Name: novel object exploration as a potential assay for higher order repetitive behaviors in mice
Uploaded: 2016-08-20T13:00:00
Description: Watch this Scientific Journal Video about Novel Object Exploration as a Potential Assay for Higher Order Repetitive Behaviors in Mice at JoVE.com

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...

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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.

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