Name: behavioral and locomotor measurements using an open field activity monitoring system for skeletal muscle diseases
Uploaded: 2014-09-29T15:00:00.000+00:00
Duration: 6 min 52 s
Description: Watch this Scientific Journal Video about Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases at JoVE.com

This publication is funded through Cure CMD, French Muscular Distrophy Association (AFM), a translational research grant from the Muscular Dystrophy Association, The National Institutes of Health (1K26RR032082, 1P50AR060836-01, 1U54HD071601, 2R24HD050846-06), the Department of Defense (W81XWH-11-1-0330, W81XWH-11-1-0782, W81XWH-10-1-0659, W81XWH-11-1-0809, W81XWH-09-1-0599) and a pilot grant from Parent Project Muscular Distrophy (PPMD).
This paper is one of several in a series of SOPs for routinely used methodologies in the congenital muscle disease field. It reflects the efforts discussed and established by over 20 experts in the field of congenital muscle disease at the recent Congenital Muscle Disease Consortium Workshop, held in April 2013 in Washington, D.C.

NOTE: Open field activity monitoring system uses an open field Plexiglas chamber with photocell emitters and receptors equally spaced along the perimeter of the chamber (Figure 1). These photocell emitters and receptors create an x-y grid of invisible infrared beams. When an animal is placed in the chamber, it moves about, causing beam breaks. Vertical sensors are also present to assess vertical activity levels (i.e. rearing behavior) as well. The analyzer records the beam break information and rapidly analyzes it. The computer software then calculates multiple activity measures over the preset time period. These measures include: horizontal activity (units), vertical activity (units), total distance traveled (cm), movement time (sec), and rest time (sec)5.
NOTE: In general the testing room should be temperature- and humidity-controlled, with even illumination. Testing chambers should be evenly distributed about the room and should not be placed in direct light, dark corners, or shadowed areas. All instrument acclimation and testing should be performed at the same time each day (e.g., in the morning) and by the same individuals. These individuals should be blinded to the animal treatment group, and genotype when possible.
The following protocol has been performed under the guidance and approval of the Children&#8217;s National Medical Center IACUC.
1. Instrument Acclimation
<ol>
	<li>Place the mice in the testing room in their home cages for approximately 10 min to acclimate. Leave the room during the acclimation period.</li>
	<li>Return to the testing room and turn on the activity chambers. Even though data are not being collected at this time, this will further mimic the testing environment.</li>
	<li>Gently remove each mouse from their home cage and immediately place them in the test chambers. If the activity chamber contains a center divider that divides the chamber into quadrants (Figure 1), place one mouse into each empty quadrant.</li>
	<li>Once all animals are loaded into the test chambers, place the lid on top of each test chamber. Leave the room during this acclimation time.</li>
	<li>Following the 60 min, return to the room. Remove the lid from each test chamber and gently return each mouse to his or her respective home cage.</li>
	<li>Clean each chamber with disinfectant and paper towels. Ensure no dirt particles are left in the chamber. 
	NOTE: If multiple sessions are being run each day, thoroughly clean each test chamber in between each session.</li>
	<li>Repeat Steps 1-6 for 4 consecutive days. 
	NOTE: Perform acclimation one week prior to initial data collection. If animals are tested multiple times throughout a study, only perform acclimation prior to the first round of testing to avoid habituation. In addition, randomly assign the animals to a new box each session. Track the box assignments throughout the duration of the study.</li>
</ol>
2. Data Collection
<ol>
	<li>Place mice in the testing room in their home cages for 10-30 min to acclimate. Leave the room during this time.</li>
	<li>Following the 10-30 min, return to the testing room. Turn on the activity chambers and open the accompanying computer software on the computer connected to the chambers. If the chamber contains a quadrant divider, insert the partition at this time. 
	NOTE: If the test chamber contains a quadrant divider, two animals can be placed in the test chamber during data collection. One animal can be placed in the front-left quadrant and one in the back-right quadrant (Figure 1). 
	NOTE: Do not place animals in all four quadrants during data collection or in the same row or column. Placing the animals in these orientations will interfere with the x-y grid of infrared beams and the animal movement will be inaccurately measured.</li>
	<li>Configure the computer software to perform a prebeam check. This configuration will allow one to run a pre-beam check following the experimental setup and prior to the insertion of animals into the test chambers (see below). 
	NOTE: When the prebeam check is run, the computer software assesses the function of the x-y infrared beams. For example, it can determine if the photocell emitters and receptors are blocked and unable to appropriately detect movement within the chamber.</li>
	<li>Set the primary data collection parameters in the computer software to collect six 10 min blocks of data (i.e. collect data for a total of 60 min), and then enter the appropriate date, filename, and Mouse ID numbers.</li>
	<li>Once all parameters are set, run the prebeam check. If a chamber does not pass the prebeam check it is most likely due to poor alignment of the center quadrant divider or test chamber. If this happens, realign the center quadrant divider and test chamber until the sensors are no longer blocked and the system states that the test chamber is ready. If this does not correct the problem, reference the instrument manual.</li>
	<li>When all test chambers are ready, gently remove the mouse from his or her home cage and immediately place him or her into the test chamber. Note the ID of the mouse and make sure it matches the one entered into computer.</li>
	<li>Once all animals are appropriately loaded into the test chambers, place the lid on top of each chamber. Then select the appropriate command in the computer software to start data collection. At this time, the analyzer and computer software will start recording activity levels according to the data collection parameters.</li>
	<li>Leave the testing room for the remainder of the testing period.</li>
	<li>Upon completion of the testing period (i.e. 60 min later), immediately return to the testing room. Save the data, and then return each animal to their respective home cage.</li>
	<li>Clean all units with a disinfectant and paper towels. 
	NOTE: If multiple sessions are being run each day, thoroughly clean each test chamber in between each session.</li>
	<li>Export data to a spreadsheet and then exit the software program.</li>
	<li>Check the data to ensure they were recorded. If the data were not recorded, or the animals slept throughout the entirety of the data collection time period, perform an extra day of data collection. 
	NOTE: An animal is considered to be &#8220;sleeping&#8221; if it does not move throughout the entire 60 min testing duration.</li>
	<li>Repeat Steps 2.1-2.12 for 4 consecutive days. 
	NOTE: If animals are tested at multiple time points throughout the duration of a study, do not perform open field activity measurements more than once a month to avoid habituation. In addition, randomly assign the animals to a new box each session. Track the box assignments throughout the duration of the study.</li>
</ol>
3. Data Analysis
<ol>
	<li>Calculate the mean horizontal activity (units), vertical activity (units), total distance traveled (cm), movement time (sec), and rest time (sec) per mouse and group. The computer software calculates and reports the total horizontal activity (units), vertical activity (units), total distance traveled (cm), movement time (sec), and rest time (sec) over the data collection period (i.e. 60 min) for each mouse. Calculate the mean for each of the aforementioned parameters from the 4 days of data collection.</li>
	<li>Prior to performing any statistical analyses, assess the normality of the data using the Shapiro-Wilk test, and check for outliers using the Grubb&#8217;s test. Remove any significant outliers (p &#60;0.05).</li>
	<li>For normally distributed data, compare means between groups using either an independent sample t-test or a one way ANOVA and post-hoc test with p-values adjusted for multiple comparisons depending on the total number of treatment groups.</li>
	<li>For non-normally distributed data, compare median values between groups using either the Wilcoxon rank sum test, or a Kruskal-Wallis test and rank sum test with resulting p-values adjusted for multiple comparisons depending on the total number of treatment groups.</li>
</ol>

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The authors have nothing to disclose.

The open field activity measurement is an in vivo assay that can be beneficial for assessing disease progression and drug efficacy in animal models of neuromuscular disease 6,7,11-14. As shown in Figure 2, it provides an assessment of activity levels that generally reflect locomotive function. This is a different measure than muscle strength, making it an ideal secondary or auxiliary outcome measure to perform in a preclinical drug study. In addition, it is a clinically relevant15, noninvasive measure, which can be performed multiple times throughout the duration of a study. However, behavioral and locomotive activity is also influenced by additional factors as well (i.e. experimenter handling, environmental conditions, and cognition) creating variation in open field activity data. The goal of this paper is to provide a well-tested and standardized protocol that reduces variation and allows results to be compared across multiple labs, in hopes of enhancing translation within our field.
A major disadvantage of this measure is that it is highly variable and influenced by numerous external factors. However, we took this into consideration when developing the protocol. We assessed a variety of testing protocols ranging in duration from 1-5 days of data collection. In the end, we determined that performing instrument acclimation prior to data collection to familiarize the animals with the test chamber environment and performing 4 days of data collection significantly reduced the amount of variation in the outcome data5. This protocol was originally designed to assess behavioral and locomotive activity levels in the mdx mouse model; however, this protocol was recently validated in the Dy2J animal model as well6. It is suggested that the protocol be standardized within your lab for each animal model prior to using it in a preclinical trial.
Open field activity varies by genetic background17, sex18-20, age18, and circadian rhythm21. This requires animals of the same age, sex, and genetic background to be assessed at the same time. During the planning stages, careful thought should be put into deciding at what age or ages open field activity levels will be assessed. Each animal model has its own distinct disease progression and locomotive and behavioral phenotype, which vary in severity and by age6,15 (Figure 2 and Figure 3). Therefore, it is important to determine clinically and pathologically relevant time points to assess open field activity measures. The total number of animals needed in each treatment group to detect statistically significant differences varies by animal model, age, and sex as well. Consequently, relevant sample size calculations should also be performed during the planning stages to determine the total number of animals needed in each treatment group to detect statistically significant differences. These calculations should also take into consideration additional outcome measures used in the study (e.g., grip strength measurements or histological analyses). Based upon our power calculations, we typically use 10-12 animals per treatment group. In addition, special attention should be paid to what control strain is used in the study. There is a tendency for improper control strains to be used in preclinical studies. For example, BL6 mice are often used as a control strain for mdx mice; however, the mdx mouse is on a BL10 background. As seen in Figure 3, BL10 mice are much more active than BL6 mice, which make it impossible to compare mdx and BL6 data. When conducting preclinical studies with mdx mice, BL10 mice should be used as the control strain. Furthermore, if a study is being conducted with the Dy2J mice, BL6 mice should be used as the control strain.
Small environmental changes can also significantly affect activity levels. These include lighting, temperature, humidity, odor, noise, and human activity4,15. Therefore, it is very important that testing be performed in a temperature- and humidity controlled room with non-direct lighting at the same time each day5. The test chambers should be evenly spaced throughout the room and not placed under direct lighting or in shadowed or dark corners5. Animals should be randomly assigned to their test chambers each day to reduce the effects of varying environmental conditions throughout the room, and they should be allowed to acclimate to the testing room for 10-30 min prior to data collection. Make sure to track the box assignment of each animal throughout the duration of the study to ensure that any influence of box/environment is equally distributed among the different treatment groups. The individuals loading the animals into the test chambers and handling the animals throughout the duration of the study should be blinded to the treatment group, and animal strain when possible. In many cases affected genotypes are markedly different from related controls and blinding is not possible. However, individuals should always be blinded between treated and untreated groups. Furthermore, all individuals should leave the room during data collection to reduce noise and distractions within the room, and all chambers should be thoroughly cleaned following each session of data collection. These actions will significantly reduce variation in the data. It is important to note that animals are also highly susceptible to adaptation15. Therefore, it is suggested that animals be removed from the test chamber directly following the 60 min of data collection each day and that open field activity levels be assessed no more than once a month.
The total distance traveled and total movement time measurements tend to be the most sensitive open field activity measurements5. In the Dy2J model, the vertical activity measurement tends to be the most sensitive open field activity measure (Figure 3); however, it can be difficult to capture accurate vertical activity measurements in smaller animals. For example, it is possible that a smaller animal will exhibit rearing behavior and the sensor will not capture it due to the height of the vertical sensor. As a result, we recommend testing animals no earlier than 5 weeks of age. It is also possible that an animal will sleep throughout the duration of a data collection session. If this is the case, it is appropriate to add an additional day of data collection. Finally, poor alignment of the quadrant divider or blockage of sensors within the box can result in inaccurate data as well. Therefore, it is very important to perform a sensor precheck, prior to testing, and review all data following the end of each data collection session.
Caution should also be taken when analyzing open field activity data. Open field activity data has a tendency to be non-normally distributed and have outliers4. Prior to performing any statistical analyses, our biostatisticians highly recommend checking the data for normality and outliers. If data are non-normally distributed, one should consider utilizing a nonparametric test when comparing means. In addition, all data should be analyzed by an individual blinded to what the treatment groups are.
Overall, the open field activity measure has major advantages: a) it is a comprehensive assessment of both locomotor and behavioral activity, which is strongly, but not always correlated with locomotive function; b) it is an easy measure to perform; c) it requires no animal handling during testing; d) it is a noninvasive measure that can be performed more than once throughout the duration of a study; e) no special training is needed to perform the test; f) multiple animals can be tested at one time; and g) it is a clinically relevant outcome measure5,16. However, when testing therapeutics, keep in mind that other factors can affect an animal&#8217;s behavior, and in turn open field activity measurements. Drugs can have CNS and or other body wide effects, and behavior can also be influenced by a stressful environment. As a result, it can be difficult to distinguish if changes in locomotive or behavioral activity levels are related to changes in muscle function, muscle strength, or are a result of side effects from the drug. Therefore, additional functional, histological and or molecular assays should be conducted as well. This standardized protocol has also been successfully utilized in other muscle diseases4,17; however, as seen in Figure 3, pilot studies should be performed initially to assess the sensitivity of the measure in the animal model.

Animal models have been useful for learning about disease mechanisms, but their utility in predicting treatment efficacy in clinical trials has frequently been challenged1-3. Numerous &#8220;promising&#8221; preclinical studies are published each year; however, very few of the proposed interventions show positive results when moved to clinical trial. These discrepancies are often attributed to publication bias, overoptimistic conclusions, and poorly designed and executed pre-clinical studies that lead to irreproducible results1-3.
With the current advances in drug development for neuromuscular disorders, there is an increasing need for well-designed preclinical trials. In particular, there is a need for rigorous methodologies that can be carried out in a standardized and blinded fashion, with validated, reproducible, and translatable outcome measures. As a member of the Congenital Muscle Disease Consortium, with the desire to conduct more rigorous preclinical studies, we share here our Standard Operating Procedure (SOP) for Open Field Activity. This SOP was previously validated4 and published as a part of TREAT-NMD&#8217;s SOPs for Duchenne muscular dystrophy (DMD) animal models5. We have used this methodology to phenotype and test the therapeutic efficacy of numerous drugs in a variety of animal models of neuromuscular disease, including Lama2dy-2J/J (Dy2J) mice, the animal model for congenital muscular dystrophy (CMD)6,7. In turn, this article is adapted from our previously published TREAT-NMD SOP5.
The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice, which can be correlated with locomotive function. The test is also widely used to assess anxiety like and exploratory behaviors 8-10. In particular, the open field is a useful tool for assessing locomotive impairment in animal models of neuromuscular disease11,12 and the efficacy of therapeutic drugs that may improve locomotion and/or motor function 6,7,13,14. The open field activity assessment provides a different measure than muscle strength, which is commonly measured with grip strength, and it shows how drugs may affect other body systems (i.e. the central nervous system) as well5. In addition, the open field activity measure, total distance traveled, mirrors the 6 min walk test, a clinical trial outcome measure, which focuses on submaximal exercise performance&#160;and quality of life15,16. Overall this makes the open field activity test a beneficial secondary or auxiliary outcome measure to use in preclinical trials. However, the open field activity monitoring system also has significant challenges associated with it. The test is behavioral and can be quite variable as it is influenced by a multitude of external factors. For example, this behavior can be influenced by exploratory drive (i.e. cognition), anxiety, sickness, circadian rhythm, environmental factors, genetic background, in addition to motor output10. As a result it is imperative to conduct this measure in a standardized fashion with a controlled environment. The protocol presented here describes our open field activity SOP in detail. It provides step-by-step procedures and further discussion of important considerations to control environmental conditions and help reduce variability, typical results, data analysis, and the assessment&#8217;s strengths and weaknesses in more detail.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>VersaMax Open Field Activity Monitoring system with acrylic test chambers, and X, Y, Z axis sensors</td><td>AccuScan Instruments, Inc. Columbus Ohio, USA</td><td></td><td>Retired</td></tr><tr><td>Fusion Open Field Activity Monitoring system with acrylic test chambers, and X, Y, Z axis sensors</td><td>Omnitech Electronics, Inc. Columbus Ohio, USA</td><td></td><td>Suggested system currently on the market</td></tr><tr><td>Computer</td><td>Dell, Inc.&amp;nbsp;</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Materials&lt;/strong&gt;</td></tr><tr><td>Virkon-S Broad spectrum disinfectant (potassium peroxymonosulfate/ sodium chloride)</td><td>Pharmacal Research Laboratories, Inc.</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Mice&lt;/strong&gt;</td></tr><tr><td>B6.WK-Lama2dy-2J/J (Dy2J)</td><td>Jackson Lab</td><td>000524</td><td></td></tr><tr><td>C57BL/6J (BL6)</td><td>Jackson Lab</td><td>000664</td><td></td></tr><tr><td>SJL/J (SJL)</td><td>Jackson Lab</td><td>000686</td><td></td></tr><tr><td>C57BL/10ScSn-Dmdmdx/J (mdx)</td><td>Jackson Lab</td><td>001801</td><td></td></tr><tr><td>C57BL/10ScSnJ (BL10)</td><td>Jackson Lab</td><td>000476</td><td></td></tr></tbody></table>

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.

When analyzing open field activity data, we focus on a few select measurements that provide an assessment of activity levels that generally reflect locomotive function. These parameters include: horizontal activity, vertical activity, movement time, rest time, and total distance traveled. In general, animals with reduced muscle function will be less active and have lower ambulatory activity. This is generally associated with decreased horizontal activity, vertical activity, total distance traveled and movement time, and increased rest time5,6,12,17. On the contrary, animals with unimpaired muscle function or those treated with therapeutics that decrease the progression of deteriorating muscle pathology are more likely to display higher activity levels6,7,14,17.
To show an example of typical results obtained using this protocol in animal models of neuromuscular disease, we provided data from a longitudinal study we previously conducted in the Dy2J congenital muscular dystrophy (CMD) animal model6. In short, the Dy2J model contains a truncated form of the LAMA2 gene that results in hindlimb paralysis, demyelination, and dystrophic skeletal muscle changes.&#160;The impact of this muscle pathology on activity levels is observed in these mice. For example, the Dy2J mice in the study tended to exhibit lower horizontal activity levels, and less distance traveled, compared to their age and gender-matched BL/6 wild-type controls throughout the duration of the study (Figure 2); however, these differences were not always significant. The lack of significance is most likely due to the small sample size, and high intra- group variation in the BL6 data. Variation is typical of open field activity data; however, these data in particular lack sufficient power to determine if these groups are statistically different from one another. Typically an n = 10-12 should be used to detect statistically significant differences5,17. For example, when a larger sample size is used, as was done in the SJL study (see the second BL6 bar in Figures 3B, 3D, and&#160;3E) significant differences between groups can be observed. The Dy2J animals also showed a complete loss of vertical activity levels, which mirrored their hindlimb paralysis, and in turn, an inability to rear (Figure 2B)6. Lastly, it is important to note the sex differences in the activity levels. For example, the females tended to be more active than males, displaying higher levels of horizontal activity, vertical activity, and total distance traveled (Figure 2); however these differences were not statistically significant.
We have also provided data from several previous studies conducted in other animal models of neuromuscular diseases, as well to highlight several additional factors that affect activity levels (Figure 3). For example, activity levels vary by genetic background10. BL10 wild type mice exhibit greater horizontal activity, vertical activity, and total distance traveled compared to age- and gender matched BL6 wild type mice (Figure 3). This is an important observation to note, as utilizing the incorrect control strains in a study can render the data unusable. Second, activity levels vary by disease model and phenotype (Figure 3). For example, the SJL mouse, the animal model for limb girdle muscular dystrophy-2B (LGMD-2B), shows the lowest level of horizontal activity and total distance traveled, followed by the Dy2J mouse and the mdx mouse, the animal model for Duchenne muscular dystrophy (Figures 3A, 3C). However, Dy2J mice, because of their hindlimb paralysis, display the lowest level of vertical activity (Figure 3B). It is also important to note that the higher level of activity in the mdx phenotype is most likely attributed to the increased activity level of the BL10 background strain. Finally, this figure highlights the importance of animal age/disease pathology at time of testing. For example, at 30 weeks of age, no differences can be detected in activity levels between mdx mice and their age- and sex matched BL10 wild type controls (Figure 3). However, at 6 weeks of age, around the peak necrotic phase in the mdx mouse model, mdx mice display a significant decrease in vertical activity, and horizontal activity. A decrease is also observed in total distance traveled, but this difference is not significant (Figure 3)4.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51785/51785fig1highres.jpg" width="500" /> 
Figure 1.&#160;Open field activity apparatuses. Two open field apparatuses with center quadrant dividers, and tops. If removable center quadrant dividers are present, animals should only be placed in the front left (1, 3) and back right chambers (2, 4) of each box during testing to obtain valid readings.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51785/51785fig2highres.jpg" width="500" /> 
Figure 2.&#160;Open field activity data. Typical open field activity data for Dy2J (grey line, n = 3) and age- and gender matched BL6 control strain (black line, n = 3) mice at 14, 19, 23, 25, and 30 weeks of age (A - F). A) Horizontal activity (arbitrary units) males, B) Horizontal activity (arbitrary units) females, C) Vertical activity (arbitrary units) males, D) Vertical activity (arbitrary units) females E) Total distance traveled (cm) males, E) Total distance traveled (cm) females. Data were collected over 4 consecutive days and averaged per mouse and group. The same mice were tested at each time point. Data represent mean &#177; SEM. Data previously published6. Dy2J and BL6 mice were compared with an Independent sample T-test at each time-point. A p-value &#60;0.05 was considered significant. * p &#60;0.05, **p &#60;0.01, ***p &#60;0.001.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51785/51785fig3highres.jpg" width="500" /> 
Figure 3.&#160;Open field activity data multiple strains. Typical behavioral activity data from male BL10 (6 weeks of age, n = 8; 25-30 weeks of age, n = 10), mdx (6 weeks of age, n = 9; 25-30 weeks of age, n = 15), BL6 (control group for Dy2J mice, n = 3; control group for SJL mice, n = 13), Dy2J (n = 3), and SJL (n = 13) mice at varying ages. A) Horizontal activity (arbitrary units) data from BL10 and mdx mice at 6 and 25-30 weeks of age, B) Horizontal activity (arbitrary units) data from BL6, Dy2J, and SJL mice at 25-30 weeks of age, C) Vertical activity (arbitrary units) data from BL10 and Mdx mice at 6 and 25-30 weeks of age, D) Vertical activity (arbitrary units) data from BL6, Dy2J, and SJL mice at 25-30 weeks of age, E) Total distance traveled (cm) data from BL10 and mdx mice at 6 and 25-30 weeks of age, and F) Total distance traveled (cm) data from BL6, Dy2J, and SJL mice at 25-30 weeks of age. Dy2J, animal model for CMD with laminin &#945;2 gene mutation on the BL6 background; SJL, animal model for limb girdle muscular dystrophy-2B (LGMD-2B); mdx, animal model for DMD on the BL10 background. There is no dysferlin-sufficient SJL control. Data are means &#177; SEM. B, C, and D contain data previously published6,17. Data were not normally distributed; therefore, data were compared using a Wilcoxon rank sums test. A p-value of p &#60;0.05 was considered significant. The following comparisons were made for each parameter: a) BL10 and mdx mice at 6 weeks of age, p &#60;0.05; b) BL10 and mdx mice at 25-30 weeks of age, not significant; c) Dy2J and BL6 matched control strain mice at 25-30 weeks of age, p &#60;0.05; d) SJL and BL6 matched control strain at 25-30 weeks of age, p &#60;0.001; e) BL10 mice at 25-30 weeks of age and BL6 (control group for Dy2J mice) mice at 25 weeks of age, p &#60;0.01; f) BL10 at 25-30 weeks of age and BL6 (control group for SJL mice) at 25-30 weeks of age, p &#60;0.001.

Watch this Scientific Journal Video about Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases at JoVE.com

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

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 ...

behavioral-locomotor-measurements-using-an-open-field-activity

Universidad San Sebastian

Research

JoVE Journal

Behavior

53.6K Views.  Children's National Medical Center. 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.

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

Methods to Quantify Pharmacologically Induced Alterations in Motor Function in Human Incomplete SCI

Spinal cord injury (SCI) is a debilitating disorder, which produces profound deficits in volitional motor control. Following medical stabilization, recovery from SCI typically involves long term rehabilitation. While recovery of walking ability is a primary goal in many patients early after injury, those with a motor incomplete SCI, indicating partial preservation of volitional control, may have the sufficient residual descending pathways necessary to attain this goal. However, despite physical interventions, motor impairments including weakness, and the manifestation of abnormal involuntary reflex activity, called spasticity or spasms, are thought to contribute to reduced walking recovery. Doctrinaire thought suggests that remediation of this abnormal motor reflexes associated with SCI will produce functional benefits to the patient. For example, physicians and therapists will provide specific pharmacological or physical interventions directed towards reducing spasticity or spasms, although there continues to be little empirical data suggesting that these strategies improve walking ability.
In the past few decades, accumulating data has suggested that specific neuromodulatory agents, including agents which mimic or facilitate the actions of the monoamines, including serotonin (5HT) and norepinephrine (NE), can initiate or augment walking behaviors in animal models of SCI. Interestingly, many of these agents, particularly 5HTergic agonists, can markedly increase spinal excitability, which in turn also increases reflex activity in these animals. Counterintuitive to traditional theories of recovery following human SCI, the empirical evidence from basic science experiments suggest that this reflex hyper excitability and generation of locomotor behaviors are driven in parallel by neuromodulatory inputs (5HT) and may be necessary for functional recovery following SCI. 
The application of this novel concept derived from basic scientific studies to promote recovery following human SCI would appear to be seamless, although the direct translation of the findings can be extremely challenging. Specifically, in the animal models, an implanted catheter facilitates delivery of very specific 5HT agonist compounds directly onto the spinal circuitry. The translation of this technique to humans is hindered by the lack of specific surgical techniques or available pharmacological agents directed towards 5HT receptor subtypes that are safe and effective for human clinical trials. However, oral administration of commonly available 5HTergic agents, such as selective serotonin reuptake inhibitors (SSRIs), may be a viable option to increase central 5HT concentrations in order to facilitate walking recovery in humans. Systematic quantification of how these SSRIs modulate human motor behaviors following SCI, with a specific focus on strength, reflexes, and the recovery of walking ability, are missing.
This video demonstration is a progressive attempt to systematically and quantitatively assess the modulation of reflex activity, volitional strength and ambulation following the acute oral administration of an SSRI in human SCI. Agents are applied on single days to assess the immediate effects on motor function in this patient population, with long-term studies involving repeated drug administration combined with intensive physical interventions.

This video demonstrates modulation of reflex activity, volitional strength and ambulation through clinical and quantitative assessments in individuals with motor incomplete SCI as a result of acute oral administration of a serotonin reuptake inhibitor (SSRI).

Spinal cord injury (SCI) is a debilitating disorder, which produces profound deficits in volitional motor control. Following medical stabilization, ...

Medicine

Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes. As such, all behaviors manifest through the motor system. In the fields of neuroscience and orthopedics, locomotion is a commonly evaluated behavior for a variety of disease models. For example, locomotor recovery after traumatic injury to the nervous system is one of the most commonly evaluated behaviors 1-3. Though locomotion can be evaluated using a variety of endpoint measurements (e.g. time taken to complete a locomotor task, etc), semiquantitative kinematic measures (e.g. ordinal rating scales (e.g. Basso Beattie and Bresnahan locomotor (BBB) rating scale, etc)) and surrogate measures of behaviour (e.g. muscle force, nerve conduction velocity, etc), only kinetics (force measurements) and kinematics (measurements of body segments in space) provide a detailed description of the strategy by which an animal is able to locomote 1. Though not new, kinematic and kinetic measurements of locomoting rodents is now more readily accessible due to the availability of commercially available equipment designed for this purpose. Importantly, however, experimenters need to be very familiar with theory of biomechanical analyses and understand the benefits and limitations of these forms of analyses prior to embarking on what will become a relatively labor-intensive study. The present paper aims to describe a method for collecting kinematic and ground reaction force data using commercially available equipment. Details of equipment and apparatus set-up, pre-training of animals, inclusion and exclusion criteria of acceptable runs, and methods for collecting the data are described. We illustrate the utility of this behavioral analysis technique by describing the kinematics and kinetics of strain-matched young adult, middle-aged, and geriatric rats.

Locomotion is often examined as a behavioural outcome in various models of disease in fields such as neuroscience and orthopedics. This video paper intends to describe a method for collecting ground reaction forces and kinematics from rats during unrestrained locomotion.

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes.  As such, all behaviors manifest through the ...

Neuroscience

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 ...

An Objective and Child-friendly Assessment of Arm Function by Using a 3-D Sensor

Progressive and irreversible muscle atrophy characterizes Spinal Muscular Atrophy (SMA) and other similar muscle disorder diseases. Objective assessment of muscle functions is an essential and important, although challenging, prerequisite for successful clinical trials. Current clinical rating scales restrain the movement abnormalities to certain predefined coarse-grained individual items. The Kinect 3-D sensor has emerged as a low-cost and portable motion sensing technology used to capture and track people&#39;s movement in many medical and research fields. A novel approach using this 3-D sensor was developed and a game-like test was designed to objectively measure the upper limb function of patients with SMA. The prototype test targeted joint movement capability. While sitting in a virtual scene, the patient was instructed to extend, flex, and lift the whole arm in order to reach and place some objects. Both kinematic and spatiotemporal characteristics of upper limb movement were extracted and analyzed, e.g., elbow extension and flexion angles, hand velocity, and acceleration. The first study included a small cohort of 18 ambulant SMA patients and 19 age- and gender-matched healthy controls. A comprehensive analysis of arm movement was achieved; however, no significant difference between the groups were found due to the mismatch of patient&#39;s capability and the test difficulty. Based on this experience, a second version of the test consisting of a modified version of the first game with increased difficulties and a second game targeting muscle endurance were designed and implemented. The new test has not been conducted in any patient groups yet. Our work has demonstrated the potential capability of the 3-D sensor in assessing such muscle function and suggested an objective approach to complement the clinical rating scales.

An objective measure of muscle functions is challenging especially in children. Based on a commercially available digital 3-D sensor, a child-friendly gaming test was developed to assess upper limb function for clinical trials.

Progressive and irreversible muscle atrophy characterizes Spinal Muscular Atrophy (SMA) and other similar muscle disorder diseases. Objective assessment ...

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 ...

Low-Cost Gait Analysis for Behavioral Phenotyping of Mouse Models of Neuromuscular Disease

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method of gait analysis demonstrated here is a simple but effective measure of gait abnormalities in murine models. Footprints are analyzed by painting a mouse&#8217;s feet with non-toxic washable paint and allowing the subject to walk through a tunnel on a sheet of paper. The design of the testing tunnel takes advantage of natural mouse behavior and their affinity for small dark places. The stride length, stride width, and toe spread of each mouse is easily measured using a ruler and a pencil. This is a well-established and reliable method, and it generates several metrics that are analogous to digital systems. This approach is sensitive enough to detect changes in stride early in phenotype presentation, and due to its non-invasive approach, it allows for testing of groups across life-span or phenotypic presentation.

Footprint analysis is a low-cost alternative to digitized gait analysis programs for researchers quantifying movement abnormalities in mice. Because of its speed, simplicity, and longitudinal potential, it is ideal for behavioral phenotyping of mouse models.

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method ...

Wireless Intramuscular EMG System for Locomotion Studies in Stroke Model Rats

A Real-Time Wearable Electromyography Measurement System for Small Animals

The intramuscular electromyography (EMG) measurement method for experimental animals has been implemented in various ways. Among these methods, tethering cables to external measurement devices can restrict the movement of experimental animals, while implantable devices may cause unwanted side effects due to the constant presence of a device with considerable size and weight. To address these issues, we propose a low-cost, wireless, detachable EMG measurement system and experimental procedure. This article focuses on the surgical installation of intramuscular wire electrodes with small connectors and the development of the wireless system. Notably, in this system, only the wire electrodes are inserted into the animal's body. Using this system, EMG measurements can be easily performed by attaching the circuit system to a connector installed on the animal's back, with real-time monitoring achievable on a laptop. The proposed method is explained in a detailed, step-by-step manner, followed by a demonstration involving the insertion of intramuscular electrodes into the hindlimbs of a rat. A treadmill experiment is conducted for a locomotion study, and the resulting electrophysiological signals are subsequently obtained and analyzed.

A protocol is presented for the installation and preparation of intramuscular electrodes, along with an animal-mountable, miniaturized measurement device for electromyography (EMG) analysis in locomotion studies. This system enables the wireless transmission of real-time EMG data for investigating locomotion recovery in stroke model rats.

The intramuscular electromyography (EMG) measurement method for experimental animals has been implemented in various ways. Among these methods, tethering ...

Engineering

Home-Based Monitor for Gait and Activity Analysis

Current outcomes in neuromuscular disorder clinical trials include motor function scales, timed tests, and strength measures performed by trained clinical evaluators. These measures are slightly subjective and are performed during a visit to a clinic or hospital and constitute therefore a point assessment. Point assessments can be influenced by daily patient condition or factors such as fatigue, motivation, and intercurrent illness. To enable home-based monitoring of gait and activity, a wearable magneto-inertial sensor (WMIS) has been developed. This device is a movement monitor composed of two very light watch-like sensors and a docking station. Each sensor contains a tri-axial accelerometer, gyroscope, magnetometer, and a barometer that record linear acceleration, angular velocity, the magnetic field of the movement in all directions, and barometric altitude, respectively. The sensors can be worn on the wrist, ankle, or wheelchair to record the subject&#8217;s movements during the day. The docking station enables data uploading and recharging of sensor batteries during the night. Data are analyzed using proprietary algorithms to compute parameters representative of the type and intensity of the performed movement. This WMIS can record a set of digital biomarkers, including cumulative variables, such as total number of meters walked, and descriptive gait variables, such as the percentage of the most rapid or longest stride that represents the top performance of patient over a predefined period of time.

This innovative device uses magneto-inertial sensors to permit gait and activity analysis in uncontrolled environments. Currently in the qualification process as an outcome measure in the European Medical Agency, one of the applications will be to serve as a clinical endpoint in clinical trials in neuromuscular diseases.

Current outcomes in neuromuscular disorder clinical trials include motor function scales, timed tests, and strength measures performed by trained clinical ...

Bioengineering

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated measurements over weeks or months of accessory respiratory muscle activity while simultaneously measuring ventilation in a non-anesthetized, freely behaving mouse. The technique includes the surgical implantation of a radio transmitter and the insertion of electrode leads into the scalene and trapezius muscles to measure the electromyogram activity of these inspiratory muscles. Ventilation is measured by whole-body plethysmography, and animal movement is assessed by video and is synchronized with electromyogram activity. Measurements of muscle activity and ventilation in a mouse model of amyotrophic lateral sclerosis are presented to show how this tool can be used to investigate how respiratory muscle activity changes over time and to assess the impact of muscle activity on ventilation. The described methods can easily be adapted to measure the activity of other muscles or to assess accessory respiratory muscle activity in additional mouse models of disease or injury.

This paper introduces a method for repeated measurements of ventilation and respiratory muscle activity in a freely behaving amyotrophic lateral sclerosis (ALS) mouse model throughout disease progression with whole-body plethysmography and electromyography via an implanted telemetry device.

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated ...

Measurements of Motor Function and Other Clinical Outcome Parameters in Ambulant Children with Duchenne Muscular Dystrophy

While the number of new treatment options tested in patients with Duchenne muscular dystrophy (DMD) is increasing, there is still no defining of the most reliable assessments regarding therapeutic efficacy. We present clinical and radiological outcome measures used in ambulatory patients participating in our trial &#34;Treatment with L-citrulline and metformin in Duchenne muscular dystrophy&#34;. The motor function measure is a validated test in patients with neuromuscular disorders that consists of 32 items and assesses all three dimensions of motor performance including standing and transfer (D1 subscore), axial and proximal motor function (D2 subscore), and distal motor function (D3 subscore). The test shows high intra- and inter-rater variability but only when strictly following guidelines of the materials, examination steps, and calculation of scores. The 6-minute walk test, timed 10-meter walk/run test, and supine-up time are commonly used timed functional tests that also sufficiently monitor changes in muscle function; however, they strongly depend on patient collaboration. Quantitative MRI is an objective and sensitive biomarker to detect subclinical changes, though the examination costs may be a reason for its limited use. In this study, a high correlation between all clinical assessments and quantitative MRI scans was found. The combinational use of these methods provides a better understanding about disease progression; however, longitudinal studies are needed to validate their reliability.

The aim of this study is to present the most reliable clinical outcome measures and their correlations with quantitative muscle MRI in ambulant patients with Duchenne muscular dystrophy.

While the number of new treatment options tested in patients with Duchenne muscular dystrophy (DMD) is increasing, there is still no defining of the most ...