We would like to thank Margriet Hulsker for her photographic assistance and help in obtaining images of mice and the reviewers for their very constructive comments. This work was supported by ZonMw, TREAT-NMD (contract number LSHM-CT-2006-036825) and the Duchenne Parent Project.

The experiments described here were approved by the Animal Ethics Committee (DEC) of the Leiden University Medical Center (LUMC). Mice were bred by the animal facility of the LUMC and kept in individually ventilated cages with 12 hr light dark cycles. They had ad libitum access to water and standard chow.
When performing any of the functional tests described below, experimental conditions have to be strictly controlled to reduce variation. Preferably, age and gender matched mice should be used, as performance differs between age and genders. Mice belonging to the same litter should be randomized over the experimental groups. Animals should be tested by the same operator, who is blinded to the experimental groups. Tests should be performed on the same time of day and weekday, same room to equalize odors, noises,&#160;etc.14&#160;Large variation between individual mice and time points can be observed for all functional tests, therefore 6-8 mice/experimental group should be used. Functional test performance can also largely differ between different inbred wild type strains. Therefore, experimental and control wild type mice should always have corresponding backgrounds (in case of mdx mice use the C57BL/10ScSnJ wild type strain). All data described here have been obtained with the C57BL/10ScSnJ wild type strain, which we refer to as wild type from here on. The tests described here can be used longitudinally from at least 1-19 months of age in mdx and wild type mice. Tests should not be repeated more than once weekly to prevent mice from losing interest and willingness to perform the task.
1. Forelimb Grip Strength Test
Use the forelimb grip strength test to measure the strength of the forelimbs. The test is based on the tendency of a mouse to instinctively grasp a grid when suspended by the tail15, and adapted from <a href="http://www.treat-nmd.eu/downloads/file/sops/dmd/MDX/dmd_m.2.2_001.pdf" target="_blank">DMD_M2.2.001.pdf</a>.
<ol>
	<li>Apparatus set up: Attach a grid to a force transducer, which measures the maximum force applied by the mouse on the grid during the pull. Make sure the setting is on Peak tension mode (T-PK) for pulling. The units of force can be adjusted in either ounces-of-force, grams-of-force, pounds-of-force, kilograms-of-force, or Newtons. 
	Note: We prefer to work with grams as unit of values. Multiple meters are commercially available, but only axial transducers give reliable outcomes as lever type force transducers are negatively influenced by the physical laws of the lever effect. Either a nonflexible grid or triangle can be used with bars that are 1-2 mm in diameter.</li>
	<li>Prior to the test, assess the body weight of the mouse, to allow normalization for body weight.</li>
	<li>Use grams as unit of values. Reset the meter at the start of each recording.</li>
	<li>Remove the mouse from its cage by grabbing the tail and moving it horizontally towards the grid.</li>
	<li>Check that the mouse grasps the grid tightly with both forepaws.</li>
	<li>Pull the mouse away from the grid so that its grasp is broken; the highest force applied to the grid will be shown on the transducer&#39;s display, which can be either manually or automatically recorded.</li>
	<li>Only take pulls into account in which the mouse shows resistance to the experimenter. Reject measures in which only one forepaw, or the hindlimbs were used and in which the mouse turned during the pull.</li>
	<li>Let the mouse pull the grid three times in a row and then return it in the cage for a resting period of at least one minute. Note: between series of pulls a resting period is necessary for the mouse to recover and avoid habit formation.</li>
	<li>Then let the mouse perform four series of pulls, each followed by a short resting period. In this way the mouse has pulled a total of 15x (3 pulls x 5 times = 15 pulls).</li>
	<li>Determine the maximum grip strength and normalize for body weight by taking the average of the three highest values out of the 15 values collected.</li>
	<li>Optional: Determine fatigue by calculating the decrement between the average of the first two and the last two series of pulls 1+2+3=A, 4+5+6=B, 10+11+12=C and 13+14+15=D. The formula: (C+D)/(A+B) gives a value of 1 for mice which are not fatigued. This can be expressed in percentages so that a mouse without fatigue has a value of 0% and a mouse which forelimbs are completely fatigued has a value of 100%.</li>
</ol>
2. Hanging Tests
With hanging tests, balance, coordination and muscle condition can be assessed. These tests are based on the knowledge that mice are eager to remain hanging on a wire or grid till exhaustion16. There are two distinctive hanging tests in which at the start of the test either only the two forelimbs or all four limbs are used, using a wire or grid respectively. The hanging test using the wire and the grid are the longest suspension time method adapted from <a href="http://www.treat-nmd.eu/downloads/file/sops/dmd/MDX/DMD_M.2.1.004.pdf" target="_blank">DMD_M.2.1.004.pdf</a> and <a href="http://www.treat-nmd.eu/downloads/file/sops/dmd/MDX/DMD_M.2.1.005.pdf" target="_blank">DMD_M.2.1.005.pdf</a> respectively. A fixed hanging limit is used of 600 sec. The majority of wild type mice can hang for 600 sec, while dystrophic mice cannot. To reduce time spend performing this test, a maximum hanging time was set in place. Mice that fall off the wire or grid before then are given up to two more tries. This is done to reinsure that mice are really unable to hang and do not fall due to clumsiness.
<ol>
	<li>Hanging test with two limbs
	<ol>
		<li>Apparatus set up: Tightly secure a 2 mm thick metal cloth hanger to a shelf with tape and maintain the hanger around 37&#160;cm above a layer of bedding.&#160;Note:&#160;alternatively, a 55 cm wide 2 mm thick metallic wire which is tightly secured between 2 vertical stands could be used. The distance of 37&#160;cm is sufficient to encourage mice to remain hanging, but also low enough to prevent mice from injuries when falling down. The wire should not vibrate or displace during the test as this could interfere with the performance of the mouse.</li>
		<li>Handle the mouse via the tail and bring it near the wire.</li>
		<li>Let the mouse grasp the wire with the two forepaws only, and lower the hindlimbs in such a way that the mouse only hangs with the two forepaws on the wire (Figure 2B).</li>
		<li>Directly start the timer when the mouse is released. After release, strong mice try to catch the wire with all the four limbs and the tail, which is allowed (Figure 2C).</li>
		<li>When a mouse shows improper behavior (like balancing on or deliberately jumping off the wire as shown in Figures 2D and 2E), directly address this by replacing the mouse on the wire without stopping the timer.</li>
		<li>When a mouse falls off the wire, stop the timer and record the hanging time.</li>
		<li>When mice are able to hang for 600 sec, take them off the wire and return them to the cage. Mice that fall before this limit are given a maximum of two more tries.</li>
		<li>Record the maximum hanging time (i.e. the longest of the trials) and use this for further analysis.</li>
	</ol>
	</li>
	<li>Hanging test with four limbs
	<ol>
		<li>Apparatus set up: Use either a hand made square or the lid of a big cage for a rat or rabbit for this test. Position the grid 25 cm above soft bedding to prevent mice from harming themselves upon falling, but also to discourage mice to intentionally jump off the grid. Tightly secure the grid so that the experimenter does not have to manually hold the grid during the experiment as these movements might interfere with the mouse&#39;s performance.</li>
		<li>Place the mouse on the grid so that it grasps it with its four paws.</li>
		<li>Invert the grid so that the mouse is hanging and directly start the timer.</li>
		<li>The test session ends for mice that are able to hang for a duration of 600 sec. Give mice that fall off the grid earlier a maximum of two more tries.</li>
		<li>Use the maximum hanging time (i.e. the longest of the trials) for further analysis.</li>
	</ol>
	</li>
</ol>
3. Rotarod Running
With the rotarod test muscle strength, coordination, balance, and condition can be determined17.
<ol>
	<li>Apparatus set up: For this test, mice have to run on a rotating tube. Ensure that the steady speed is set at 5 rotations per min (rpm), and that the speed increases from 5-45 rpm in the first 15 sec&#160;when started. After this it has to maintain its speed.</li>
	<li>Place the mice on the tube of the rotarod when it rotates at a slow steady speed of 5 rpm. Five mice can be tested simultaneously.</li>
	<li>Start the run once all mice are positioned. Within the first 15 sec&#160;the speed of the tube accelerates from 5-45 rpm after which it maintains that speed.</li>
	<li>Monitor the run. The running time is continuously recorded by the software. Running time stops automatically when a mouse falls off the tube as this activates the time bar positioned below the tube.&#160;Reposition mice that turn around facing the opposite direction on the tube while running without stopping the tube to rotate.</li>
	<li>End the test session for mice that are able to run for a duration of 500 sec. Give mice a maximum of two more tries allowing them to improve their running time, when they fall earlier.&#160;</li>
	<li>Use the maximum running time (i.e. the longest of&#160;the trials) for further analysis.&#160;</li>
</ol>
4. Treadmill Exercise
The treadmill can be used in three ways as a tool in pre clinical research. Firstly, forced treadmill running can be used to exacerbate disease pathology as described in this protocol (see also: <a href="http://www.treat-nmd.eu/downloads/file/sops/dmd/MDX/DMD_M.2.1.001.pdf" target="_blank">DMD_M2.1.001.pdf</a>). Secondly, the maximal running capability of mice and the effects of treatments on this can be assessed (See for the method to let mice run till exhaustion <a href="http://www.treat-nmd.eu/downloads/file/sops/dmd/MDX/DMD_M.2.1.003.pdf">DMD_M.2.1.003.pdf</a>). Finally, treadmill running can be used prior to another functional test to exhaust the mouse so that it performs less well in the second test13. This is done by exercising mice twice or three times weekly as described below, directly followed by either one of the functional tests described in protocol 1-3.
<ol>
	<li>Apparatus set up: There are several treadmills commercially available on which several mice can run simultaneously and for which elevation, duration and speed can be adjusted. Some treadmills are equipped with a grid to deliver low intensity shocks to encourage mice to run. However,&#160;mdx mice are sensitive to stress and can easily be motivated in a friendlier manner by a gentle push with the hand in the running direction. Therefore, it is strongly encouraged to NOT use the shock grid. Generally, stimulation with the hand is only needed during the first running session.</li>
	<li>Place the mice on the horizontal treadmill.</li>
	<li>Start the treadmill at a running speed of 12 m/min. Lower speeds (8 m/min) have to be used in old mice (&#62;15 months), where higher speeds easily lead to exhaustion.</li>
	<li>During the first session, encourage mice to run by gently pushing them when they are near the end of the belt.</li>
	<li>When the mice have run for a duration of 30 min, place them back into their cage.</li>
	<li>Repeat this twice weekly for e.g. 12 weeks.</li>
	<li>Allow resting periods when needed. For example, some mdx mice have to stop running and should be allowed to rest for a few min. If this happens, turn the belt off, give all mice a resting period of two min, turn the belt on for two min at 4 m/min. After this, increase speed to 12 m/min and allow the mice to finish the protocol. It is important that all mice complete the entire running protocol. Note: In case mdx mice need resting periods, consider a warm up before the 30 min exercise protocol. This warm up session consists of: a 2&#160;min&#160;acclimatization period at a speed of 4 m/min, immediately followed by an 8 min warmup at 8 m/min. In our hands 4-16 week old female mdx mice are able to complete the 30 min&#160;exercise protocol without resting. Others have reported that in age matched male mdx mice 45% of the mice do need resting periods to finish the exercise. The warm up protocol reduces the amount of stops12.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophin:+the+protein+product+of+the+Duchenne+muscular+dystrophy+locus.">Dystrophin: the protein product of the Duchenne muscular dystrophy locus.</a> Cell. 51, 919-928 (1987).</li><li>Bushby, 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=Diagnosis+and+management+of+Duchenne+muscular+dystrophy,+part+1:+diagnosis,+and+pharmacological+and+psychosocial+management.">Diagnosis and management of Duchenne muscular dystrophy, part 1: diagnosis, and pharmacological and psychosocial management.</a> Lancet Neurol. 9, 77-93 .</li><li>Bulfield, G., Siller, W. G., Wight, P. A., Moore, K. 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Genome. 11, 725-728 (2000).</li><li>Chapillon, P., Lalonde, R., Jones, N., Caston, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+development+of+synchronized+walking+on+the+rotorod+in+rats.+Effects+of+training+and+handling.">Early development of synchronized walking on the rotorod in rats. Effects of training and handling.</a> Behav. Brain Res. 93, 77-81 (1998).</li><li>Massett, M. P., Berk, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-dependent+differences+in+responses+to+exercise+training+in+inbred+and+hybrid+mice.">Strain-dependent differences in responses to exercise training in inbred and hybrid mice.</a> Am. J. Physiol. Regul. Integr. Comp. 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The authors have nothing to disclose.

The&#160;functional tests presented here are reproducible, easy to perform and applicable to wild type and dystrophic mice independent of their age. The tests provide useful tools to pre clinically assess muscle function, strength, condition, and coordination. When testing the effects of a compound on the natural history of the disease, the noninvasive tests described here (forelimb grip strength, both hanging tests and the rotarod test) can be nicely combined in a functional test regime where these tests are performed o...

Duchenne muscular dystrophy (DMD) is the most common neuromuscular disorder affecting 1:5,000 newborn boys. This severe and progressive muscle wasting disease is caused by mutations in the DMD gene that disrupt the open reading frame and prevent the synthesis of functional dystrophin protein. Muscle fibers lacking dystrophin are vulnerable to exercise induced damage. Upon exhaustion of the muscle&#39;s regenerative capacity, and due to chronic inflammation of damaged muscle, fibers are replaced by connective tissue and fat, subsequently leading to a loss of function. Generally, DMD patients lose ambulation of the lower limbs early in the second decade. Later, also the muscles of the arms and shoulder girdle are affected and patients often develop thoracolumbar scoliosis due to asymmetric weakening of the muscles supporting the spinal cord. Assisted ventilation is generally required in the late teens or early twenties. Respiratory and heart failure lead to death in the third or fourth decade1.
Although the causative gene has been discovered over 25 years ago2, there is no cure available for DMD. However, improved health care and the use of corticosteroids have increased life expectancy in the Western world3. With the use of animal models like the mdx mouse, major steps forward into the discovery of potential therapeutic strategies have been made. The mdx mouse is the most commonly used DMD mouse model. It has a point mutation in exon 23 of the murine Dmd gene and consequently lacks dystrophin4. Over the last couple of years, many proposed strategies have progressed into clinical trials5-9. In these trials, improvement of muscle function is the primary endpoint, underlying the importance of testing the benefit of compounds on muscle function in mice during the pre clinical stage of testing.
Like DMD patients, also the dystrophin negative muscle fibers of mdx mice are vulnerable to exercise induced damage and their muscle function is impaired compared to C57BL/10ScSnJ wild type mice. This impairment can be assessed with a variety of functional tests. Some of these tests are noninvasive and do not interfere with muscle pathology (e.g. forelimb grip strength, hanging tests and rotarod running). Therefore they can be used to monitor the natural history of the disease or determine the effects of compounds on disease progression. To get an in depth picture of the influence of compounds on muscle function in mdx mice, a functional test regime that does not interfere with disease progression consisting of all of these tests can be used10.
Alternatively, forced treadmill running can be used to intentionally exacerbate disease progression and test the protective capacities of compounds11. The treadmill can also be used as outcome measure in which running time till exhaustion is measured12, or as a tool to fatigue mdx mice so that they perform less well in a subsequent functional test ensuring larger differences in performance between treatment groups13. When choosing functional tests, their effect on disease progression should be kept in mind especially when testing dystrophic mice like the mdx mouse14.
We here describe in detail how to perform the most commonly used functional tests in a reliable and reproducible manner based on available standard operating procedures from the TREAT-NMD network. <a href="http://www.treat-nmd.eu/resources/research-resources/dmd-sops/" target="_blank">Click here to visit TREAT-NMD</a>.

<table><tbody><tr><td>Mouse grip strength meter</td><td>Chatillon DFE (resold by Columbus Instruments)</td><td># 80529</td><td></td></tr><tr><td>Hanging wire 2 limbs device</td><td>Cloth hanger or custom made device</td><td></td><td></td></tr><tr><td>Hanging wire 4 limbs device</td><td>Lid of rat cage or custom made device</td><td></td><td></td></tr><tr><td>Rotarod</td><td>Ugo Basil</td><td># 47600</td><td></td></tr><tr><td>Treadmill for mice Exer 3/6</td><td>Columbus Instruments</td><td># 1055SRM</td><td></td></tr></tbody></table>

assessing functional performance in the mdx mouse model

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential pharmaceutical compounds and gene therapy approaches have progressed into clinical trials. With improvement in muscle function being the most important end point in these trials, a lot of emphasis has been placed on setting up reliable, reproducible, and easy to perform functional tests to pre clinically assess muscle function, strength, condition, and coordination in the mdx mouse model for DMD. Both invasive and noninvasive tests are available. Tests that do not exacerbate the disease can be used to determine the natural history of the disease and the effects of therapeutic interventions (e.g. forelimb grip strength test, two different hanging tests using either a wire or a grid and rotarod running). Alternatively, forced treadmill running can be used to enhance disease progression and/or assess protective effects of therapeutic interventions on disease pathology. We here describe how to perform these most commonly used functional tests in a reliable and reproducible manner. Using these protocols based on standard operating procedures enables comparison of data between different laboratories.

The forelimb grip strength of wild type and mdx mice increases between the age of 4-12 weeks and reduces again in older mice. Impairments in force can already be observed in young mdx mice. Representative data of 9 week old female mice are shown in Figures 1A and 1B. Although fatigue does not differ between the strains yet at this age, mdx mice are weaker than wild type mice. We do not have data yet on fatigability in older mdx and wild type mice....

Watch this Scientific Journal Video about Assessing Functional Performance in the Mdx Mouse Model at JoVE.com

Assessing Functional Performance in the Mdx Mouse Model

The primary outcome measure in clinical trials for neuromuscular disorders is generally improved muscle function. Therefore, assessing the effect of potential therapeutic compounds on muscle performance pre clinically in mouse models is of great importance. We here describe several functional tests to address this.

Assessing Functional Performance in the Mdx Mouse Model

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential ...

assessing-functional-performance-in-the-mdx-mouse-model

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Behavior

32.3K Views.  Leiden University Medical Center. The primary outcome measure in clinical trials for neuromuscular disorders is generally improved muscle function. Therefore, assessing the effect of potential therapeutic compounds on muscle performance pre clinically in mouse models is of great importance. We here describe several functional tests to address this.

Video: Assessing Functional Performance in the Mdx Mouse Model

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  ...

Measuring the Strength of Mice

Kondziela7 devised the inverted screen test and published it in 1964. It is a test of muscle strength using all four limbs. Most normal mice easily score maximum on this task; it is a quick but insensitive gross screen, and the weights test described in this article will provide a finer measure of muscular strength.
There are also several strain gauge-based pieces of apparatus available commercially that will provide more graded data than the inverted screen test, but their cost may put them beyond the reach of many laboratories which do not specialize in strength testing. Hence in 2000 a cheap and simple apparatus was devised by the author. It consists of a series of chain links of increasing length, attached to a "fur collector" a ball of fine wire mesh sold for preventing limescale build up in hard water areas. An accidental observation revealed that mice could grip these very tightly, so they proved ideal as a grip point for a weight-lifting apparatus. A common fault with commercial strength meters is that the bar or other grip feature is not thin enough for mice to exert a maximum grip. As a general rule, the thinner the wire or bar, the better a mouse can grip with its small claws.
This is a pure test of strength, although as for any test motivational factors could potentially play a role. The use of scale collectors, however, seems to minimize motivational problems as the motivation appears to be very high for most normal young adult mice.

Deficits in muscular strength occur in many clinical conditions such as motor neuron disease. The inverted screen and weight lifting tests described here measure strength in mice almost exclusively, with minimal influence of factors such as coordination.

Kondziela7 devised the inverted screen test and published it in 1964. It is a test of muscle strength using all four limbs. Most normal mice easily score ...

Medicine

Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing locomotive impairment in animal models of neuromuscular disease and efficacy of therapeutic drugs that may improve locomotion and/or muscle function. The open field activity measurement provides a different measure than muscle strength, which is commonly assessed by grip strength measurements. It can also show how drugs may affect other body systems as well when used with additional outcome measures. In addition, measures such as total distance traveled mirror the 6 min walk test, a clinical trial outcome measure. However, open field activity monitoring is also associated with significant challenges: Open field activity measurements vary according to animal strain, age, sex, and circadian rhythm. In addition, room temperature, humidity, lighting, noise, and even odor can affect assessment outcomes. Overall, this manuscript provides a well-tested and standardized open field activity SOP for preclinical trials in animal models of neuromuscular diseases. We provide a discussion of important considerations, typical results, data analysis, and detail the strengths and weaknesses of open field testing. In addition, we provide recommendations for optimal study design when using open field activity in a preclinical trial.

Open field activity levels are used to assess locomotive and behavioral activity levels. This protocol provides a well-designed, standardized protocol to use in preclinical trials for neuromuscular disorders.

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing ...

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment

The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer&#8217;s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer&#8217;s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.

This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences.

 The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, ...

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 ...

Neuroscience

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation in mouse models is a non-invasive and reproducible activity assay that is directly analogous to measures of patient ambulation such as the 6-minute walk test and related mobility scores. Many common methods for testing mouse ambulation speed and distance are based on the open field test, where an animal&#39;s free movement within an arena is measured over time. One major downside to this approach is that commercial software and equipment for high-resolution motion tracking is expensive and may require transferring mice to specialized facilities for testing. Here, we describe a low-cost, video-based system for measuring mouse ambulation that utilizes free and open-source software. Using this protocol, we demonstrate that voluntary ambulation in the dystrophin-null mdx mouse model for Duchenne muscular dystrophy (DMD) is decreased relative to wild-type mouse activity. In mdx mice expressing the utrophin transgene, these activity deficits are not observed and the total distance traveled is indistinguishable from wild-type mice. This method is effective for measuring changes in voluntary ambulation associated with dystrophic pathology, and provides a versatile platform that can be readily adapted to diverse research settings.

This protocol describes a flexible, low-cost system for measuring mouse ambulation in an open field activity assay. We show that a 6-minute ambulation assay based on this system detects a decrease in voluntary movement in mdx mice, and accurately distinguishes improvement in a muscle-specific rescue of these animals.

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation ...

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 ...

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice.

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating ...

Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the application of these techniques to identify gait deficits in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). Paralysis and motor deficits in mice subjected to EAE are typically assessed using a clinical scoring scale. However, this scale yields only ordinal data that provides little information about the precise nature of the motor deficits. EAE disease severity has also been assessed by rotarod performance, which provides a measure of general motor coordination. By contrast, kinematic gait analysis of the hind limb in the sagittal plane generates highly precise information about how movement is impaired. To perform this procedure, reflective markers are placed on a hind limb to detect joint movement while a mouse is walking on a treadmill. Motion analysis software is used to measure movement of the markers during walking. Kinematic gait parameters are then derived from the resultant data. We show how these gait parameters can be used to quantify impaired movements of the hip, knee, and ankle joints in EAE. These techniques may be used to better understand disease mechanisms and identify potential treatments for MS and other neurodegenerative disorders that impair mobility.

Kinematic gait analysis in the sagittal plane yields highly precise information about how movement is executed. We describe the application of these techniques to identify gait deficits for mice subjected to autoimmune-mediated demyelination. These methods may also be used to characterize gait deficits for other mouse models featuring impaired locomotion.

Assessing Functional Performance in the Mdx Mouse Model

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Rozdziały w tym wideo

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

Measuring the Strength of Mice

Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment

Measuring Motor Coordination in Mice

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

Strategies for Assessing Autistic-Like Behaviors in Mice

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis