The authors wish to thank A.M. for animal identification assistance. This work was supported by grants from the US National Institutes of Health (R01 7 RF1 AG057264 to A.R.L.S. and C.J.C. and R01 NS100023 to A.R.L.S) and the Muscular Dystrophy Association (Basic Research Grant to A.R.L.S., Development Grant to C.J.C.).

All testing conducted with mice was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Duke University. Personnel responsible for testing and scoring must be blinded to animal genotype or experimental condition until gait analysis and scoring of papers has been completed for the entire cohort.
1. Testing material preparation
<ol>
	<li>Conduct testing with a tunnel built from 3 pre-cut clear acrylic panels that are 0.375 inches thick. Assemble tunnel by gluing panels together with a sealant that specifically bonds acrylic and will not emit odors when dried.
	<ol>
		<li>For standard C57BL/6 mice, use the following tunnel measurements: 2.5 in. wide, 3 in. high, and 13 in. long. Mice must be able to comfortably walk through the tunnel and take enough steps (&#62;4) so that gait can be measured.</li>
	</ol>
	</li>
	<li>Construct the goal chamber with pre-cut gray acrylic panels 0.375 inches thick, glued together with the same sealant as used on the tunnel. The interior measurements of the chamber are 4 in. wide, 4 in. long, and 3 in. tall. Match the opening of this chamber to the opening of the tunnel (2.5 in. wide x 3.0 in. tall). Because the mice naturally prefer darkened spaces to well-lit spaces, use material that is opaque and dark in color.</li>
	<li>Use paper for tracking steps that is thick and smooth (watercolor paper works well). Cut individual papers strips to be slightly wider and longer than the width and length of the tunnel. If using the tunnel dimensions described here, cut papers to 15 in. long by 3.5 in. wide.</li>
	<li>Use two contrasting colors (e.g., green and purple) of non-toxic washable water-based paint. Assign one color for hind-limbs, the second one for forelimbs. Mice will lick the remaining paint from their feet after testing, so the selected paint must be completely non-toxic.</li>
	<li>Use two round barrel paintbrushes, one for each paint color (~0.5 cm in diameter, tapered/pointed brush tip).</li>
	<li>Select a ruler with markings down to millimeters, and a caliper with measurements down to 0.1 mm. Pencil is recommended to write on the scoring papers.</li>
	<li>Optional: For animals with high anxiety or low motivation, provide a behavioral incentive in the goal chamber. This can include small amounts of sterilized sunflower seeds (placed in the home cage 2 days prior to testing to allow habituation). On the day of testing, place sunflower seeds inside the goal chamber to encourage mice to walk through without stopping.</li>
</ol>
2. Data collection
<ol>
	<li>If testing is performed in a separate room, acclimate the mice to the new room for 30 minutes and then start the behavioral assays. Additionally, because mice are naturally nocturnal, ensure all mice are fully awake and alert for at least 5 minutes before testing.</li>
	<li>Prepare the testing setup by positioning the tunnel over the paper and marking the paper with mouse ID and testing date. Position the goal chamber at the end of the tunnel, connecting both open ends. Add sunflower seeds at the end of the tunnel (inside the goal chamber) for motivation if needed.</li>
	<li>Remove the mouse to be tested from its cage and grip it firmly by its scruff, making sure to grip the tail to stabilize movement of its hind limbs.</li>
	<li>Paint forepaws so the entire underside of all toes and the center of the foot are fully covered in paint. Repeat this with a contrasting color of paint on its hind paws. Wipe off any paint that the mouse gets on other parts of its body with a clean damp cloth to prevent smudges that may interfere with data collection. 
	NOTE: Mouse handling must be performed by experienced researchers to minimize animal stress.</li>
	<li>Place the mouse at the start of the tunnel and allow it to walk all the way into the goal chamber, and then retrieve the mouse, gently wipe off its feet with a water-dampened cloth, and return it to its home cage.</li>
	<li>Allow paper with footprints to dry fully before scoring. Wipe down the testing area and tunnel with ethanol or an equivalent cleaning solution in between each animal.</li>
</ol>
3. Scoring criteria
<ol>
	<li>Use steps that are consistently spaced with clear, non-smudged footprints for scoring. Figure 1B is a good example of a footprint sequence that can be scored. In order to generate sufficient scoring data, there must be at least 2 consecutive steps from each foot, but 4-6 steps per foot is recommended. Do not include the first and last footprints on the paper, as they are unlikely to represent normal gait because the mouse is changing its walking speed.</li>
	<li>Use stride length, stride width, and toe spread as three different measures of gait that can be analyzed using this method. 
	NOTE: Stride length and width require clear sequential prints where the forefoot region is well defined in paint. Toe spread does not require sequential prints for scoring, only clear prints of the first and last toes on a single foot. However, if a given footprint is not included in measurements of stride length or width, it cannot not be scored for toe spread. All three measures are assessed in centimeters.
	<ol>
		<li>Define stride length as the distance between two sequential footprints created by the same foot (i.e., one stride) (Figure 1A, 1B).
		<ol>
			<li>With a pencil, draw a 2-4 mm circle around the fore-foot region of both forelimb footprints (identified by assigned color above) in a single stride and draw a line between them using a ruler.</li>
			<li>Record the distance between two prints from the middle of each circle (i.e. center of each foot pad) as Right-Fore 1 (RF1) or Left-Fore 1 (LF1).</li>
			<li>Repeat for all steps that can be scored (RF2, LF2, RF3, LF3 and so on).</li>
			<li>Repeat for right and left hind-limb footprints.</li>
			<li>Average all individual recorded stride distances for each limb. For statistical analysis, individual cohort members can be averaged together.</li>
		</ol>
		</li>
		<li>Define stride width as the measure of distance between left and right forelimbs or hind-limbs (Figure 1A, 1B).
		<ol>
			<li>To assess this distance, draw and measure a line from the circled forefoot region of one hind-limb that intersects perpendicularly with the line for stride length on the contralateral hind-limb.</li>
			<li>Repeat this for all hind-limb prints that can be scored, and then average the measurements. The method of calculation for stride width is the same for fore- and hind-limbs.</li>
		</ol>
		</li>
		<li>Define toe spread as the distance between the first and last toes on a single fore- or hind-limb footprint (Figure 1A, 1B).
		<ol>
			<li>Use calipers to measure the distance between the tip of the first toe print and the tip of the last toe print.</li>
			<li>Repeat for all hind-limb prints that can be scored and average the measurements. The method of calculation for toe spread is the same for fore- and hind-limbs.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>If the paper cannot be scored, allow the animal to rest for 10 minutes before trying again.</li>
</ol>

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Q., 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=RNA+interference+improves+motor+and+neuropathological+abnormalities+in+a+Huntington's+disease+mouse+model.">RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (16), 5820-5825 (2005).</li><li>Lin, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurological+abnormalities+in+a+knock-in+mouse+model+of+Huntington's+disease.">Neurological abnormalities in a knock-in mouse model of Huntington's disease.</a> Human Molecular Genetics. 10 (2), 137-144 (2001).</li><li>Sopher, B. L., 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=Androgen+receptor+YAC+transgenic+mice+recapitulate+SBMA+motor+neuronopathy+and+implicate+VEGF164+in+the+motor+neuron+degeneration.">Androgen receptor YAC transgenic mice recapitulate SBMA motor neuronopathy and implicate VEGF164 in the motor neuron degeneration.</a> Neuron. 41 (5), 687-699 (2004).</li><li>Tillerson, J. L., Caudle, W. M., Reveron, M. E., Miller, G. 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A., Collins, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Policy:+NIH+to+balance+sex+in+cell+and+animal+studies.">Policy: NIH to balance sex in cell and animal studies.</a> Nature. 509 (7500), 282-283 (2014).</li><li>Maricelli, J. W., Lu, Q. L., Lin, D. C., Rodgers, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trendelenburg-Like+Gait,+Instability+and+Altered+Step+Patterns+in+a+Mouse+Model+for+Limb+Girdle+Muscular+Dystrophy+2i.">Trendelenburg-Like Gait, Instability and Altered Step Patterns in a Mouse Model for Limb Girdle Muscular Dystrophy 2i.</a> PLoS One. 11 (9), e0161984 (2016).</li><li>Castelhano-Carlos, M. J., Sousa, N., Ohl, F., Baumans, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+methods+in+newborn+C57BL/6+mice:+a+developmental+and+behavioural+evaluation.">Identification methods in newborn C57BL/6 mice: a developmental and behavioural evaluation.</a> Lab Animals. 44 (2), 88-103 (2010).</li><li>Lakes, E. H., Allen, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gait+analysis+methods+for+rodent+models+of+arthritic+disorders:+reviews+and+recommendations.">Gait analysis methods for rodent models of arthritic disorders: reviews and recommendations.</a> Osteoarthritis Cartilage. 24 (11), 1837-1849 (2016).</li></ol>

The authors have nothing to disclose.

Using the low-cost gait analysis method described above, we show successful identification of several parameters of gait dysfunction at post-symptomatic ages in the BAC fxAR121 mouse model of SBMA. Decreases in stride length are consistent with prior SBMA studies of mouse models and human patients9. We also show for the first time that there are significant differences in hind-limb toe spread in symptomatic SBMA mice compared to non-transgenic littermate controls. Interestingly, decreases in hind toe spreading can be caused by weakness in paw extensor muscles, tightness in paw flexor muscles, or poor nerve innervation2,19, which is also consistent with the etiology of SBMA.
The mice should readily run to the goal chamber due to their natural behavioral preference for small dark spaces, but some mice may not continuously move through the tunnel. If a mouse jumps, stops, or turns around within the tunnel (see examples in Figure 1C), repeat the assay after a rest period on a new scoring paper. The results may be salvageable if a mouse stops at the very beginning of the tunnel since it can often be gently prodded into running to the goal box.
Applying too much or too little paint to a mouse&#8217;s feet can produce unusable results. Excess paint can lead to smudged or distorted prints, while insufficient paint can produce faint or unidentifiable prints (Figure 1C). In either case, repeat the assay on a clean scoring paper to prevent inaccurate measurements.
Very young mice (&#60;3 months old) are more likely to jump forward in the tunnel, whereas older (&#62;8 months old) or very phenotypic mice are more likely to stop or resist forward movement entirely. Adding a behavioral incentive (sunflower seeds) in the goal chamber can help decrease the frequency of problematic behaviors by encouraging unmotivated mice to traverse the tunnel without stopping.
Tunnel dimensions should reflect the dimensions of the subject; if using mice that are significantly larger or smaller than an average lab mouse (due to age, diet, or genetic mutations), we recommend changing the tunnel and goal chamber dimensions to match the animal&#8217;s size. In the tunnel, the mice should be able to walk comfortably in a straight line, but should have some difficulty turning around to discourage this behavior. The goal chamber should match the height of the tunnel and mice should fit comfortably inside the chamber.
Researchers who use the toe-clipping method of identification for their mice may not be able to collect data on toe spread, but other measures of gait like stride length and stride width can still be collected. Toe-clipping does not significantly impact gait in mice as long as no more than two toes are clipped per mouse20.
This gait analysis method does not reflect cognitive function, so it should not be used as a measure of cognition. Others intending to use this method should consider the neuromuscular groups affected in their mouse model, and then choose fore- or hind limb metrics accordingly. This method of gait analysis is not recommended for researchers who study pain responses requiring footpad injections, or for studies requiring biomechanical measures of locomotion that cannot be described by footprints alone, like temporal measurements of limb motion or joint rotation21.

Locomotion requires complex neurological and musculoskeletal coordination, and deficits in a single aspect of motor pathways can produce observable gait abnormalities1,2. Gait analysis is a critical tool for researchers testing mouse models because it provides quantifiable behavioral data on how a given disease, injury, or drug impacts an animal&#8217;s movement3. However, digitized gait analysis requires the purchase of a treadmill, a camera, and associated software, which can be prohibitively expensive for researchers. Gait analysis is often used intermittently to track longitudinal changes in motor function, hence it may be difficult to justify the expenditure if sporadically used4. Although digitized analyses may provide more detailed gait metrics than simple footprint analysis, these more complex measures are not always necessary or relevant for the characterization of a behavioral phenotype5.
Here we present a low-cost manual footprint analysis method as a quick and sensitive alternative to digitized gait analysis programs6,7. Manual footprint analysis has been demonstrated to detect significant gait differences in a multitude of murine disease models4,7,8,9,10,11,12,13,14,15,16,17, and in at least one case, this low-cost method identified changes in gait that were not detected by a common digitized gait analysis program12. The total cost of materials is nominal, and it can be easily adapted to other rodent research models.
While there are many different gait metrics from which data can be drawn, the method we describe focuses on three specific metrics: stride length, stride width (a.k.a. &#8220;track width&#8221;), and toe spread. It is important to note that the parameters to be assessed should be determined on a model-by-model basis. This method of gait analysis is not designed to measure cognitive function, and it is not recommended for studies that require complex biomechanical measurements of gait16.
We present behavioral data from a cohort of pre- and post-symptomatic mice modeling X-linked Spinal and Bulbar Muscular Atrophy (SBMA), a neuromuscular disease characterized by motor neuron degeneration and muscle atrophy. These mice develop progressive deficits in gait that coincide with the onset of other disease-specific phenotypes. This demonstrates the validity and specificity of this method, and confirms that it can reliably discriminate between affected and non-affected animals.
The experimental mice in this study were 2.5 (pre-symptomatic) and 9-month-old (post-symptomatic) BAC fxAR121 transgenic mice on a C57BL/6 background (nexpt=12). This model was generated in our lab and has been fully characterized as a powerful mouse model of SBMA9. Non-transgenic littermates were used as controls (nctrl=8). SBMA is a sex-limited disease which fully manifests in males only, so male mice were used exclusively for this study. During planning stages, researchers must take into account the National Institutes of Health&#8217;s considerations of sex as a biological variable to determine group sizes and composition18.

<table>
	<tbody>
		<tr>
			<td>Caliper</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>must have markings down to 0.1 mm</td>
		</tr>
		<tr>
			<td>Craft Glue</td>
			<td>E6000</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td>Footprint Paint (Tempera Paint)</td>
			<td>Artmind</td>
			<td>n/a</td>
			<td>must be non-toxic</td>
		</tr>
		<tr>
			<td>Round Barrel Paintbrushes</td>
			<td>Symply Simmons</td>
			<td>n/a</td>
			<td>0.5 cm diameter</td>
		</tr>
		<tr>
			<td>Ruler</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>must have markings down to millimeters</td>
		</tr>
		<tr>
			<td>Scoring Paper (Watercolor Pads)</td>
			<td>Canson</td>
			<td>n/a</td>
			<td>cut to size</td>
		</tr>
		<tr>
			<td>Tunnel and Goal Chamber</td>
			<td>Interstate Plastics</td>
			<td>n/a</td>
			<td>cut to size</td>
		</tr>
	</tbody>
</table>

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.

With sufficient numbers of animals, this procedure is capable of detecting gait differences between mouse genotypes, within the same strain over time. Figure 1B shows representative traces of footprint images collected in our lab, using a mouse model of X-linked Spinal and Bulbar Muscular Atrophy (SBMA), a neurodegenerative disorder affecting lower motor neurons and skeletal muscle. We have previously reported that male BAC fxAR121 transgenic mice develop significant weight loss, impairments in grip strength, and shortened stride length at post-symptomatic ages when compared to non-transgenic littermate controls9.
Here we present gait analysis results from a cohort of pre-symptomatic (2.5 months of age) and post-symptomatic (9 months of age) BAC fxAR121 transgenic and littermate control male mice (Figure 2). Prior to disease onset, BAC fxAR121 transgenic mice display similar stride length, stride width, and toe spread compared to their littermate non-transgenic controls. After disease onset, BAC fxAR121 transgenic mice display significantly shorter stride length (pforelimb= 0.001, phind-limb= 0.009) (Figure 2A). Similar longitudinal analysis revealed no differences in stride width at either age tested (p2.5months=0.709, p9 months=0.204) (Figure 2B). Post-symptomatic BAC fxAR121 transgenic mice also have significantly narrower hind toe spread (p=0.01) than age-matched littermate controls (Figure 2C). BAC fxAR121 mice model a neuromuscular disease that primarily affects hind-limbs, so detailed measures of forelimb gait were not collected. We encourage researchers using this gait analysis method to consider the phenotype of their mouse models and choose forelimb or hind-limb gait metrics accordingly.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59878/59878fig01.jpg" /> 
Figure 1: Gait Analysis Measures and Troubleshooting. 
A. Schematic representation of gait analysis on mice, depicting stride length, stride width, and toe spread information. B. Representative example of a gait analysis footprint sequence that can be scored, depicting measurement of all three parameters. C. Representative examples of problematic gait analysis footprint sequences that cannot be scored. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59878/59878fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59878/59878fig02.jpg" /> 
Figure 2: SBMA BAC fxAR121 transgenic mice exhibit a progressive, neurodegenerative gait phenotype that can be detected via gait analysis. 
A. Despite no differences at pre-symptomatic ages (2.5 months, nctl=11, nexpt=12), BAC fxAR121 mice develop significantly reduced stride length compared to their non-transgenic littermate controls at post-symptomatic stages (9 months, nctl=8, nexpt=12). B. No changes were detected in stride width at either age. C. Symptomatic SBMA BAC fxAR121 transgenic mice display significantly reduced hind limb toe spread compared to non-transgenic littermate controls. N= 8-12/group. ANOVA with post-hoc Tukey test * p &#60; 0.05, ** p &#60; 0.01. Error bars represent SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59878/59878fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Low-Cost Gait Analysis for Behavioral Phenotyping of Mouse Models of Neuromuscular Disease at JoVE.com

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

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 ...

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Research

JoVE Journal

Behavior

16.1K Views.  Duke University School of Medicine. 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.

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

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  ...

Nest Building as an Indicator of Health and Welfare in Laboratory Mice

The minimization and alleviation of suffering has moral and scientific implications. In order to mitigate this negative experience one must be able to identify when an animal is actually in distress. Pain, illness, or distress cannot be managed if unrecognized. Evaluation of pain or illness typically involves the measurement of physiologic and behavioral indicators which are either invasive or not suitable for large scale assessment. The observation of nesting behavior shows promise as the basis of a species appropriate cage-side assessment tool for recognizing distress in mice. Here we demonstrate the utility of nest building behavior in laboratory mice as an ethologically relevant indicator of welfare. The methods presented can be successfully used to identify thermal stressors, aggressive cages, sickness, and pain. Observation of nest building behavior in mouse colonies provides a refinement to health and well-being assessment on a day to day basis.

We demonstrate the utility of nest building behavior in laboratory mice as an indicator of welfare. Nest scoring is a sensitive technique that is altered by temperature, illness, and aggression. The time to integrate into nest test (TINT) is a simple cage-side assessment that can detect postoperative pain.

The minimization and alleviation of  suffering has moral and scientific implications. In order to mitigate this  negative experience one must be able to ...

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

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

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

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

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

Behavioral Assessment of the Aging Mouse Vestibular System

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of studies show changes in balance phenotype with age in rodents, very few isolate the vestibular contribution to balance under either normal conditions or during senescence. We use two standard behavioral tests to characterize the balance performance of mice at defined age points over the lifespan: the rotarod test and the inclined balance beam test. Importantly though, a custom built rotator is also used to stimulate the vestibular system of mice (without inducing overt signs of motion sickness). These two tests have been used to show that changes in vestibular mediated-balance performance are present over the murine lifespan. Preliminary results show that both the rotarod test and the modified balance beam test can be used to identify changes in balance performance during aging as an alternative to more difficult and invasive techniques such as vestibulo-ocular (VOR) measurements.

Motor control and balance performance are known to deteriorate with age. This paper presents a number of standard noninvasive behavioral tests with the addition of a simple rotary stimulus to challenge the vestibular system and show changes in balance performance in a murine model of aging.

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of ...

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied1,2.
In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells.
The protocol is most advantageous for parallel analysis of multiple samples (e.g. single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies.

This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays.

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific ...

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of current interventions to adequately treat fatigue in all patients leaves a need for new treatment options. Despite the therapeutic need and importance of preclinical research in helping identify promising novel treatments, few preclinical assays of fatigue are available. Moreover, the most common preclinical assay used to assess fatigue-like behavior, voluntary wheel running, is not suitable for use with some strains of mice, may not be sensitive to drugs that reduce fatigue, and has relatively low throughput. The current protocol describes a novel, non-voluntary preclinical assay of fatigue-like behavior, the treadmill fatigue test, and provides evidence of its efficacy in detecting fatigue-like behavior in mice treated with a chemotherapy drug known to cause fatigue in humans and fatigue-like behavior in animals. This assay may be a beneficial alternative to wheel running, as fatigue-like behavior and potential interventions can be assessed in a greater number of mice over a shorter time frame, thus permitting faster discovery of new therapeutic options.

Fatigue is a common, undertreated and frequently poorly-understood symptom in many diseases and disorders. New preclinical assays of fatigue may help to improve current understanding and future treatment of fatigue. To that end, the current protocol provides a novel means of measuring fatigue-like behavior in the mouse.

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of ...

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 ...

Clinical-oriented Three-dimensional Gait Analysis Method for Evaluating Gait Disorder

Three-dimensional gait analysis (3DGA) is shown to be a useful clinical tool for the evaluation of gait abnormality due to movement disorders. However, the use of 3DGA in actual clinics remains uncommon. Possible reasons could include the time-consuming measurement process and difficulties in understanding measurement results, which are often presented using a large number of graphs. Here we present a clinician-friendly 3DGA method developed to facilitate the clinical use of 3DGA. This method consists of simplified preparation and measurement processes that can be performed in a short time period in clinical settings and intuitive results presentation to facilitate clinicians&#39; understanding of results. The quick, simplified measurement procedure is achieved by the use of minimum markers and measurement of patients on a treadmill. To facilitate clinician understanding, results are presented in figures based on the clinicians&#39; perspective. A Lissajous overview picture (LOP), which shows the trajectories of all markers from a holistic viewpoint, is used to facilitate intuitive understanding of gait patterns. Abnormal gait pattern indices, which are based on clinicians&#39; perspectives in gait evaluation and standardized using the data of healthy subjects, are used to evaluate the extent of typical abnormal gait patterns in stroke patients. A graph depicting the analysis of the toe clearance strategy, which depicts how patients rely on normal and compensatory strategies to achieve toe clearance, is also presented. These methods could facilitate implementation of 3DGA in clinical settings and further encourage development of measurement strategies from the clinician&#39;s point of view.

In this study, a clinician-friendly three-dimensional gait analysis method, which was designed to be performed in the rehabilitation clinic, is presented. The method consists of a simplified measurement method and intuitive figures to facilitate clinicians&#39; understanding of the results.

Three-dimensional gait analysis (3DGA) is shown to be a useful clinical tool for the evaluation of gait abnormality due to movement disorders. However, ...

Automated, Long-term Behavioral Assay for Cognitive Functions in Multiple Genetic Models of Alzheimer's Disease, Using IntelliCage

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive decline, such as Alzheimer&#39;s disease (AD), have become a promising tool to elucidate the underlying mechanisms and promote the therapeutic advances. An important step is the validation and characterization of expected behavioral abnormality in the models, in the case of AD, cognitive decline. The long-term behavioral investigations of laboratory animals to study the effect of aging demand substantial efforts from researchers. The IntelliCage system is a high-throughput and cost-effective test battery for mice that eliminates the need for daily human handling. Here, we describe how the system is utilized in the long-term phenotyping of a genetic Alzheimer&#39;s disease model, specifically focusing on the cognitive functions. The experiment employs repeated battery of tests that assess spatial learning and executive functions. This cost-effective age-dependent phenotyping allows us to identify the transient and/or permanent effects of genes on various cognitive aspects.

This paper describes a protocol for cognitive assessments for genetic models of the Alzheimer&#39;s disease using the IntelliCage system, which is a high throughput automated behavioral monitoring system with operant conditioning.

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive ...

Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress

Gait disturbance is frequently observed in patients with movement disorders. In mouse models used for movement disorders, gait analysis is important behavioral test to determine whether the mice mimic the symptoms of patients. Motor deficits are often induced by stress when no spontaneous motor phenotype is observed in the mouse models. Therefore, gait analysis followed by stress loading would be a sensitive method for evaluating the motor phenotype in mouse models. However, researchers face the requirement of an expensive apparatus to obtain quantitative results automatically from gait analysis. For stress, stress loading by simple methods without expensive apparatuses required for electric shock and forced running is desirable. Therefore, we introduce a simple and low-cost protocol consisting of footprint analysis with paper and ink, hanging box test to evaluate motor function, and stress loading defined by restraint with a conical tube. The motor deficits of mice were successfully detected by this protocol.

The low-cost protocol consisting of footprint analysis and hanging box test after restraint stress is useful for evaluating the movement disorders of mouse model.

Gait disturbance is frequently observed in patients with movement disorders. In mouse models used for movement disorders, gait analysis is important ...