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>

<ol>
	<li>Chamberlain, S. R., Blackwell, A. D., Fineberg, N. A., Robbins, T. W., Sahakian, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuropsychology+of+obsessive+compulsive+disorder:+the+importance+of+failures+in+cognitive+and+behavioural+inhibition+as+candidate+endophenotypic+markers.">The neuropsychology of obsessive compulsive disorder: the importance of failures in cognitive and behavioural inhibition as candidate endophenotypic markers.</a> Neurosci. Biobehav. Rev. 29, 399-419 (2005).</li><li>McGuire, J. F., Lewin, A. B., Horng, B., Murphy, T. K., Storch, E. 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+nature,+assessment,+and+treatment+of+obsessive-compulsive+disorder.">The nature, assessment, and treatment of obsessive-compulsive disorder.</a> Postgrad. Med. 124, 152-165 (2012).</li><li>Fineberg, N. A., Brown, A., Reghunandanan, S., Pampaloni, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence-based+pharmacotherapy+of+obsessive-compulsive+disorder.">Evidence-based pharmacotherapy of obsessive-compulsive disorder.</a> Int. J. Neuropsychopharmacol. 15, 1173-1191 (2012).</li><li>Dove, D., 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=Medications+for+adolescents+and+young+adults+with+autism+spectrum+disorders:+a+systematic+review.">Medications for adolescents and young adults with autism spectrum disorders: a systematic review.</a> Pediatrics. 130, 717-726 (2012).</li><li>Doyle, C. A., McDougle, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacotherapy+to+control+behavioral+symptoms+in+children+with+autism.">Pharmacotherapy to control behavioral symptoms in children with autism.</a> Expert Opin. Pharmacother. 13, 1615-1629 (2012).</li><li>Albelda, N., Joel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+animal+models+of+obsessive+compulsive+disorder:+an+update.">Current animal models of obsessive compulsive disorder: an update.</a> Neuroscience. 211, 83-106 (2012).</li><li>Albelda, N., Joel, D. <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+obsessive-compulsive+disorder:+Exploring+pharmacology+and+neural+substrates.">Animal models of obsessive-compulsive disorder: Exploring pharmacology and neural substrates.</a> Neurosci. Biobehav. Rev. 36, 47-63 (2012).</li><li>Wang, L., Simpson, H. B., Dulawa, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+validity+of+current+mouse+genetic+models+of+obsessive-compulsive+disorder.">Assessing the validity of current mouse genetic models of obsessive-compulsive disorder.</a> Behav. Pharmacol. 20, 119-133 (2009).</li><li>Joel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+animal+models+of+obsessive+compulsive+disorder:+a+critical+review.">Current animal models of obsessive compulsive disorder: a critical review.</a> Prog. Neuropsychopharmacol. Biol. Psychiatry. 30, 374-388 (2006).</li><li>Witkin, 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=Animal+models+of+obsessive-compulsive+disorder.">Animal models of obsessive-compulsive disorder.</a> Curr. Protoc. Neurosci. 30, 1-9 (2008).</li><li>Deacon, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digging+and+marble+burying+in+mice:+simple+methods+for+in+vivo+identification+of+biological+impacts.">Digging and marble burying in mice: simple methods for in vivo identification of biological impacts.</a> Nat. Protoc. 1, 122-124 (2006).</li><li>Moy, S. S., Nadler, J. J., Magnuson, T. R., 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=Mouse+models+of+autism+spectrum+disorders:+the+challenge+for+behavioral+genetics.">Mouse models of autism spectrum disorders: the challenge for behavioral genetics.</a> Am. J. Med. Genet. C Semin. Med. Genet. 142, 40-51 (2006).</li><li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+behavioral+assays+relevant+to+the+symptoms+of+autism.">Mouse behavioral assays relevant to the symptoms of autism.</a> Brain Pathol. 17, 448-459 (2007).</li><li>Silverman, J. L., Yang, M., Lord, C., Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+phenotyping+assays+for+mouse+models+of+autism.">Behavioural phenotyping assays for mouse models of autism.</a> Nat. Rev. Neurosci. 11, 490-502 (2010).</li><li>Angoa-Perez, 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=Genetic+depletion+of+brain+5HT+reveals+a+common+molecular+pathway+mediating+compulsivity+and+impulsivity.">Genetic depletion of brain 5HT reveals a common molecular pathway mediating compulsivity and impulsivity.</a> J. Neurochem. 121, 974-984 (2012).</li><li>Kane, M. J., 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=Mice+genetically+depleted+of+brain+serotonin+display+social+impairments,+communication+deficits+and+repetitive+behaviors:+possible+relevance+to+autism.">Mice genetically depleted of brain serotonin display social impairments, communication deficits and repetitive behaviors: possible relevance to autism.</a> PLoS One. 7, (2012).</li><li>Deacon, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+nest+building+in+mice.">Assessing nest building in mice.</a> Nat. Protoc. 1, 1117-1119 (2006).</li><li>Thomas, D. M., Angoa Perez, ., Francescutti-Verbeem, D. M., Shah, M. M., Kuhn, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+endogenous+serotonin+in+methamphetamine-induced+neurotoxicity+to+dopamine+nerve+endings+of+the+striatum.">The role of endogenous serotonin in methamphetamine-induced neurotoxicity to dopamine nerve endings of the striatum.</a> J. Neurochem. 115, 595-605 (2010).</li><li>Gyertyan, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+marble+burying+response:+marbles+serve+to+measure+digging+rather+than+evoke+burying.">Analysis of the marble burying response: marbles serve to measure digging rather than evoke burying.</a> Behav. Pharmacol. 6, 24-31 (1995).</li><li>Thomas, A., 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=Marble+burying+reflects+a+repetitive+and+perseverative+behavior+more+than+novelty-induced+anxiety.">Marble burying reflects a repetitive and perseverative behavior more than novelty-induced anxiety.</a> Psychopharmacology. 204, 361-373 (2009).</li><li>Hayashi, E., Kuratani, K., Kinoshita, M., Hara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologically+distinctive+behaviors+other+than+burying+marbles+during+the+marble+burying+test+in+mice.">Pharmacologically distinctive behaviors other than burying marbles during the marble burying test in mice.</a> Pharmacology. 86, 293-296 (2010).</li></ol>

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 ...

marble-burying-nestlet-shredding-as-tests-repetitive-compulsive-like

Research

JoVE Journal

Behavior

28.0K visualizações.  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.

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

Shallow Water (Paddling) Variants of Water Maze Tests in Mice

When Richard Morris devised his water maze in 19817, most behavioral work was done in rats. However, the greater understanding of mouse genetics led to the mouse becoming increasingly important. But researchers found that some strains of mutant mice were prone to problems like passively floating or diving when they were tested in the Morris water maze11. This was unsurprising considering their natural habitat; rats swim naturally (classically, the "sewer rat"), whereas mice evolved in the dry areas of central Asia.
To overcome these problems, it was considered whether shallow water would be a sufficient stimulus to provide escape motivation for mice. This would also avoid the problems of drying the small creatures with a towel and then putting them in a heated recovery chamber to avoid hypothermia, which is a much more serious problem than with rats; the large ratio of surface area to volume of a mouse makes it particularly vulnerable to rapid heat loss.
Another consideration was whether a more natural escape strategy could be used, to facilitate learning. Since animals that fall into water and swim away from the safety of the shore are unlikely to pass on their genes, animals have evolved a natural tendency to swim to the edge of a body of water. The Morris water maze, however, requires them to swim to a hidden platform towards the center of the maze - exactly opposite to their evolved behavior. Therefore the paddling maze should incorporate escape to the edge of the apparatus. This feature, coupled with the use of relatively non-aversive shallow water, embodies the "Refinement" aspect of the "3 Rs" of Russell and Burch8.
Various types of maze design were tried; the common feature was that the water was always shallow (2 cm deep) and escape was via a tube piercing the transparent wall of the apparatus. Other tubes ("false exits") were also placed around the walls but these were blocked off. From the inside of the maze all false exits and the single true exit looked the same. Currently a dodecagonal (12-sided) maze is in use in Oxford, with 12 true/false exits set in the corners. In a recent development a transparent paddling Y-maze has been tested successfully.

Shallow Water Paddling Variants of Water Maze Tests in Mice

Mice can swim, but many strains appear to find this activity stressful. To overcome this problem mazes have been devised where escape from shallow water is used to motivate behaviour. These have been demonstrated to support learning at least as good as the traditional and widely used Morris water maze.

When Richard Morris devised his water maze in 19817, most behavioral work was done in rats. However, the greater understanding of mouse genetics led to ...

Nest Building as an Indicator of Health and Welfare in Laboratory Mice

The minimization and alleviation of suffering has moral and scientific implications. In order to mitigate this negative experience one must be able to identify when an animal is actually in distress. Pain, illness, or distress cannot be managed if unrecognized. Evaluation of pain or illness typically involves the measurement of physiologic and behavioral indicators which are either invasive or not suitable for large scale assessment. The observation of nesting behavior shows promise as the basis of a species appropriate cage-side assessment tool for recognizing distress in mice. Here we demonstrate the utility of nest building behavior in laboratory mice as an ethologically relevant indicator of welfare. The methods presented can be successfully used to identify thermal stressors, aggressive cages, sickness, and pain. Observation of nest building behavior in mouse colonies provides a refinement to health and well-being assessment on a day to day basis.

We demonstrate the utility of nest building behavior in laboratory mice as an indicator of welfare. Nest scoring is a sensitive technique that is altered by temperature, illness, and aggression. The time to integrate into nest test (TINT) is a simple cage-side assessment that can detect postoperative pain.

The minimization and alleviation of  suffering has moral and scientific implications. In order to mitigate this  negative experience one must be able to ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

A Protocol of Manual Tests to Measure Sensation and Pain in Humans

Numerous qualitative and quantitative techniques can be used to test sensory nerves and pain in both research and clinical settings. The current study demonstrates a quantitative sensory testing protocol using techniques to measure tactile sensation and pain threshold for pressure and heat using portable and easily accessed equipment. These techniques and equipment are ideal for new laboratories and clinics where cost is a concern or a limiting factor. We demonstrate measurement techniques for the following: cutaneous mechanical sensitivity on the arms and legs (von-Frey filaments), radiant and contact heat sensitivity (with both threshold and qualitative assessments using the Visual Analog Scale (VAS)), and mechanical pressure sensitivity (algometer, with both threshold and the VAS). The techniques and equipment described and demonstrated here can be easily purchased, stored, and transported by most clinics and research laboratories around the world. A limitation of this approach is a lack of automation or computer control. Thus, these processes can be more labor intensive in terms of personnel training and data recording than the more sophisticated equipment. We provide a set of reliability data for the demonstrated techniques. From our description, a new laboratory should be able to set up and run these tests and to develop their own internal reliability data.

The goal of this procedure is to demonstrate a battery of quantitative techniques for sensory and pain measurement in humans. The equipment and techniques described are commonly found in pain clinics or are easy to obtain.

Numerous qualitative and quantitative techniques can be used to test sensory nerves and pain in both research and clinical settings. The current study ...

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

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

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

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

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 ...

Battery of Behavioral Tests Assessing General Locomotion, Muscular Strength, and Coordination in Mice

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the animal&#39;s wellbeing and health. There are numerous behavioral tests that may be applied. We selected a test for general locomotion, the open field test (OFT); a test for muscular strength, the mesh test (MT); and a test for coordination, the rotarod test (RR). Testing can be accomplished on a weekly or monthly basis. As a test for general locomotion, the OFT works by objectively monitoring movement parameters while the mouse is in an open field apparatus. The field is generally a 2&#39; x 2&#39; box, and the movements are recorded through laser sensing or through video capture. The mouse is placed in the center of the open field and allowed to move freely for the test. The MT uses the latency for a mouse to fall off an inverted screen as a measure of muscular strength. A mouse is placed on a screen, which is inverted over a clear box, and is timed for their latency to fall. Three trials are performed, with the best of the three trials scored for that day. A score of 60 s is the maximum time a mouse is left inverted. Mice are given a 5-min rest period between mesh test trials. Lastly, an accelerated protocol on the RR assesses motor coordination and endurance. During a trial, a mouse walks on a rotating rod as it increases in speed from 4 rpm to 40 rpm over 5 min. The trial ends when the mouse touches the magnetized pressure sensor upon falling. Each mouse undergoes three trials, and the best trial is scored for that day. This combined behavioral data allows for the global assessment of mobility, coordination, strength, and movement of the test animals. At least two out of the three behavioral testing measures must show improvement for an animal to qualify as having overall improved motor function.

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects of a disease or treatment on an animal's wellbeing. In order to globally assess motor functioning, we selected tests for general locomotion, muscular strength, and coordination: the open field test, the mesh test, and the rotarod test, respectively.

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the ...

Assessment of Social Transmission of Food Preferences Behaviors

Olfactory recognition deficits are suggested to be able to serve as clinical marker to differentiate Alzheimer&#39;s disease (AD) subjects from healthy aging groups. For example, olfactory dysfunction in AD can present as impairment in olfactory recognition, emerging during early stages of the disease and worsening while the disease progresses. The social transmission of food preferences (STFP) task is based on a rudimentary form of communication between rodents concerning distant foods dependent on the transmission of olfactory cues. Healthy wild-type mice would prefer to eat a novel, flavored food that was previously cued by a conspecific, and this food preference would be hampered in transgenic AD mice, such as the APP/PS1 model. Indeed, a strong preference for the cued food in C57Bl6/J mice of 3 months of age was found, and this was reduced in 3 months old transgenic APP/PS1 mice. In summary, STFP task could be a powerful measure to be integrated in present subclinical detection assays of AD.

This paper presents a protocol for the investigation of social transmission of food preference in mice. The advantages and possible applications for this procedure, for instance, in detecting early changes in AD mouse models, are highlighted. To conclude, interpretation of the results in light of critical details are discussed.

Olfactory recognition deficits are suggested to be able to serve as clinical marker to differentiate Alzheimer&#39;s disease (AD) subjects from healthy ...

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.

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents ...

Nestlet Shredding: Nest Building Test in an Angelman Syndrome Mouse Model

Take a cotton nestlet with forceps and record its weight manually using scales. Place the nest lit randomly in a cage but on the opposite side of the water supply. Weigh each nestlet for the next four days manually using scales.Ensure that the nestlet is dry when weighed. If not dry on a heating pad. If the nestlet is torn into several parts, weigh the largest part.Record the weights on paper or in a pre-made spreadsheet. The UBE3A mutant animals used significantly less material to build their nests and this difference was particularly prominent between transgenic females and their control counterparts.