The authors thank Dr. Cora SW Lai from the School of Biomedical Sciences, the University of Hong Kong, for lending the elevated plus maze test, and the Department of Anesthesiology from the University of Hong Kong for lending the rotarod test apparatus.

All methods described here have been approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR), the University of Hong Kong.
1. General protocol
NOTE: This section is based on Deacon19.
<ol>
	<li>Behavioral room setup
	<ol>
		<li>Get rid of unrelated stimulation/distraction, including direct bright light on the experimental apparatus, odor, noise, and other irrelevant animals, in the behavioral room (which should be about 10 m2 with adjustable lighting and, preferably, have an anteroom). 
		NOTE: Since the mouse is a nocturnal animal, lighting under 15 lux in the open-field test, novel object recognition test, and social interaction test could minimize the interference/stress from the light and help the mouse to focus on the test.</li>
		<li>Set the camera for video recording at least 1.5 m above the floor, ensuring that it is out of the sight of the testing mouse.</li>
	</ol>
	</li>
	<li>Housing and habituation
	<ol>
		<li>Group-house the mice in an animal unit under observation (e.g., group-housing no more than four adult mice). 
		NOTE: Here, 3-month-old male C57BL/6N mice were used and housed in a 1144B cage. Rule out sick, injured, or severely stressed mice. Experiences of starvation, thirst, or being bullied may affect the performance of the mouse.</li>
		<li>Arrange for the same animal handler to conduct all the behavioral tests, to diminish variability. Perform the transportation, handling, and experiment during the light cycle from 7:00 a.m. to 7:00 p.m. If possible, arrange all the other handling, such as the administration of any drug/toxin (e.g., intranasal instillation of silica nanoparticles) or cage-cleaning, after the test.</li>
		<li>Relabel the cages with random numbers to blind the experimenter before each experiment. Habituate the mice to the experimental environment in the behavioral room for 15 to 30 min in their home cages. Keep the home cages in the behavioral room during the entire experiment.</li>
		<li>Before starting the experiment, put a nonexperimental C57BL/6N mouse in the apparatus so that the experimental condition for the first mouse is the same as the rest. Then, clean the apparatus as follows: remove the urine and feces with a clean paper towel, clean the experimental device with tap water, and then, cover the odor left by the mouse by wiping the apparatus with a paper towel lightly sprayed with 70% ethanol.</li>
		<li>To minimize the distraction caused by the experimenter, ask the experimenter to leave the behavioral room during video recording, or stay behind the curtain during the Morris water maze test.</li>
	</ol>
	</li>
	<li>Behavioral test arrangement
	<ol>
		<li>Arrange the behavioral test in the order as shown in Figure 1A. Plan to perform the tests spaced out by 24 h, except for the elevated plus maze test. 
		NOTE: Since the entire procedure requires up to 2 weeks, simplify the battery by choosing among similar tests before application in an acute or short-term study. 
		CAUTION: The 2 day open-field test ensures proper habituation for the novel object recognition test. Test the forced swimming test last as it may cause stress in C57BL/6N mice.</li>
	</ol>
	</li>
</ol>
2. BehavioralTest Protocol
<ol>
	<li>Open-field test8,18,19,20
	<ol>
		<li>Perform the open-field test in a 60 cm (length [L]) x 60 cm (width [W]) x 40 cm (height [H]) nontransparent white plastic arena. 
		NOTE: Using multiple arenas can increase the throughput of the test.</li>
		<li>Start the camera recording and gently put the mouse next to the middle of a wall of the arena, facing that same wall. Record the behavior of the mouse for 10 min before returning it to the home cage. Clean the apparatus as described in step 1.2.4. 
		NOTE: The start point in the open-field test can also be the center of the arena. Be consistent among the mice.</li>
		<li>Repeat until all the mice finish the protocol. Counterbalance the testing order between groups.</li>
		<li>Perform the data analysis as follows.
		<ol>
			<li>Divide the arena into four squares by four squares (imaginary grid) on the computer screen. To assess locomotor function, count the number of lines crossed by the mouse in the arena19. 
			NOTE: The definition of &#8220;crossing a line&#8221; is when both hind limbs cross it. This definition also applies to the elevated plus maze test.</li>
			<li>Measure the time spent in the central area as an indicator of anxiety. The central area is the four squares in the center of the arena. 
			NOTE: Additional parameters, such as rearing (both front paws off the ground, with front paws against a wall or standing), latency to the first rear, and grooming and freezing indicate the emotionality of the mouse.</li>
			<li>Alternatively, use a tracking software, as described in detail by Seibenhener and Wooten8, to measure the distance traveled, the speed, and the time spent in the center area.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Accelerating rotarod test18
	<ol>
		<li>Perform the 3 day training of the accelerating rotarod test before any treatment of drugs or toxin/modeling/onset of disease and test the motor function for 1 day, as planned in Figure 1A. 
		NOTE: The mouse receives three trials per day during the training and testing. Each trial starts with the rotation of the rod and ends with the drop of the mouse.</li>
		<li>Place the rotarod apparatus on the bench in the behavioral room. Avoid direct lighting to the equipment. Program the equipment as starting from 4 rpm and accelerating to 40 rpm within 5 min.</li>
		<li>In each trial, put the mouse on the static rod, facing the wall of the machine. Start the device when the mouse is settled. Stop the device once the mouse drops and record the time the mouse spent on the rod. Immediately repeat for another two trials before returning the mouse back to the home cage.</li>
		<li>Repeat the procedure on the other mice.</li>
		<li>Measure the average time spent on the rod of three trials during testing to estimate motor function. 
		NOTE: The average time on the rod during the third day of training is the baseline of motor function.</li>
	</ol>
	</li>
	<li>Social interaction test18
	<ol>
		<li>For the social interaction test, use an open-field arena with two identical transparent chambers (8 cm [L] x 6 cm [W] x 12 cm [H]) with holes on the surface&#8212;and a novel mouse (helper) which is a same-sex juvenile conspecific that has had no previous contact with the subject mouse. Figure 1B shows the scheme of the procedure. Clean the arena and the chambers as described in step 1.2.4. 
		NOTE: The novel mouse cannot be a littermate or cage mate of the subject mouse. It is group-housed and healthy. Habituate the novel mouse to the behavioral room for 15 to 30 min as described in step 1.2.3.</li>
		<li>Separately place the two chambers in the middle of two opposite walls of the arena. Introduce the subject mouse into the arena as described in step 2.1.2 and shown in Figure 1B, for a 3 min exploration. Return the subject mouse to the home cage and remove any urine or feces in the arena.</li>
		<li>Put the helper in one of the chambers. Reintroduce the subject mouse to the arena and record for 3 min. Afterward, return both mice to each of their own home cages. Repeat the procedures with other subject mice as described above. 
		NOTE: Counterbalance the side of the helper or randomly assign it within the group.</li>
		<li>From the video, estimate the parameter describing the social interaction activity of the mice as thelper/tempty, which means the ratio of time interacting with the helper chamber (thelper) and the empty one (tempty), or use the recognition index thelper/(thelper + tempty). 
		NOTE: An interaction between the subject mouse and the chamber is defined as when the mouse&#8217;s nose is within 2 cm of the chamber and pointing toward it.</li>
	</ol>
	</li>
	<li>Elevated plus maze test10
	<ol>
		<li>Conduct the elevated plus maze test on the same day after all mice are tested in the open-field test. Clean the apparatus as described in step 1.2.4. 
		NOTE: The configuration of the elevated plus maze is a &#8220;+&#8221;-shape. It has two open arms (30 cm x 5 cm x 0.5 cm) across from each other and perpendicular to two closed arms (30 x 5 x 16 cm) with a center platform (5 cm x 5 cm x 0.5 cm). The maze is elevated 40 cm from the ground.</li>
		<li>Place the mouse at the junction of the open and closed arms, facing the open arm that is opposite to the experimenter (Figure 1C). Record the behavior for 5 min before returning the mouse to the home cage. Repeat till all mice are tested. 
		NOTE: Entering the maze with its face to the open arm could increase the mouse&#8217;s exploration of the open arm.</li>
		<li>Measure the time the mouse spent in the open arms (topen) and in the closed arms (tclose) based on the video: topen/tclose indicates the level of anxiety.</li>
	</ol>
	</li>
	<li>Forced swim test11
	<ol>
		<li>The apparatus of the forced swim test is a cylindrical tank that is 30 cm high and 20 cm in diameter. Fill the tank up to 15 cm high with tap water at room temperature (23 - 25 &#176;C). 
		NOTE: Use fresh water for each mouse.</li>
		<li>Start the video recording and gently put the mouse in the water, in the center of the apparatus. Record the video for 6 min before putting the mouse back in its home cage under infrared light. 
		NOTE: Do not disturb the mouse by drowning it or twisting its tail.</li>
		<li>Measure the immobility time in the last 5 min of the recorded video. Mobility means any movements other than those required to balance the body and to keep the head above the water.</li>
	</ol>
	</li>
	<li>Novel object recognition test17,18
	<ol>
		<li>Set up the novel object recognition test to include 2 days of habituation, 1 day of familiarization, and 1 day of testing (Figure 1D); each session is 10 min per mouse, and the intersession interval is 24 h. 
		NOTE: The habituation using the open-field test is performed as described in section 2.1. The mouse interacts with two identical objects (old objects) in familiarization. In the test, the mouse interacts with one of the old objects and a new object, both placed in the same place as the objects in familiarization. The apparatus of the novel object recognition test includes an open-field arena and two sets of objects. Each set contains two identical objects (objects A and A and objects B and B). Objects A and B are similar in size but different in texture (glass/plastic/paper), shape (round/cubic), and color (bright/dark). The objects should be odor-free and big enough for the mouse to explore within 10 min. The appropriate size for adult C57BL/6N mice is 8 cm tall and 5 cm wide/in diameter.</li>
		<li>Mark the positions of the two objects in familiarization and test, which are 5 cm away from the side and 7 cm away from the top of the arena. 
		NOTE: Mark the position on the evening before the familiarization to avoid the smell of the marker.</li>
		<li>In the familiarization, the mouse interacts with one set of identical objects. Clean the arena and objects as described in step 1.2.4 before placing the mouse in the arena, facing the middle of the wall as shown in Figure 1D. Record for 10 min before returning the mouse to the home cage. Repeat until all the mice are finished and return all the cages to the animal unit. 
		NOTE: Counterbalance the objects used in familiarization within the group to diminish bias (e.g., mice No. 1 and 2 explore objects A and A, and mice No. 3 and 4 explore objects B and B; in this way, the novel object is object B for mice No. 1 and 2 and object A for mice No. 3 and 4).</li>
		<li>Perform the test 24 h after the familiarization. Use the same procedure as for the familiarization, except replace one of the objects with one from another set (Figure 1D). Repeat until all the mice have performed the test and, afterward, return all the cages to the animal unit. 
		NOTE: Counterbalance the side of the new object within the group to diminish bias (e.g., introduce mice No. 1 and 3 to objects A and B, and mice No. 2 and 4 to objects B and A). In this way, the novel object shows at the right side for mice No. 1 and 4, and at the left side for mice No. 2 and 3. Here, the left side is the left side of the experimenter when facing the arena.</li>
		<li>Measure the time that each mouse interacts with the new object (tnew) and the old object (told) separately, from the video footage in the test phase. An interaction between the animal and the object is described in the note following step 2.3.4. Calculate the memory of the mouse as the preference to the novel object = tnew/told; or tnew/(tnew + told). 
		NOTE: tnew/told equals to 1 or tnew/(tnew + told) equals to 0.5 means the mouse has no preference for the novel object (i.e., memory impairment). The time interacting with objects in the familiarization can serve as a control of the experiment. The total time indicates the exploration activity of the mouse, and tleft/tright suggests spatial bias.</li>
	</ol>
	</li>
	<li>Morris water maze test16
	<ol>
		<li>Set up the apparatus as follows.
		<ol>
			<li>Put the water maze, a circular pool (of 120 cm in diameter and 60 cm deep), in the center of a behavioral room, and mark the position of the maze to ensure the position remains the same during the entire experiment.</li>
			<li>Divide the maze into four equal imaginary quadrants. Hang the visual cues (e.g., circle, square, triangle, and pentagon) in the center of each quadrant, 130 cm above the floor and 53 cm away from the wall of the maze. 
			NOTE: The maze and the cue must stay in the same position during the entire test, so the mouse can form accurate spatial memory.</li>
			<li>Place a platform 25 cm away from the wall, in the center of the fourth quadrant, and mark the position. The platform for the mouse is 10 cm in diameter. 
			NOTE: The position and diameter of the platform determine the difficulty of the task. The nearer it is to the wall of the maze, or the bigger the platform, the easier the task.</li>
			<li>Fill the water maze with water (with a temperature of 23 to 25 &#176;C, colored into white and made opaque by milk powder/food whitening powder) until the water level is 1 cm higher than the platform. Bring the mice into the behavioral room for 15 to 30 min of habituation as shown in step 1.2.3 and turn on the infrared light above the cages, which will be used to dry the mice. 
			NOTE: Cover the top of the platform with white cloth and net so that the mouse can easily climb onto it. Make sure there is no direct lighting above the water.</li>
		</ol>
		</li>
		<li>Conduct the training phase as follows.
		<ol>
			<li>The training phase takes 5 days, four trials per day. Semi-randomly arrange the starting points on each day as demonstrated in literature16. This effort prevents the mouse from establishing associative memory, which is the most common way to &#8220;cheat&#8221; in the test.</li>
			<li>At the beginning of each trial, start the video recording and gently put the mouse into the maze. 
			NOTE: Do not drop the mouse into the tank or twist its tail, which may cause extra stress and disorientation.</li>
			<li>Ask the experimenter to stay out of sight of the mouse and to return to take the mouse back to its home cage when any of the following happens: (i) the mouse cannot locate the platform within 60 s; (ii) the mouse finds the platform within 60 s and stays on it for 10 s. In circumstance (i), ask the experimenter to place the mouse on the platform and let it stay there for 10 s. 
			NOTE: Point (ii) means the mouse successfully located the platform.</li>
			<li>Stop the video and put the mouse back in the home cage under infrared light. 
			NOTE: Maintaining the mice&#8217;s body temperature is critical for their performance because hypothermia stresses mice and may affect the following tests.</li>
			<li>Repeat the procedure with another mouse.</li>
			<li>Based on the video, record the escape latency, which is the duration of the period the mouse spends in the maze, from entering the maze till the moment it successfully locates the platform. If the mouse cannot find the platform or stays there for less than 10 s in 60 s, the escape latency counts as 60 s. Plot the learning curve against the training days with the average escape latency per day. 
			NOTE: The escape latency does not include the 10 s spend on the platform.</li>
		</ol>
		</li>
		<li>Perform the probe phase as follows.
		<ol>
			<li>On the sixth day of the Morris water maze test, set up the apparatus as described in step 2.7.1., take a picture of the maze to record the position of the platform, and then remove the platform from the tank.</li>
			<li>Start the video recording, and gently put the mouse into the maze in the quadrant diagonally opposite to the target quadrant.</li>
			<li>Ask the experimenter to stay out of sight of the mouse during the 1 min video recording. Afterward, have the experimenter take the mouse out of the maze and put it back in the home cage.</li>
			<li>Use the image taken at step 2.7.3.1. as a reference to measure the duration of any platform crossing. Time the duration the mouse stays in the target quadrant (ttarget), according to the video. The total time is ttotal. Measure the preference to the target quadrant as ttarget /ttotal.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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V., 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=Behavioural+battery+testing:+evaluation+and+behavioural+outcomes+in+8+inbred+mouse+strains.">Behavioural battery testing: evaluation and behavioural outcomes in 8 inbred mouse strains.</a> Physiology &amp; Behavior. 99 (3), 301-316 (2010).</li><li>Powell, T. R., Fernandes, C., Schalkwyk, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depression-Related+Behavioral+Tests.">Depression-Related Behavioral Tests.</a> Current Protocols in Mouse Biology. 2 (2), 119-127 (2012).</li><li>Paylor, R., Spencer, C. M., Yuva-Paylor, L. A., Pieke-Dahl, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+behavioral+test+batteries,+II:+effect+of+test+interval.">The use of behavioral test batteries, II: effect of test interval.</a> Physiology &amp; Behavior. 87 (1), 95-102 (2006).</li><li>Lee, K. M., Coehlo, M., McGregor, H. A., Waltermire, R. S., Szumlinski, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binge+alcohol+drinking+elicits+persistent+negative+affect+in+mice.">Binge alcohol drinking elicits persistent negative affect in mice.</a> Behavioural Brain Research. 291, 385-398 (2015).</li><li>Shiotsuki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rotarod+test+for+evaluation+of+motor+skill+learning.">A rotarod test for evaluation of motor skill learning.</a> Journal of Neuroscience Methods. 189 (2), 180-185 (2010).</li></ol>

The authors have nothing to disclose.

Behavioral analysis of mice is critical for neurodegeneration research. While cognitive function is often the most susceptible domain of behavior affected in neurodegenerative diseases, mood dysfunction, such as depression and anxiety, is often comorbid. Moreover, motor function often affects the interpretation of the results in some tests, such as the novel object recognition test, the elevated plus maze test, and the social interaction test. Based on these thoughts, a comprehensive behavioral test battery is required f...

Neurodegenerative diseases featured devastating behavioral symptoms, including cognitive impairment, mood dysfunctions such as anxiety and depression, or motor dysfunction1. The pathogenesis of various kinds of neurodegenerative diseases is unclear2. Accumulative studies indicate that genetic and environmental factors might both contribute to the pathogenesis of neurodegenerative diseases. Identifying the risk factor of neurodegeneration requires behavioral analysis. Although each type of neurodegenerative disease has its signature behavioral symptom (e.g.,&#160;Alzheimer&#39;s disease [AD] is featured with cognitive impairment and Parkinson&#39;s disease [PD] with motor dysfunction). With the progression of the disease, the patients manifest comorbidity of different behavioral abnormalities3. For example, AD patients show symptoms of mood dysfunction in the advanced stage4,5. PD patients may progress into PD-related dementia and develop cognitive impairment6. Based on these features, the behavioral analysis in neurodegeneration models is usually comprehensive and repeated.
To achieve this goal, a battery which contains classical and widely used behavioral tests with excellent validity was designed for behavioral analyses in motor, mood, and cognition. The motor function can be tested by the open-field test7,8 and the accelerating rotarod test. Mood dysfunction, including social dysfunction, depression, and anxiety, are most commonly seen in neurodegenerative diseases5. Hence, this battery includes a social interaction test for sociability9, the elevated plus maze test for anxiety10, and the forced swim test for depression11. Cognitive impairment is one of the most characteristic symptoms in neurodegenerative diseases such as AD and frontotemporal lobar dementia12. Cognitive domains, including short-term memory and episodic memory, are susceptible to neurodegeneration13,14,15. Therefore, the Morris water maze test for spatial learning and memory16 and the novel object recognition test for short-term memory17 are included in the battery. These tests are compatible with each other. The order of the tests was designed to maximize habituation and to minimize interference, to further increase the compatibility within the battery. Since each function is tested by at least two independent tests that are different in principle and method, the results of each test can be further validated. Moreover, the protocols of some tests are highlighted for repeated testing, facilitating the longitudinal study of the development of neurodegenerative diseases. Therefore, this behavior test battery studies different subdomains of behavioral changes seen in various stages of neurodegeneration while costing a minimal number of animals. This battery has been used in a longitudinal study which evaluated the behavioral changes in young adult (3-month-old) male C57BL/6N mice after respiratory exposure to silica nanoparticles, an occupational hazard that is a potential risk factor of neurodegeneration18. However, other strains or models, such as aged mice and genetically manipulated mice, may behave differently than young C57BL/6N mice. Therefore, caution may be required when using this battery in these mice.

<table><tbody><tr><td>chambers in social interaction test</td><td>home made</td><td></td><td>(8 cm (L) x 6 cm (W) x 12 cm (H)), transparant with holes, plastic</td></tr><tr><td>cylindrical tanks used in forced swimming test</td><td>home made</td><td></td><td>30 cm height, 20 cm diameters, glass</td></tr><tr><td>elevated plus maze</td><td>home made</td><td></td><td>open arms (30 cm x 5 cm x 0.5 cm) ,closed arms (30 cm x 5 cm x 16 cm), center platform (5 cm x 5 cm x 0.5 cm), 40 cm tall. Plastic, nontransparant</td></tr><tr><td>IITC Roto-Rod Apparatus</td><td>IITC life science Inc.</td><td>755, series 8</td><td></td></tr><tr><td>open field arena</td><td>home made</td><td></td><td>60 cm (L) x 60 cm (W) x 40 cm (H), plastic, nontransparant</td></tr><tr><td>water maze</td><td>home made</td><td></td><td>120 cm in diameter, 60 cm deep, steel</td></tr></tbody></table>

a behavioral test battery for the repeated assessment of motor skills, mood, and cognition in mice

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.

This behavioral test battery was designed for the comprehensive and valid behavioral analysis of motor, mood, and cognition, which are commonly affected in neurodegeneration5. We have applied this battery to study the behavioral changes in young adult C57BL/6N mice after respiratory exposure to silica nanoparticles for 1 month and 2 months18. The results revealed that C57BL/6N mice exposed to silica nanoparticles showed various behavioral ch...

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A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

A comprehensive behavioral test battery of motor skills, mood&#8212;including social interaction, depression, and anxiety&#8212;and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and ...

a-behavioral-test-battery-for-repeated-assessment-motor-skills-mood

Universidad San Sebastian

Research

JoVE Journal

Behavior

18.9K Views.  Children's Hospital of Nanjing Medical University. A comprehensive behavioral test battery of motor skills, mood—including social interaction, depression, and anxiety—and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.

Video: A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the &#34;cloud&#34; and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in&#160;cabinet; two such cabinets (16 environments) may be controlled by one computer.

Fully automated system for measuring physiologically meaningful properties of the mechanisms mediating spatial localization, temporal localization, duration, rate&#160;and probability estimation, risk assessment, impulsivity, and the accuracy and precision of memory, in order to assess the effects of genetic and pharmacological manipulations on foundational mechanisms of cognition in mice.

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and ...

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of chronic CNS disorders, there is an increasing demand for murine models with high validity at the behavioral level. However, multiple pathogenic mechanisms and complex functional deficits often impose challenges to reliably measure and interpret behavior of chronically sick mice. Therefore, the assessment of peripheral pathology and a behavioral profile at several time points using a battery of tests are required. Video-tracking, behavioral spectroscopy, and remote acquisition of physiological measures are emerging technologies that allow for comprehensive, accurate, and unbiased behavioral analysis in a home-base-like setting. This report describes a refined phenotyping protocol, which includes a custom-made monitoring apparatus (Integrated Behavioral Station, INBEST) that focuses on prolonged measurements of basic functional outputs, such as spontaneous activity, food/water intake and motivated behavior in a relatively stress-free environment. Technical and conceptual improvements in INBEST design may further promote reproducibility and standardization of behavioral studies.

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station INBEST

Prolonged and comprehensive monitoring of mice in a home-cage environment provides a deeper understanding of aberrant behavior in murine models of brain diseases. This paper describes the Integrated Behavioral Station (INBEST) as the key component of contemporary behavioral analysis.

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of ...

Assessment of Motor Balance and Coordination in Mice using the Balance Beam

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice. Fine motor coordination and balance can be assessed by the beam walking assay. The goal of this test is for the mouse to stay upright and walk across an elevated narrow beam to a safe platform. This test takes place over 3 consecutive days: 2 days of training and 1 day of testing. Performance on the beam is quantified by measuring the time it takes for the mouse to traverse the beam and the number of paw slips that occur in the process. Here we report the protocol used in our laboratory, and representative results from a cohort of C57BL/6 mice. This task is particularly useful for detecting subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the Rotarod.

Deficits in fine motor coordination can be assessed with the balance beam test. Performance on the beam is quantified by the speed at which the beam is traversed and the number of times the mouse slips on the beam.

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice.  Fine motor coordination and ...

Neuroscience

Measuring Motor Coordination in Mice

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such as emotionality or cognition can be assessed, however, it is vital to determine whether the motor capabilities of e.g. a mutant or lesioned mouse allow such an assessment. Performance on a maze task requiring strength and coordination, such as the Morris water maze, might well be impaired in a mouse by motor, rather than cognitive, impairments, so it is essential to selectively dissect the latter from the former. For example, sensorimotor impairments caused by NMDA antagonists have been shown to impair water maze performance2. 
Motor coordination has traditionally been assessed in mice and rats by the rotarod test, in which the animal is placed on a horizontal rod that rotates about its long axis; the animal must walk forwards to remain upright and not fall off. Both set speed and accelerating versions of the rotarod are available.
The other three tests described in this article (horizontal bar, static rods and parallel bars) all measure coordination on static apparatus. The horizontal bar also requires strength for adequate performance, particularly of the forelimbs as the mouse initially grips the bar just with the front paws. 
Adult rats do not perform well on tests such as the static rods and parallel bars (personal observations); they appear less well coordinated than mice. I have only tested male rats, however, and male mice seem generally less well coordinated than females. Mice appear to have a higher strength:weight ratio than rats; the Latin name, Mus musculus, seems entirely appropriate. The rotarod, the variations of the foot fault test12 or the Catwalk (Noldus)15 apparatus are generally used to assess motor coordination in rats.

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

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 ...

The Tail Suspension Test

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that are expected to affect depression related behaviors. Mice are suspended by their tails with tape, in such a position that it cannot escape or hold on to nearby surfaces. During this test, typically six minutes in duration, the resulting escape oriented behaviors are quantified. The tail-suspension test is a valuable tool in drug discovery for high-throughput screening of prospective antidepressant compounds. Here, we describe the details required for implementation of this test with additional emphasis on potential problems that may occur and how to avoid them. We also offer a solution to the tail climbing behavior, a common problem that renders this test useless in some mouse strains, such as the widely used C57BL/6. Specifically, we prevent tail climbing behaviors by passing mouse tails through a small plastic cylinder prior to suspension. Finally, we detail how to manually score the behaviors that are manifested in this test.

The tail-suspension test is validated as an experimental procedure to assess antidepressant efficacy of drug treatments in mice. Mice are suspended by their tails for six minutes and escape-related behaviors are assessed. We describe procedures used in conducting the tail suspension test.

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that ...

Behavioral Assessments of Spontaneous Locomotion in a Murine MPTP-induced Parkinson's Disease Model

Parkinson&#8217;s disease (PD) is a common neurodegenerative disorder disease, causing the phenomenon of shaking, rigidity, slowness of movement and dementia. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can lead to some Parkinson&#8217;s-like symptoms by destroying dopaminergic neurons in the substantia nigra of the brain. It has been thus used to establish PD models in various animal studies. Here, mice receive MPTP injections (20 mg/kg/day) for seven days and the behavioral tests are performed on the eighth day. This model is adapted efficiently in the study of PD. The behavioral tests here include the cylinder test and the open field test. The cylinder experiment is used to detect the animals&#8217; ability to lift their front paws when put into a different environment. As the PD model mice show arching&#8212;the mouse arches its back&#8212;the number of paw liftings decrease. This test is easy to execute. The open field test is used to detect the amount of time the mice spend on running, walking, and remaining immobile. We analyze animals&#8217; movements in open field using software and obtain data. Lastly, we use L-DOPA, one of the most commonly used PD drugs, as one example to show how to apply this model to the study of PD drugs. Our results indicate that MPTP neurotoxicity induces motor deficit which can be mitigated by L-DOPA.

We describe establishment of a murine model for Parkinson's disease using MPTP, and behavioral assessments using cylinder and open field tests to measure motor function. We then use L-DOPA as one example to show how to apply this model in the study of PD drugs.

Parkinson&#8217;s disease (PD) is a common neurodegenerative disorder disease, causing the phenomenon of shaking, rigidity, slowness of movement and ...

Behavioral Assays for Autism Spectrum Disorder in Mouse Models

Strategies for Assessing Autistic-Like Behaviors in Mice

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social communication impairments and restrictive or repetitive behaviors, such as hand flapping or lining up objects. These behavioral patterns can be reliably observed in mouse models with ASD-linked genetic mutations, making them highly useful tools for studying the underlying cellular and molecular mechanisms in ASD. Understanding how genetic changes affect the neurobiology and behaviors observed in ASD will facilitate the development of novel targeted therapeutic compounds to ameliorate core behavioral impairments. Our lab has employed several protocols encompassing well-described training and testing procedures that reflect a wide range of behavioral deficits related to ASD. Here, we detail two assays to study the core features of ASD in mouse models: self-grooming (a measure of repetitive behavior) and the three-chamber social interaction test (a measure of social interaction approach and preference for social novelty).

Rodent models are valuable tools for studying core behaviors related to autism spectrum disorder (ASD). In this article, we expound on two behavioral tests for modeling the core features of ASD in mice: self-grooming, which assesses repetitive behavior, and the three-chamber social interaction test, which documents social impairments.

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social ...

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, standardized battery of neonatal mouse motor tests. These tests can validate injury or disease models, determine treatment efficacy and/or assess motor behaviors in new transgenic strains. This paper presents a series of neonatal motor tests to evaluate general motor function, including ambulation, hindlimb foot angle, surface righting, negative geotaxis, front- and hindlimb suspension, grasping reflex, four limb grip strength and cliff aversion. Mice between the ages of post-natal day 2 to 14 can be used. In addition, these tests can be used for a wide range of neurological and neuromuscular pathologies, including cerebral palsy, hypoxic-ischemic encephalopathy, traumatic brain injury, spinal cord injury, neurodegenerative diseases, and neuromuscular disorders. These tests can also be used to determine the effects of pharmacological agents, as well as other types of therapeutic interventions. In this paper, motor deficits were evaluated in a novel neonatal mouse model of cerebral palsy that combines hypoxia, ischemia and inflammation. Forty-eight hours after injury, five tests out of the nine showed significant motor deficits: ambulation, hindlimb angle, hindlimb suspension, four limb grip strength, and grasping reflex. These tests revealed weakness in the hindlimbs, as well as fine motor skills such as grasping, which are similar to the motor deficits seen in human cerebral palsy patients.

Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between groups.

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, ...