Name: battery of behavioral tests assessing general locomotion, muscular strength, and coordination in mice
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Description: Watch this Scientific Journal Video about Battery of Behavioral Tests Assessing General Locomotion, Muscular Strength, and Coordination in Mice at JoVE.com

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 experime...

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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="42" style="height:42px;">Square Open Field 45 x 45 x 40 cm (grey color). Other floor color available: white, black.</td>
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			<td height="42" style="height:42px;">&#160;Open Field Preconfigured Module</td>
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			<td height="42" style="height:42px;">Smart V3.0 Software Platform&#160;</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 ...

The overall goal of this battery of behavioral testing is to observe the general locomotion, muscular strength, and coordination in mice. These methods can help answer key questions about the implementation of different treatments in multiple preclinical trials, such as the benefit of treatment outcomes on motility in mice. The main advantage of this technique is that multiple facets and dimensions of movement are measured and assessed by this battery of behavioral testing.Demonstrating the procedure will be Meera, a master student in our lab. Begin by turning on the open field test program, and connecting the computer to the camera or open field box. Start the selective SmartWare software in the Open Field icon.Select okay. If applicable, choose the Digital Analog Converter source from the camera options. Follow the instructions to make the appropriate dimensions to cover the open field box.Next, make sure that the mouse is being detected within the confines of the open field by calibrating the appropriate part of the software. Click time to preset an amount of time to test the mice. In this case, set five minutes.Enter the subjects, by clicking and writing the appropriate name for the files. For example, phase one, session one. Next, place a mouse in the center of the open fields, and click the start icon to prompt the program to begin recording.After five minutes, observe the program 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 of the program. Then click start.Finally, to analyze the data click on the analyze icon at the top right of the screen. Click analyze again, and then summary report. Begin by placing a mouse in the center of a mesh.And invert the mesh at lease 20 centimeters above a clear container. Once inverted, start the timer. Record the time of latency for the mouse to fall off the mesh.If the mouse does not fall within 60 seconds, give it a score of 60 seconds, and conduct no further trials. Then, allow the mouse to rest for at least five minutes. Next turn on the rotarod and the computer.And ensure that they are properly connected for data recording. In order to run the accelerated protocol, use codes on the keypad to set the desired length of time on the rotarod to 300 seconds, the number of lanes to five, the starting and end speed to four and forty rotations per minute, respectively, and the ramp time to 300 seconds. Then set the unit to mouse forward mode.Ensure that all magnetized lanes are balanced before beginning the trial, and place one mouse in each lane of the rotarod. Next press start to initiate the program. As the mice fall, remove them from their lanes promptly to ensure that they do not trip the other lanes.Finally, allow the mice to rest for a 10 minute intertrial interval. During this interval, begin the trial for the next set of mice. This data demonstrates the use of the rotarod as a means to see how the treated mice are behaving compared to the normal heterozygous mice.The divergence of the two groups at 40 weeks denotes the beginning of the treatment group's decreased coordination in comparison to normal controls. Over monthly behavioral testing the treated mice performed similarly to the normal heterozygous controls, the open field test, rotarod, and mesh test. At 12 weeks, treated animals outperform untreated animals on time moving in the open field test and on the latency to fall in the mesh test.Once mastered, this technique can be completed in two and half hours to complete 20 mice through the protocol, if performed properly. While attempting this procedure, it's important to remember to clean the apparatus between each mouse's trial, in order to avoid possible contaminates across mice and cages. Following this procedure, other methods such as further behavioral and biochemical assays can be performed in order to answer additional questions concerning treatment efficiency, such as biodistribution assays, enzyme activity assays, histology assays, or memory and learning tests.After it's development, this technique paved the way for researchers in the fields of psychology and later in neurosciences to explore the affect of a variety of treatments and conditions have on model organisms. After watching the video, you should have a good understanding of how to perform behavior testing for general locomotion muscular strength and coordination. Don't forget that working with mice can be hazardous, and precautions such as wearing proper personal protective equipment should always be taken while performing this procedure.

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