We would like to thank The New Hope Resarch&#160;Foundation for their continued support and funding of our research. We would also like to thank IITC for partial financial support for this work.

All protocols herein described were performed in accordance with, and were approved by, the Queen&#39;s University Animal Care Committee. The frequency of behavioral testing can be weekly or monthly, or a combination of the two (i.e., weekly when one expects to see the biggest difference, and monthly elsewhere).
NOTE: Murine behavioral studies are largely sex/age/strain- dependent, and therefore it is essential to collect the proper time points and to remain consistent across experimental groups. Environmental factors (e.g., scents, noise, temperature, humidity lighting, etc.) greatly affect behavior and anxiety in mice. Try to reduce these environmental factors as much as possible.
1. Open Field Test
NOTE: Prior to behavioral testing, the animals should be habituated to the room where the testing is to take place for 10 - 15 min prior to the beginning of testing. Ensure that proper personal protective equipment and all local facility guidelines are followed for the behavioral testing protocols.
NOTE: No training prior to the test.
<ol>
	<li>Ensure that the computer and OFT program are on, running, and properly connected to the camera or open field box. Click on the smartware program, the open field icon, and &#34;OK.&#34; Chose the &#34;digital analog converter&#34; source from the camera options. Click on &#34;detection&#34; and make sure that the whole open field space is visualized and that the dimensions are 30 cm x 30 cm. 
	NOTE: Equipment layout may vary depending on space constraints, but ensure that the computer and open field apparatus are near to each other in a well-lit room for proper video capture.</li>
	<li>Click &#34;calibration,&#34; &#34;snapshot,&#34; and then &#34;OK&#34; to make sure that the mouse is being detected within the confines of the open field. Click &#34;time&#34; to pre-set an amount of time to test the mice; in this case, set 5 min.</li>
	<li>Enter the subjects by clicking the &#34;subjects&#34; icon. Click &#34;scheduler&#34; and highlight all test subjects and then click the double green arrows and &#34;phase one, session one;&#34; this will bring in all of the subjects in order to begin collecting data.</li>
	<li>Place a mouse in the center of the open field and click the &#34;start&#34; icon to prompt the program to begin recording. Test only one mouse at a time.</li>
	<li>Once 5 min is complete, the program will automatically move to the next subject. In this case, remove the mouse that has completed the test and place the next subject into the open field. Click &#34;start.&#34;</li>
	<li>Wipe down the equipment. Repeat steps 1.3 - 1.4 for the remaining test subjects.</li>
	<li>In order to analyze the data once all the subjects have gone through testing, click on the &#34;analyze&#34; icon at the top right of the screen. Click &#34;analyze&#34; again and then &#34;summary report.&#34; Optionally, export the data onto a USB.</li>
</ol>
2. Mesh Test
NOTE: The surface area and size of the mesh on the MT may influence performance. On the MT, if a mouse falls off within the first 10 s during the first 2 collection time points, they will be put back on the mesh immediately for a second attempt. The mesh used herein was a wire mesh, with the mesh holes approximately 1.5 mm in diameter.
NOTE: Running these tests at the same time of day is recommended, since physiological and biochemical parameters fluctuate throughout the day. Bodyweight may influence performance in this test and therefore must be taken into consideration.
<ol>
	<li>Place a mouse in the center of a mesh and invert the mesh at least 20 cm above a clear container. Once inverted, start the timer.</li>
	<li>Time and record the time of latency to fall. If the mouse does not fall within 60 s, give it a score of 60 s and conduct no further trials. Allow the mouse to rest for at least 5 min. This is the end of trial 1.</li>
	<li>Wipe down the equipment and during the 5-min inter-trial interval, test a second mouse, if required. Repeat steps 2.1 - 2.2 for trials 2 and 3, if necessary (as long as the mouse does not score 60 s, it should undergo three trials).</li>
</ol>
3. Rotarod Test
NOTE: The surface and diameter of the rod on the RT may influence performance. On the RT, if a mouse falls off within the first 10 s during the first 2 collection time points, put it back on the rod immediately for a second attempt.
NOTE: Running these tests at the same time of day is recommended, since physiological and biochemical parameters fluctuate throughout the day. Bodyweight may influence performance in this test and therefore must be taken into consideration.
<ol>
	<li>Turn on the IITC rotarod and computer. Ensure that they are properly connected for data recording.</li>
	<li>Make sure that the program is running the accelerated protocol. Set the desired length of the rotarod test to xxx (the valid range is 000 through 999); in this case, press 300*. Set the number of lanes to be used to xxx (the valid range is 001 through 005); in this case, press 005*. Set the starting speed to xxx rpms (enter 3005 for 5 rpms; the valid range is 001 through 045); in this case, press 004*.</li>
	<li>Set the top speed to xxx rpms (enter 4,030 for 30 rpms; the valid range is 001 through 045); in this case, press 040*. Set the ramp speed to xxx in s (enter 5,030 for a 30-s ramp to top speed; the valid range is 000 through 999); in this case press 300*. Set the unit to mouse mode (enter 6001 for mouse forward and 6002 for mouse reverse); in this case, press 6001*.</li>
	<li>Ensure that all magnetized lanes are balanced before beginning the trial. Place one mouse in each lane of the rotarod.</li>
	<li>Press start to initiate the program. As the mice fall, remove them from their lanes promptly to ensure that they do not &#34;trip&#34; the other lanes.</li>
	<li>Allow the mice to rest for a 10-min inter-trial interval. This is the end of trial 1. During the inter-trial interval, wipe down the equipment and begin the trial for the next set of mice.</li>
	<li>Repeat steps 4 - 6 as necessary. If a mouse remains on the rotarod for the full 5-min trial, no further trials are required for that mouse.</li>
</ol>

<ol>
	<li>Osmon, K. 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=Systemic+Gene+Transfer+of+a+Hexosaminidase+Variant+Using+an+scAAV9.47+Vector+Corrects+GM2+Gangliosidosis+in+Sandhoff+Mice.">Systemic Gene Transfer of a Hexosaminidase Variant Using an scAAV9.47 Vector Corrects GM2 Gangliosidosis in Sandhoff Mice.</a> Human Gene Therapy. 27 (7), 497-508 (2016).</li><li>Brooks, S. P., Dunnett, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tests+to+assess+motor+phenotype+in+mice:+a+user's+guide.">Tests to assess motor phenotype in mice: a user's guide.</a> Nature Reviews, Neuroscience. 10, 519-529 (2009).</li><li>Ma, M., Basso, D. M., Walters, P., Stokes, B. T., Moser, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+and+histological+outcomes+following+graded+spinal+cord+contusion+injury+in+the+C57Bl/6+mouse.">Behavioural and histological outcomes following graded spinal cord contusion injury in the C57Bl/6 mouse.</a> Exp. Neurol. 169, 239-254 (2001).</li><li>Menalled, L. B., 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=Early+motor+dysfunction+and+striosomal+distribution+of+huntingtin+microaggregates+in+Huntington's+disease+knock-in+mice.">Early motor dysfunction and striosomal distribution of huntingtin microaggregates in Huntington's disease knock-in mice.</a> J.Neurosci. 22, 8266-8276 (2002).</li><li>Grusser, C., Grusser-Cornehls, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+in+motor+performance+of+Weaver+mutant+mice+following+lesion+of+the+cerebellum.">Improvement in motor performance of Weaver mutant mice following lesion of the cerebellum.</a> Behav. Brain Res. 97, 189-194 (1998).</li><li>De Leonibus, E., 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=Spatial+deficits+in+a+mouse+model+of+Parkinson's+disease.">Spatial deficits in a mouse model of Parkinson's disease.</a> Psychopharmacology (Berl). 194, 517-525 (2007).</li><li>Morris, M., Koyama, A., Masliah, E., Mucke, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+Reduction+Does+Not+Prevent+Motor+Deficits+in+Two+Mouse+Models+of+Parkinson's+Disease.">Tau Reduction Does Not Prevent Motor Deficits in Two Mouse Models of Parkinson's Disease.</a> PLoS ONE. 6 (12), 1-7 (2011).</li><li>Abo-ouf, 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=Deletion+of+tumour+necrosis+factor-&#945;+ameliorates+neurodegeneration+in+Sandhoff+disease+mice.">Deletion of tumour necrosis factor-&#945; ameliorates neurodegeneration in Sandhoff disease mice.</a> Human Molecular Genetics. , 1-16 (2013).</li><li>Dumont, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+Phenotyping+of+Mouse+Models+of+Neurodegeneration.">Behavioral Phenotyping of Mouse Models of Neurodegeneration.</a> Neurodegeneration : Methods and Protocols, Methods in Molecular Biology. 793, 229-237 (2011).</li><li>Cabe, P. A., Tilson, H. A., Mitchell, C. L., Dennis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+recording+grip+strength+device.">A simple recording grip strength device.</a> Pharmacol. Biochem. 8, 101-102 (1978).</li><li>Barn&#275;oud, P., Loliver, J., Sanger, D., Moser, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+motor+assessment+in+FALS+mice:+a+longitudinal+study.">Quantitative motor assessment in FALS mice: a longitudinal study.</a> Neuroreport. 8, 2861-2865 (1997).</li><li>Lui, Y., 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+model+of+GM2+activator+deficiency+manifests+cerebellar+pathology+and+motor+impairment.">Mouse model of GM2 activator deficiency manifests cerebellar pathology and motor impairment.</a> Proc. Natl. Acad. Sci. 94, 8133-8143 (1997).</li><li>Sango, K., 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+models+of+Tay-Sachs+and+Sandhoff+diseases+differ+in+neurologic+phenotype+and+ganglioside+metabolism.">Mouse models of Tay-Sachs and Sandhoff diseases differ in neurologic phenotype and ganglioside metabolism.</a> Nature Genetics. 11 (2), 170-176 (1995).</li><li>Jones, B. J., Roberts, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rotarod+suitable+for+quantitative+measurements+of+motor+inco-ordination+in+na&#239;ve+mice.">A rotarod suitable for quantitative measurements of motor inco-ordination in na&#239;ve mice.</a> Nauyn-Schmiedebergs Arch. Pharmacol. 259 (2), 211 (1968).</li><li>Deacon, R. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+Strength+of+Mice.">Measuring the Strength of Mice.</a> Journal of Visualized Experiments JoVE. (76), (2013).</li></ol>

The authors have nothing to disclose.

For all tests, Clean and disinfect the surface to minimize contagions and distractors to the mice.&#160;Also, make certain that the mice are given sufficient rest periods between trials and between behavioral testing when using the OFT, RR, and MT.
In the OFT, it is critical that each mouse is placed in the middle of the open field and that the program is started upon the release of the mouse into the center. Be sure that there are no hand movements the sensors may register upon starting the p...

The goal of behavioral testing is to measure phenotypic characterization, whether in studies for a new mouse model of diseases affecting the central nervous system or for a trial of a new treatment. There are a variety of behavioral tests to choose from. The rationale for using a combination of behavioral tests is often to fully assess the pre-clinical treatment outcomes. The battery of behavioral testing herein includes the open field test (OFT), the mesh test (MT), and the rotarod test (RR).
The OFT is used as a measure of general locomotor activity1,2,3,4,5,6,7. This assessment allows the mouse to move freely in an open field, and the measurements taken are the movements of the mouse over the course of the behavioral trial. The quantitative analyses made using an OFT include rearing, distance moved, time spent moving (e.g., walking and running), and changes in activity over time (e.g., jumping and slower/hyperactive movements), which is why it is a useful and thorough analysis of rodent behavior.
The MT is used to assess the strength of the mice1,2,8,9,10,11. The use of all four limbs to hang off of a wire grid mesh allows for the non-invasive measurement of the ability of the mice to show sustained limb tension while opposing the effects of gravity. This assessment is done by placing a mouse on an inverted mesh and timing its latency to fall. This test assesses the muscular strength of the mouse by measuring how long it can hold itself onto the inverted screen before falling off.
The RR assesses the coordination of a mouse while using the accelerating protocol1,2,7,8,9,12,13. The rotarod apparatus consists of a cylindrical beam that rotates in the air starting at 4 rpm. The mouse can learn this motor coordination skill and is then challenged by slow, incremental increases up to 40 rpm. Classically, the constant speed protocol is a measure of muscular strength, whereas the accelerating protocol for the RR assesses coordination, endurance, and muscular strength1,2,7,14.
The advantages of these three behavioral tests are that they are widely accepted throughout the rodent behavioral testing community2,7,8,9, and when taken not just as the sum of the individual tests, but collectively, they can be used as a global assessment of phenotypic behavior when investigating the different outcomes of experimental treatments. The OFT is one of the only tests for overall locomotor activity2. The RR is also one of the only ways to assess coordination2, other than to use gait analysis software, which will not be discussed here. There are a few ways in which the muscular strength of a mouse can be assessed; however, using the MT, or a wire hang test, has been prevalently used in the literature and is accepted as a reliable method for testing muscular strength2,8,9,10,11. Many other techniques for measuring general locomotion and coordination are extremely time consuming and may not produce reliable data (e.g., manually timing mouse movement or manually painting paws for gait analysis). The automated systems for the OFT and the RR increase the reliability and validity of the data produced and save time and effort.
For example, given the neurodegeneration of the Sandhoff disease mouse model, when performing this battery of tests, the OFT would be the first of these tests used to assess the motor abnormalities that are usually seen in this mouse model starting at 3 months of age. Deficits in motor coordination should also become apparent beginning at 12 weeks of age. Deficits in muscular strength should begin around 14 weeks of age, but they ultimately become much more significant by 16 weeks.
These tests are useful when studying diseases that cause significant motor impairments, including neurodegenerative diseases9 such as Sandhoff disease1,8,13, Parkinson&#39;s disease7, Huntington&#39;s disease, multiple sclerosis. The tests can also be used to study aging and to make comparative geriatric assessments or measurements of health. Gait disturbances and changes in locomotion and balance are seen in many of these disease states. However, the usefulness of these tests is not limited to diseases with prevalent neuromuscular symptoms.
Please note that there have been no confounding variables noted with respect to motivation or the novelty of any of the described behavioral testing measures. It is imperative for certain disease states and pathologies that the researchers acquire preliminary behavioral data before the mouse shows any phenotypic symptoms; the researcher should then periodically (weekly) ensure that all motor impairments are accounted for. Many diseases have a quick onset of phenotypic symptoms, and without weekly testing, these symptoms would not be accounted for.

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			<td height="21" style="height:21px;width:216px;">IITC Life Sciences Rotarod with Series 8 computing software&#160;</td>
			<td style="width:127px;">IITC Life Sciences</td>
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			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;">Square Open Field 45 x 45 x 40 cm (grey color). Other floor color available: white, black.</td>
			<td style="width:127px;">Harvard Aparatus (PanLab)</td>
			<td align="right">760190</td>
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			<td height="42" style="height:42px;">&#160;Open Field Preconfigured Module</td>
			<td style="width:127px;">Harvard Aparatus (PanLab)</td>
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			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;">Smart V3.0 Software Platform&#160;</td>
			<td style="width:127px;">Harvard Aparatus (PanLab)</td>
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			<td height="42" style="height:42px;">Video converter (analog/digital)</td>
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			<td height="42" style="height:42px;">Roof fixing sys for camera</td>
			<td style="width:127px;">Harvard Aparatus (PanLab)</td>
			<td align="right">760277</td>
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			<td height="42" style="height:42px;">Analog Sony Camera &#38; lenseNTSC</td>
			<td style="width:127px;">Harvard Aparatus (PanLab)</td>
			<td align="right">760505</td>
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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.

The study animals may undergo the behavioral testing paradigm weekly or monthly to accommodate the necessary time points required for analysis. See Figure 1 for examples on behavioral testing timing. In the representative data below, taken from Osmon et al. (2016), the mice underwent the battery of behavioral testing monthly, beginning at 12 weeks of age, until their humane endpoint around 40 weeks of age. The timing of behavioral testing is sometimes critical fo...

Watch this Scientific Journal Video about Battery of Behavioral Tests Assessing General Locomotion, Muscular Strength, and Coordination in Mice at JoVE.com

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 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 ...

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Research

JoVE Journal

Behavior

13.4K Views.  Queen's University. 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. 

Video: Battery of Behavioral Tests Assessing General Locomotion, Muscular Strength, and Coordination 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 ...

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

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

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

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

Operant Procedures for Assessing Behavioral Flexibility in Rats

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these processes is the ability to adjust behavior in response to changes in one&#8217;s environment (i.e., behavioral flexibility). These processes are essential to normal human behavior, and may be disrupted in diverse neuropsychiatric conditions, including schizophrenia, alcoholism, depression, stroke, and Alzheimer&#8217;s disease. Understanding of the neurobiology of executive functions has been greatly advanced by the availability of animal tasks for assessing discrete components of behavioral flexibility, particularly strategy shifting and reversal learning. While several types of tasks have been developed, most are non-automated, labor intensive, and allow testing of only one animal at a time. The recent development of automated, operant-based tasks for assessing behavioral flexibility streamlines testing, standardizes stimulus presentation and data recording, and dramatically improves throughput. Here, we describe automated strategy shifting and reversal tasks, using operant chambers controlled by custom written software programs. Using these tasks, we have shown that the medial prefrontal cortex governs strategy shifting but not reversal learning in the rat, similar to the dissociation observed in humans. Moreover, animals with a neonatal hippocampal lesion, a neurodevelopmental model of schizophrenia, are selectively impaired on the strategy shifting task but not the reversal task. The strategy shifting task also allows the identification of separate types of performance errors, each of which is attributable to distinct neural substrates. The availability of these automated tasks, and the evidence supporting the dissociable contributions of separate prefrontal areas, makes them particularly well-suited assays for the investigation of basic neurobiological processes as well as drug discovery and screening in disease models.

The ability to assess executive functions such as behavioral flexibility in rats is useful for investigating the neurobiology of cognition in both intact animals and disease models. Here we describe automated tasks for assessing strategy shifting and reversal learning, which are particularly sensitive to disruptions in prefrontal cortical networks.

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these ...

Asymmetric Walkway: A Novel Behavioral Assay for Studying Asymmetric Locomotion

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 ...

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence2. In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

This protocol is intended to enable researchers to conduct experiments designed to test these aspects of addiction using the conditioned place preference and locomotor behavioral sensitization assays.

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

Assessing Working Memory in Children: The Comprehensive Assessment Battery for Children &#8211; Working Memory (CABC-WM)

The Comprehensive Assessment Battery for Children - Working Memory (CABC-WM) is a computer-based battery designed to assess different components of working memory in young school-age children. Working memory deficits have been identified in children with language-based learning disabilities, including dyslexia1,2 and language impairment3,4, but it is not clear whether these children exhibit deficits in subcomponents of working memory, such as visuospatial or phonological working memory. The CABC-WM is administered on a desktop computer with a touchscreen interface and was specifically developed to be engaging and motivating for children. Although the long-term goal of the CABC-WM is to provide individualized working memory profiles in children, the present study focuses on the initial success and utility of the CABC-WM for measuring central executive, visuospatial, phonological loop, and binding constructs in children with typical development. Immediate next steps are to administer the CABC-WM to children with specific language impairment, dyslexia, and comorbid specific language impairment and dyslexia.

Working memory predicts a significant amount of variance for a variety of cognitive tasks, including speaking, reading, and writing. However, few tools are available to assess working memory in children. We present an innovative, computer-based battery that comprehensively assesses different components of working memory in school-age children.

The Comprehensive Assessment Battery for Children - Working Memory (CABC-WM) is a computer-based battery designed to assess different components of ...

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal experimentation whenever possible. When there is no alternative to animal experimentation, the total number of laboratory animals used should be the minimum needed to obtain valuable data. Moreover, appropriate refinement measures should be applied to minimize pain, suffering, and distress accompanying the experimental procedure. The categories used to classify the degree of pain, suffering, and distress are non-recovery, mild, moderate, or severe (EU Directive 2010/63). To determine which categories apply in individual cases, it is crucial to use scientifically sound tools.
The well-being-assessment protocol presented here is designed for procedures during which general anesthesia is used. The protocol focuses on home cage activity, the Mouse Grimace Scale, and luxury behaviors such as burrowing and nest building behavior as indicators of well-being. It also uses the free exploratory paradigm for trait anxiety-related behavior. Fecal corticosterone metabolites as indicators of acute stress are measured over the 24-h post-anesthetic period.
The protocol provides scientifically solid information on the well-being of mice following general anesthesia. Due to its simplicity, the protocol can easily be adapted and integrated in a planned study. Although it does not provide a scale to classify distress in categories according to the EU Directive 2010/63, it can help researchers estimate the degree of severity of a procedure using scientifically sound data. It provides a way to improve the assessment of well-being in a scientific, animal-centered manner.

We developed a protocol to assess well-being in mice during procedures using general anesthesia. A series of behavioral parameters indicating levels of well-being as well as glucocorticoid metabolites were analyzed. The protocol can serve as a general aid to estimate the degree of severity in a scientific, animal-centered manner.

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

Assessing Activity-based Anorexia in Mice

Rodents develop activity-based anorexia (ABA) when exposed to a restricted feeding schedule and allowed free access to a running wheel. These conditions lead to a life-threatening reduction in body weight. However, rodents exposed to only one of these conditions ultimately adapt to re-establish normal body weight. Although increased running coupled with reduction in voluntary food intake appear paradoxical under ABA conditions, ABA behavior is observed across numerous mammalian species. 
The ABA paradigm provides an animal model for anorexia nervosa (AN), an eating disorder with severe dysregulation of appetite-behavior. Subjects are singly housed with free access to a running wheel. Each day, the subject is offered food for a limited amount of time. During the course of the experiment, a subject's body weight decreases from high activity and low caloric intake. The duration of the study varies based on how long food is offered daily, the type of food offered, the strain of mouse, if drugs are being tested, and environmental factors.
A lack of effective pharmacological treatments for AN patients, their low quality of life, high cost of treatment, and their high mortality rate indicate the urgency to further research AN. We provide a basic outline for performing ABA experiments with mice, offering a method to investigate AN-like behavior in order to develop novel therapies. This protocol is optimized for use in Balb/cJ mice, but can easily be manipulated for other strains, providing great flexibility in working with different questions, especially related to genetic factors of ABA.

Mice individually housed with a running wheel while given limited access to food develop reductions in food consumption and increase activity on the running wheel. This experimental phenomenon is called activity-based anorexia. This paradigm provides an experimental tool for studying the neurobiology and behaviors underlying aspects of anorexia nervosa.

Rodents develop activity-based anorexia (ABA) when exposed to a restricted feeding schedule and allowed free access to a running wheel. These conditions ...

The Unpredictable Chronic Mild Stress Protocol for Inducing Anhedonia in Mice

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by many patients prompts the need to develop new therapeutic alternatives and to better understand the etiology of the disorder. Pre-clinical models with translational merits are rudimentary for this task. Here we present a protocol for the unpredictable chronic mild stress (UCMS) method in mice. In this protocol, adolescent mice are chronically exposed to interchanging unpredictable mild stressors. Resembling the pathogenesis of depression in humans, stress exposure during the sensitive period of mice adolescence instigates a depressive-like phenotype evident in adulthood. UCMS can be used for screenings of antidepressants on the variety of depressive-like behaviors and neuromolecular indices. Among the more prominent tests to assess depressive-like behavior in rodents is the sucrose preference test (SPT), which reflects anhedonia (core symptom of depression). The SPT will also be presented in this protocol. The ability of UCMS to induce anhedonia, instigate long-term behavioral deficits and enable reversal of these deficits via chronic (but not acute) treatment with antidepressants strengthens the protocol&#39;s validity compared to other animal protocols for inducing depressive-like behaviors.

Here we present the unpredictable chronic mild stress protocol in mice. This protocol induces a long-term depressive-like phenotype and enables to assess the efficacy of putative antidepressants in reversing the behavioral and neuromolecular depressive-like deficits.

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by ...