This work was supported by grants from the Department of Veterans Affairs and the National Institutes of Health.

All protocols using animals were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Care and Use Committee of Wayne State University (Permit Number A3310-01).
1.&#160;Preparation of Cages for Marble Burying and Nestlet Shredding
<ol>
	<li>Use standard polycarbonate rat cages (26 cm x 48 cm x 20 cm) with fitted filter-top covers for the marble burying test. Add fresh, unscented mouse bedding material to each cage to a depth of 5 cm and level bedding surface by inserting another cage of the same size onto the surface of the bedding. This has the added advantage of impressing a template of parallel lines on the bedding surface that can be used for marble placement.</li>
	<li>Place standard glass toy marbles (assorted styles and colors, 15 mm diameter, 5.2 g in weight) gently on the surface of the bedding in 5 rows of 4 marbles (see Figure 1A). Wash marbles in mild laboratory detergent, rinse exhaustively in distilled-deionized water, and dry prior to each use.</li>
	<li>Use standard polycarbonate rat cages (19 cm &#160;x 29 cm x 13 cm) with fitted filter-top covers for the nestlet shredding test. Add fresh, unscented mouse bedding material to each cage to a depth of 0.5 cm and pack bedding by insertion of another cage of the same size onto the surface of the bedding.</li>
	<li>Weigh commercially available cotton fiber nestlets (5 cm x 5 cm, 5 mm thick, ~2.5 g each) on an analytical balance and place one nestlet is placed on top of the bedding in each test cage (see Figure 3A).</li>
	<li>Set stop watches or timers for timing session lengths (30 min for each test).</li>
</ol>
2.&#160;Animal Handling Prior to Testing
<ol>
	<li>House mice individually or in groups in a room with a 12 hr light/dark cycle. Provide all mice with food and water ad libitum. Handle mice gently for three to five days prior to testing. Use mice of either sex for both tests.</li>
	<li>Perform behavioral testing between 9:00 am and 4:00 pm and transport all home cages containing mice to the testing room 60 min before the start of testing.</li>
</ol>
3.&#160;Marble Burying Test
<ol>
	<li>Place one mouse into a corner of the cage containing marbles, being careful to place the mouse on bedding as far from marbles as possible, and place the filter-top cover on the cage. Withhold food and water during the test. Allow mouse to remain in the cage undisturbed for 30 min.</li>
	<li>Remove the mouse and return it to its home cage after test completion, taking extreme care not to move or dislodge the marbles in the process of removing the subject from the cage.</li>
	<li>Task scorers (2-3) blind to the treatment conditions or genotype of the mouse being tested to count the number of marbles buried. Score a marble as buried if two-thirds of its surface area is covered by bedding. Average scores for the number of marbles buried for each mouse.</li>
	<li>Retrieve all 20 marbles from the bedding. Dispose of bedding.</li>
</ol>
4.&#160;Nestlet Shredding Test
<ol>
	<li>Place one mouse into a cage containing a single, preweighed nestlet and place the filter-top cover on the cage. Withhold food and water during the test. Leave mouse undisturbed in the cage with the nestlet for 30 min.</li>
	<li>Remove mouse and return to it to its home cage after test completion. Remove the remaining intact nestlet material from the cage with forceps and allow to dry O/N.</li>
	<li>Weigh remaining unshredded nestlet and divide this weight by the starting weight to calculate percentage&#160;of nestlet shredded. If desired, score the form of the nest produced by the subject mouse using a definitive 5-point nest rating scale17 but realizing that this scale is generally intended for scoring nest building, not obsessive-like shredding.</li>
	<li>Discard remaining shredded nestlet material and bedding.</li>
</ol>

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The authors declare that they have no competing financial interests.

The marble burying and nestlet shredding tests are good examples of behavioral methods for studying repetitive and compulsive-like behaviors in mice. Both tests show excellent face, construct and predictive validity for the human disorders they model6-11. These repetitive behaviors are natural and spontaneous in rodents and their frequency and intensity can vary after genetic modifications, as shown presently, or after head injury and treatment of subjects with drugs. The value of these two tests derives from ...

Mouse models of human behaviors, and particularly psychiatric disorders, constitute important and essential experimental approaches to the study of disease mechanisms and to the development of new therapies. Many psychiatric illnesses remain extremely difficult to model in animals (e.g.&#160;schizophrenia). However, animals exhibit a large number of natural and distinctive behaviors that can readily be linked to similar behaviors in humans. Movement or locomotor activity is one example of a behavior in animals that has a counterpart in humans. Rodents often engage in a variety of repetitive behaviors and examples include grooming or digging. One psychiatric condition that involves the exhibition of recurrent and unwanted behaviors in humans is obsessive-compulsive disorder (OCD). Behaviors that are highly characteristic of OCD include checking, counting, and excessive washing or grooming. OCD is a highly prevalent (affects 1-3% of the population) and debilitating psychiatric disorder1. A large number of pharmacological and behavioral approaches have been used to treat OCD. Many of these fall short of providing long-term relief and relapse is common at the end of a treatment regimen2,3. Another psychiatric condition that involves the expression repetitive behaviors is autism spectrum disorder (ASD). ASDs are characterized by deficits in communication and social interaction&#160;and repetitive, stereotyped behaviors to include spinning, rocking, finger flicking&#160;or flapping, and complex body movements. The Centers for Disease Control and Prevention has estimated that about 1 in 88 children are diagnosed with ASDs. Treatment options for this disorder are extremely limited in number and efficacy4,5. The intensity and frequency of occurrence of repetitive behaviors in OCD and ASD can become great enough to displace most other normal behaviors and social interactions in patients with these conditions. In light of the prevalence and debilitating nature of psychiatric conditions characterized by repetitive behaviors, and considering the poor understanding of their causes and limited treatment options, the use of animal models to study these behaviors takes on added urgency.
A large number of animal models of repetitive behaviors as seen in OCD and ASDs have been developed over the past 30 years and numerous excellent reviews describing these models have been published6-14. In general, these established models can be classified as genetic, pharmacological, neurodevelopmental and behavioral and most show good face, construct and predictive validity6,7. We have used two behavioral models- marble burying and nestlet shredding- to assess repetitive behaviors in mice genetically depleted of serotonin15,16. These methods have numerous advantages not the least of which are their ease of use, the accuracy with which the behaviors can be scored, the spontaneous exhibition of these behaviors in rodents, and their use in high-throughput screening of genetically modified mice for abnormal behavioral phenotypes. Furthermore, each test is carried out in standard and familiar rodent housing cages containing the same bedding used in subjects&#39; home cages, minimizing the need for habituation of mice to novel testing chambers. Food and/or water restriction are not required to &#34;motivate&#34; mice to perform. The marble burying test takes advantage of the proclivity of mice to dig in natural settings (e.g.&#160;burrows, escape tunnels) and in standard cage bedding, and the nestlet shred test capitalizes on the fact that mice are nesting animals by nature that build nests for protection of themselves and their offspring against environmental conditions (e.g.&#160;sound, light, temperature). Using nonspecialized and commonly available rodent housing materials, the marble burying and nestlet shredding tasks are simple and powerful behavioral assays of repetitive, compulsive-like behaviors in mice.

<table border="0" cellpadding="0" cellspacing="0" width="614">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass toy marbles</td>
			<td style="width:112px;">local toy store</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">assorted styles, colors, clear, and opaque</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nestlets</td>
			<td style="width:112px;">Ancare</td>
			<td style="width:119px;">NES3600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N10 series mouse cage</td>
			<td style="width:112px;">Ancare</td>
			<td style="width:119px;">N10HT</td>
			<td>standard rodent cage with wire top</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">R20 series rat cage</td>
			<td style="width:112px;">Ancare</td>
			<td style="width:119px;">R20</td>
			<td>standard rodent cage with wire top</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bed-o&#39;Cobs</td>
			<td style="width:112px;">The Andersons</td>
			<td style="width:119px;">8B</td>
			<td>standard rodent bulk bedding, 1/8 in</td>
		</tr>
	</tbody>
</table>

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.

A primary advantage of the marble burying and nestlet shredding tests is the fact that each depends on natural and spontaneous behaviors of mice that can be quantified after test completion, allowing a single investigator to set up an experiment and then remain outside of the testing room until sessions end. A critical component of the marble burying test is the careful removal of the subject from the cage after testing. Mice typically attempt to avoid capture and, in the process, can dislodge or move marbles that were p...

Watch this Scientific Journal Video about Marble Burying and Nestlet Shredding as Tests of Repetitive, Compulsive-like Behaviors in Mice at JoVE.com

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 disorder.

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

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27.7K Views.  Wayne State University School of Medicine. 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.