a greater degree of virtual aphasia in healthy subjects, measured by delay in reaction time to picture naming.

Repetitive transcranial magnetic stimulation (rTMS) is widely used for several neurological conditions, as it has gained acknowledgement for its potential ...

Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous research determined a profound impact of gentle handling protocols on the well-being of laboratory mice. By introducing clicker training to the keeping of mice, not only do we promote the amicable treatment of mice, but we also enable them to experience cognitive enrichment. Clicker training is a form of positive reinforcement training using a conditioned secondary reinforcer, the "click" sound of a clicker, which serves as a time bridge between the strengthened behavior and an upcoming reward. The effective implementation of the clicker training protocol with a cohort of 12 BALB/c inbred mice of each sex proved to be uncomplicated. The mice learned rather quickly when challenged with tasks of the clicker training protocol, and almost all trained mice overcame the challenges they were given (100% of female mice and 83% of male mice). This study has identified that clicker training for mice strongly correlates with reduced fear in the mice during human-mice interactions, as shown by reduced anxiety-related behaviors (e.g., defecation, vocalization, and urination) and fewer depression-like behaviors (e.g., floating). By developing a reliable protocol that can be easily integrated into the daily routine of the keeping of laboratory mice, the lifetime experience of welfare in the mice can be improved substantially.

The development of new refinement strategies for laboratory mice is a challenging task that contributes towards fulfilling the 3R principle. This protocol introduces clicker training as a cognitive enrichment program for laboratory mice.

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous ...

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 ...

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 ...

Defining the Role Of Language in Infants' Object Categorization with Eye-tracking Paradigms

Assessing infant category learning is a challenging but vital aspect of studying infant cognition. By employing a familiarization-test paradigm, we straightforwardly measure infants&#8217; success in learning a novel category while relying only on their looking behavior. Moreover, the paradigm can directly measure the impact of different auditory signals on the infant categorization across a range of ages. For instance, we assessed how 2-year-olds learn categories in a variety of labeling environments: in our task, 2-year-olds successfully learned categories when all exemplars were labeled&#160;or the first two exemplars were labeled, but they failed to categorize when no exemplars were labeled or only the final two exemplars were labeled. To determine infants&#8217; success in such tasks, researchers can examine both the overall preference displayed by infants in each condition and infants&#8217; pattern of looking over the course of the test phase, using an eye-tracker to provide fine-grained time-course data. Thus, we present a powerful paradigm for identifying the role of language, or any auditory signal, in infants&#8217; object category learning.

Defining the Role Of Language in Infants&#039; Object Categorization with Eye-tracking Paradigms

Here we present a protocol for familiarization-test paradigms which provide a direct test of infant categorization and help to define the role of language in early category learning.

Assessing infant category learning is a challenging but vital aspect of studying infant cognition. By employing a familiarization-test paradigm, we ...

Nest Building Behavior as an Early Indicator of Behavioral Deficits in Mice

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents synthetic and/or natural materials (such as twine, tissue, cotton, paper, and hay) as a gauge of their overall well-being and as an ancillary assessment to predict the possible decline in cognition. Typically, changes in nesting behaviors, such as failure to create a nest, indicate a change in health or welfare. In addition, nesting behavior is sensitive to many environmental and physiological challenges, as well as many genetic mutations underlying pathological disease states. The following protocol describes a nesting behavior paradigm that explores the usage of four types of nesting material. In addition, the protocol utilizes intraclass correlations to demonstrate that inter-rater reliability is higher when nests are constructed out of shredded paper compared to other common nesting materials such as cotton squares, paper twists, and soft cob bedding. The chosen methodology and statistical considerations (i.e., intraclass correlation) for this assay may be of interest for those conducting experiments assessing the quality of living of mice.&#160;

Here, we present a protocol to quantify nest building behavior in mice, which is known to be impaired in several neurological disorders and diseases. This protocol examines the utility of four materials and offers the opportunity to quantify the rater agreement in scoring, improving the validity and reliability of the assay.