This work was supported by JSPS (Japan Society for the Promotion of Science) KAKENHI (Grant-in-Aid for Scientific Research C), Grant number 18K07373 (H.S.) and Subsidies for Private Universities.

All animal experiments were conducted in a humane manner. The Institutional Animal Experiment Committee of Jichi Medical University approved the study. The study was conducted in accordance with the Institutional Regulation for Animal Experiment and Fundamental Guideline for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the MEXT of Japan. Mice used in this protocol have been described previously21.
1. Hanging Box Test
<ol>
	<li>Record the weight of each mouse. Mark the tail by marking pen for individual discrimination (e.g., a line, double lines, and triple lines). 
	NOTE: Growth curves are used for an index of general health22.</li>
	<li>Place the mice in the experimental room at least 30 min before the behavioral test. Set the hanging box, which consists of a clear box (25 x 25 x 40 cm3) with a rotatable mesh lid on the top (Figure 1). The mesh lid can be rotated along a central bar so that the top is flipped 180 degrees.</li>
	<li>Put a mouse in the center of the mesh lid. Carefully turn the mesh lid up side down.</li>
	<li>Measure the fall latency (hanging time) of the mouse from the mesh lid. 
	NOTE: If the mouse does not fall within 5 min, record the latency as 5 min.</li>
	<li>Return the mouse to the home cage. Clean the hanging box with 70% ethanol after every test.</li>
</ol>
2. Footprint Analysis
NOTE: Following the hanging box test, perform the footprint analysis.
<ol>
	<li>Set up the Runway (Figure 2A).
	<ol>
		<li>Cut a piece of white paper (29.7 cm x 42 cm x 0.09 mm) longitudinally into three lengths of equal width. Set a piece of the white paper (9.9 cm x 42 cm) on the table.</li>
		<li>Put the dark goal box at the distal end of the paper. Put other boxes (approximately the same length as that of the paper) with the walls on both sides of the runway, preventing the escape of mice.</li>
		<li>Put black ink and red ink into separate Petri dishes (35 mm in diameter).</li>
	</ol>
	</li>
	<li>Training session. 
	NOTE: Perform the training session only at 4 weeks of age.
	<ol>
		<li>Put a mouse on the proximal end of the paper (Face the head toward the goal box). Let the mouse walk from the proximal end to the goal box. Remove the mouse from the goal box. If the mouse stops on the paper, gently push the mouse to the goal box by finger.</li>
		<li>Hold the mouse by grasping the scruff between the thumb and forefinger to limit the movement of forelimbs. Then, grasp the back and the tail between the ball of the thumb and the other fingers to limit the movement of hindlimbs. 
		NOTE: Insufficient holding of a mouse results in blots of ink on clothing.</li>
		<li>Immerse the bottoms of forelimbs in red ink and the bottoms of hindlimbs in black ink. Immediately put the mouse on the proximal end of the paper (Face the head toward the goal box). Let the mouse walk from the proximal end to the goal box. If the mouse stops on the paper, gently push the mouse to the goal box by finger.</li>
		<li>Remove the mouse from the goal box. Go to the test session.</li>
	</ol>
	</li>
	<li>Test Session.
	<ol>
		<li>Following the training session, set up the runway for footprints with a new cut piece of white paper.</li>
		<li>Hold the mouse by grasping the scruff between the thumb and forefinger to limit the movement of forelimbs. Then, grasp the back and the tail between the ball of the thumb and the other fingers to limit the movement of hindlimbs.</li>
		<li>Immerse the bottoms of forelimbs in red ink and the bottoms of hindlimbs in black ink. Immediately put the mouse on the proximal end of the paper. Let the mouse walk from the proximal end to the goal box. 
		NOTE: Because mice prefer the dark, walking becomes steadier as the mouse approaches the dark goal box. If the mouse stops on the paper, gently push the mouse to the goal box by finger. Then, if reliable footprints are not obtained for analysis (see step 2.4. Analysis of footprints for details) because the mouse stopped, retry the test session.</li>
		<li>Return the mouse to the home cage from the goal box. Clean the goal box with 70% ethanol after each test session. Air-dry the foot-printed paper.</li>
	</ol>
	</li>
	<li>Analysis of footprints
	<ol>
		<li>Obtain three measurements of each parameter (stride lengths of forelimbs and hindlimbs, front and hind base widths, overlap between forelimb and hindlimb, Figure 2B) with a ruler from foot-printed paper. 
		NOTE: Because footprints of proximal and distal ends frequently show large variations because of stopping or running, choose the part with a steady gait pattern of footprints. The middle part of the foot-printed paper will usually be suitable for the analysis.
		<ol>
			<li>For the stride length, measure the distances between the same parts of the paw (e.g., paw pad or toe).</li>
			<li>For the front base width, draw a line between consecutive right (or left) front footprints. Then, measure the length of the vertical line from the pad of the left (or right) front footprint to the line drawn between the right (or left) footprints.</li>
			<li>For the hind base width, draw a line between consecutive right (or left) hind footprints. Then, measure the length of the vertical line from the pad of the left (or right) hind footprint to the line drawn between the right (or left) footprints.</li>
			<li>For overlap, measure the distance between pads of left (or right) front and hind footprints.</li>
		</ol>
		</li>
		<li>Average the three measurements for each individual. Use the individual average of each parameter for the statistical analysis.
		<ol>
			<li>For the stride length, use the average of the individual averages of the left and right strides.</li>
			<li>For asymmetry of stride length, use the absolute value of the difference between individual averages of left limb and right limb stride length.</li>
			<li>For the statistical analysis of the other parameters (front base width, hind base width, and overlap), use the individual average directly.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Restraint Stress Loading
<ol>
	<li>Preparation of Restraint Tubes. 
	<ol>
		<li>Make 16 holes (approximately 2 mm in diameter) in a 50 ml conical tube (30 mm in diameter x 115 mm in length) along the scale marks (5, 10, 15, 20, 25, 30, 35, 40 mL) and the backside of each scale mark by square drill (Figure 3). Make a hole on the tip of the 50 mL conical tube (approximately 5 mm in diameter) for breathing by cutting off the tip. Make a hole (approximately 4 mm in diameter) in the tube cap to pass the tail of mice.</li>
	</ol>
	</li>
	<li>Stress Loading
	<ol>
		<li>Place the mice in the experimental room.</li>
		<li>Hold a mouse by grasping the scruff between the thumb and the forefinger. Enter the mouse into the restraint tube from the head. Pass the tail through the hole in the cap. Close the cap. 
		NOTE: Limit the forelimb movement, because mice reject entering the tube by the forelimbs.</li>
		<li>Keep the mouse enclosed for 2 h on a desk at room temperature. Remove the mouse from the restraint tube and return to the home cage. 
		NOTE: The restraint tubes can be reused after a wash and dry.</li>
	</ol>
	</li>
</ol>
4. Experimental Schedule (Figure 4):
<ol>
	<li>Perform the hanging box test and the footprint analysis on the same day at 4 weeks of age (see step 1. Hanging box test and step 2. Footprint analysis for details) as a baseline measurement on all mice prior to the grouping into &#8216;stress group&#8217; and a &#8216;non-stress group&#8217;. 
	NOTE: About 8-10 mice in 2-3 litters may be suitable to use in an experiment. Footprint analysis at 4 weeks of age consists of training and test sessions.</li>
	<li>Randomly divide the mice into a &#8216;stress group&#8217; and a &#8216;non-stress group&#8217;. 
	NOTE: When the mice are used consisting of several litters, divide littermates evenly into both groups. Number in each group consists of about 4-5 mice.</li>
	<li>Apply the restraint stress to the &#8216;stress group&#8217; 6 times over the course of two weeks (see step 3. Restraint stress loading for details). 
	NOTE: 6 times of restraint are applied every two weeks, followed by a hanging box test and footprint analysis from 6-12 weeks of age. Do not apply the restraint stress on the test day of the hanging box test and the footprint analysis.</li>
	<li>Perform the hanging box test and the test session of footprint analysis on the same day at 6, 8, 10, and 12 weeks of age.</li>
</ol>

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The authors have nothing to disclose.

The footprint analysis and the hanging box test are simple and inexpensive behavioral tests for the motor function of mice. The neurobehavioral phenotypes in several mouse models have been successfully detected by these tests. For example, shortened stride length in amyotrophic lateral sclerosis24, increased length of asymmetrical stride in ataxia-telangiectasia25, increased length of overlap in Huntington&#39;s disease26 and dystonia27, and widened base in ataxia28,29, Angelman syndrome30 and dystonia31 were demonstrated by the footprint analysis. Additionally, shorter hanging time than that of the control mice was observed in mouse model of Duchenne muscular dystrophy32. Thus, both tests described in this paper would be useful for evaluating motor dysfunction of mice.
There is a critical step of this protocol to obtain the sharp footprints. It is very important to put a mouse on paper immediately after immersing the bottoms of limbs in ink to avoid drying. It is necessary to finish the immersing steps (steps 2.2.3 and 2.3.3) in a short time. A disadvantage of this protocol of footprint analysis is that the parameters obtained are limited to simple ones that do not include temporal information (e.g., duration of gait cycle and step sequence of each foot) and physical information (e.g., pressure of footprint based on touch-sensitive LED panel). When temporal and physical information is required, an automated gait analysis apparatus must be used. Alternatively, temporal information of gait pattern can be obtained by high-speed camera33. Another disadvantage is that measuring the parameters on foot-printed paper is more laborious than automated gait analysis. For future application, the development of a program for semiautomated or automated data collection from foot-printed paper will be required to decrease the effort expended measuring parameters.
For the stress loading, total number and duration per session can be varied in this protocol. Restraint stress has been conducted with various durations and number of applications (e.g., single for 5 min34, single for 24 h35, 12 h x 5 sessions36, and 6 h x 31 sessions37, reviewed in ref. 20). To our knowledge, no systematic study concerns the effects of total number and duration per session on the motor function of mice. The activity of mice in the open field is affected by single stress loading of 1 h38 or 2 h but not by that of 15 min19. Prolonged duration of stress loading may lead to severe motor deficits. For the number of stress loadings, the stance of mice becomes wider than that of non-stressed mice after 36 times of stress loading (1 h twice weekly), but not after 30 times39. Chronic restraint stress (6 h per day for 31 consecutive days) aggravates rotarod performance in the mouse model of Parkinson&#39;s disease37. Our results showed the hanging time of &#39;stressed&#39; Atp1a3+/&#8722; mice became shorter than that of the &#39;stressed&#39; wild-type mice after 24 times of stress loading (at 12 weeks of age), but not after 18 times or less (at 4, 6, 8, and 10 weeks of age). Therefore, repeated stress loading is required for induction of certain motor deficits in mice, although the induction of motor deficits after a long period from the start of a single or several times of stress loading cannot be excluded. When mice do not show a neurobehavioral phenotype in this protocol, the recommendation is to increase the total number and duration of stress loading per session. By contrast, when the mice show gait abnormality from a single or several times of stress loading, duration and number can be decreased.
Finally, performance of walking and hanging can be affected by emotional behavioral characters (e.g., anxiety and activity) other than those of motor dysfunction. Mice with high anxiety and with hyperactivity often run (instead of walk) to the goal box. Mice showing depression-like behavior may walk with frequent stops. Therefore, an aberrant footprint pattern may not be due to motor deficits. Thus, to confirm the gait abnormality resulting only from motor deficits, performing additional tests that evaluate emotional characteristics (activity: open field test; anxiety: open field and elevated plus maze test; depression-like behavior: forced swim test) after this protocol as described previously18 is advisable.

Movement disorders are defined as disturbances of the nervous system showing an excess or paucity of voluntary or automatic movements1. In particular, gait disturbance is frequently documented among patients with movement disorders2,3,4. Therefore, gait analysis is a suitable behavioral test for the validation of animal models of movement disorders. In mice, automated gait analyses have been performed for walking at natural speed5 and at adjustable speeds by treadmill6,7. These analyses provide quantitative results of gait automatically. An alternative method to detect gait disturbance is called footprint analysis. After labeling the bottoms of the feet with ink, mice walk on paper, and the footprints are analyzed. Initially, Vaseline and powdered charcoal were used to visualize the footprint8, and then were replaced by ink on polygraph paper9 and photographic developer on photographic paper10. A cheaper and less toxic method using ink and paper than the other methods remains to date11. Footprint analysis is less expensive compared with automated analysis5,6,7 and would be useful to evaluate the movement disorders in mouse models for the researchers without abundant research funds.
The hanging box test is a kind of four limb hanging tests using wire cage lid12 and wire mesh screen13. The box is an apparatus with rotatable mesh lid on the top of box along a center bar. In addition to gait analysis, the test can be inexpensively and easily performed. Therefore, we conducted the hanging box test to evaluate grip strength and balance, in addition to the footprint analysis in this protocol.
Stress induces the symptoms of movement disorders14,15. Motor deficits are often induced by several chronic stresses even when no spontaneous motor phenotype is observed in the mouse models of a movement disorder16,17,18. Restraint is one of the commonly used methods for stress loading in mice, because the animal is not physically harmed19 and cost is less compared with other methods such as electric shock with dedicated apparatus and forced running with use of a treadmill. Restraint by a tube, which is performed by confining a mouse in a holed 50 mL conical tube, is easier than other methods such as wire mesh strainer, taped limb, and wrapping of animal with gauze (reviewed20). In this paper, we summarize the protocols of footprint analysis and the hanging box test after restraint by a tube. This protocol would help us to use mouse models of movement disorders without spontaneous motor phenotype.

<table><tbody><tr><td>Hanging box</td><td>O&amp;rsquo;hara &amp;amp; Co.</td><td>http://ohara-time.co.jp/products/wire-hanging-test/</td><td></td></tr><tr><td>Marking pen</td><td>ZEBRA</td><td>MO-120-MC-BK</td><td></td></tr><tr><td>Goal box</td><td>O&amp;rsquo;hara &amp;amp; Co.</td><td>http://ohara-time.co.jp/products/balanced-beam-test/</td><td>Accessory for apparatus of balanced beam test</td></tr><tr><td>Boxes</td><td>O&amp;rsquo;hara &amp;amp; Co.</td><td>-</td><td>Side wall of runway</td></tr><tr><td>Black ink</td><td>Shin-asahi</td><td>-</td><td></td></tr><tr><td>Red ink</td><td>Maruyamakogyo</td><td>BC-6</td><td></td></tr><tr><td>Disposable Petri Dish</td><td>Corning</td><td>351008</td><td>Petri dishes (35 mm in diameter)</td></tr><tr><td>Askul Multipaper Super White J Monochrome A3</td><td>Askul</td><td>701-712</td><td>White paper (29.7 cm x 42 cm x 0.09mm)</td></tr><tr><td>50 mL Conical tube</td><td>Corning</td><td>430829</td><td></td></tr><tr><td>Square drill</td><td>KAKURI Corporation</td><td>DIY FACTORY (K32-0313)</td><td></td></tr></tbody></table>

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 heterozygous male mice of Atp1a3 (Atp1a3+/&#8722;) that are the mouse model for rapid onset dystonia parkinsonism and wild-type littermates were used in this protocol. Atp1a3+/&#8722;&#160;showed significantly shorter stride lengths of forelimb and hindlimb than those of the wild type at 4 weeks of age (Figure 5A and Figure 5B, open circle and square). &#39;Stressed&#39; Atp1a3+/&#8722;&#160;showed significantly shorter stride lengths of both limbs than those of &#39;non-stressed&#39; Atp1a3+/&#8722; at 8 weeks of age (Figure 5A and Figure 5B, closed and open circle). Asymmetries of stride lengths of both limbs were not significantly different in all groups of mice at all ages (Figure 5C and Figure 5D). The front base and overlaps of both limbs were also similar in all groups at all ages (Figure 5E, G, H). The hind base was significantly wider in the &#39;stressed&#39; Atp1a3+/&#8722; than that in the &#39;stressed&#39; wild-type mice at 10 weeks of age (Figure 5F, closed circle and square). Thus, restraint stress caused motor deficits (short stride and wide base) of Atp1a3+/&#8722;.
The hanging box test was performed to evaluate the grip strength and balance on the test day of footprint analysis. No significant differences in hanging time were observed in 4 to 10 weeks old mice (Figure 6). At 12 weeks of age, the hanging time of &#39;stressed&#39; wild-type mice was significantly longer than that of the other groups (Figure 6, closed square). Restraint stress prolonged hanging time in wild-type mice only, but not in Atp1a3+/&#8722;. Thus, the motor deficit of Atp1a3+/&#8722; was distinguishable from wild-type mice by restraint stress.
Body weight is an index of general health22. We measured the body weight of mice on the test day of the hanging box test and footprint analysis, and no significant differences were observed in all groups of mice at all ages (Figure 7). Thus, restraint stress did not affect the general health of mice.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59027/59027fig1.jpg" /> 
Figure 1: Hanging box apparatus with a rotatable mesh lid. A mouse was placed in the center of the mesh lid, and then the mesh lid was turned up side down. The fall latency of the mouse from the mesh lid is to be measured. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59027/59027fig1large.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/59027/59027fig2.jpg" /> 
Figure 2: Footprint analysis. (A) Runway for footprint analysis. A foot-painted mouse (forelimbs: red ink; hindlimbs: black ink) was allowed to walk from the proximal end to the goal box. (B) Representative image of footprint and measurement of parameters.&#160;Three measurements of each parameter (forelimb and hindlimb stride lengths, widths of front and hind base, overlap between forelimb and hindlimb) were obtained from foot-printed paper. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59027/59027fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59027/59027fig3.jpg" /> 
Figure 3: Restraint stress by holed 50 mL conical tube. The restraint tube has a hole for breathing, a hole for passing the tail, and 16 holes for air circulation. Mouse was kept in the tube for 2 h at room temperature. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59027/59027fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59027/59027fig4.jpg" /> 
Figure 4: Experimental schedule of the hanging box test and the footprint analysis with the chronic restraint stress loading. Footprint analysis and the hanging box test were performed at 4 weeks of age. Then, the mice were split into &#39;stressed&#39; and &#39;non-stressed&#39; groups. For the &#39;stressed&#39; group, the restraint stress was applied 6 times in 2 weeks until 12 weeks of age. For both groups, footprint analysis and the hanging box test were performed once per 2 weeks until 12 weeks of age. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59027/59027fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59027/59027fig5.jpg" /> 
Figure 5: Representative results of footprint analyses. Footprint analysis was performed with 4-week-old Atp1a3+/&#8722; (a3) and wild-type (wt) mice (N = 19 and 17, respectively). Then, Atp1a3+/&#8722; and wild-type mice were split into &#39;stressed&#39; and &#39;non-stressed&#39; groups. Footprint analysis was conducted for &#39;non-stressed&#39; Atp1a3+/&#8722;, &#39;non-stressed&#39; wild-type, &#39;stressed&#39; Atp1a3+/&#8722;, and &#39;stressed&#39; wild-type mice from 6-12 weeks of age (open circles, N = 9; open squares, N = 8; solid circles, N = 10; solid squares, N = 9, respectively). (A) Stride length of forelimb. (B) Stride length of hindlimb. (C) Asymmetry of forelimb strides length. (D) Asymmetry of hindlimb strides length. (E) Width between forelimbs. (F) Width between hindlimbs. (G) Overlap between left forelimb and hindlimb. (H) Overlap between right forelimb and hindlimb. Data are the mean &#177; SD. Statistical analyses (t-test for 4-week-old mice and pairwise t-test with the Holm adjustment method for 6-12-week-old mice) were performed by R23. # p &#60; .05, for &#39;non-stressed&#39; Atp1a3+/&#8722; and &#39;non-stressed&#39; wild-type mice. * p &#60; .05, for &#39;stressed&#39; Atp1a3+/&#8722; and &#39;stressed&#39; wild-type mice. + p &#60; .05, for &#39;non-stressed&#39; and &#39;stressed&#39; Atp1a3+/&#8722; mice. Figure 5A-F has been modified from reference18 with permission of Elsevier. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59027/59027fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59027/59027fig6.jpg" /> 
Figure 6: Representative results of hanging box tests. The hanging box test was performed with Atp1a3+/&#8722; (a3) and wild-type (wt) mice at 4 weeks of age. Then, Atp1a3+/&#8722; and wild-type mice were split into &#39;stressed&#39; and &#39;non-stressed&#39; groups. Hanging time of 4-week-old Atp1a3+/&#8722; (open circles, N = 19) and wild-type (open squares, N = 17) mice and hanging time of 6-12-week-old &#39;non-stressed&#39; Atp1a3+/&#8722; (open circles, N = 9), &#39;non-stressed&#39; wild-type (open squares, N = 8), &#39;stressed&#39; Atp1a3+/&#8722; (solid circles, N = 10), and &#39;stressed&#39; wild-type (solid squares, N = 9) mice were plotted on a logarithmic scale. Data are the mean &#177; SD. Statistical analyses (t-test for 4-week-old mice and pairwise t-test with the Holm adjustment method for 6-12-week-old mice) were performed by R23. * p &#60; .05, for &#39;stressed&#39; Atp1a3+/&#8722; and &#39;stressed&#39; wild-type mice. $ p &#60; .05, for &#39;non-stressed&#39; and &#39;stressed&#39; wild-type mice. + p &#60; .05, for &#39;non-stressed&#39; Atp1a3+/&#8722; and &#39;stressed&#39; wild-type mice. Data has been reprinted from reference18&#160;with permission of Elsevier. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59027/59027fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59027/59027fig7.jpg" /> 
Figure 7: Representative results of growth curves. Body weights were measured of Atp1a3+/&#8722; (a3) and wild-type (wt) mice at 4 weeks of age (open circles, N = 19; open squares, N = 17, respectively). Then, Atp1a3+/&#8722; and wild-type mice were split into &#39;stressed&#39; and &#39;non-stressed&#39; groups. Body weights between 6 and 12 weeks of age of &#39;non-stressed&#39; Atp1a3+/&#8722; (open circles, N = 9), &#39;non-stressed&#39; wild-type (open squares, N = 8), &#39;stressed&#39; Atp1a3+/&#8722; (solid circles, N = 10), and &#39;stressed&#39; wild-type (solid squares, N = 9) mice were measured. Data are the mean &#177; SD. Statistical analyses (t-test for 4-week-old mice and pairwise t-test with the Holm adjustment method for 6-12-week-old mice) were performed by R23. Figure 7 has been modified from reference18&#160;with permission of Elsevier. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59027/59027fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress at JoVE.com

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

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 ...

low-cost-protocol-footprint-analysis-hanging-box-test-for-mice

Nanyang Technological University

Research

JoVE Journal

Behavior

18.2K Views.  Jichi Medical University. 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.

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

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a living animal&#8217;s brain. Moreover, the lengthy recovery from anesthesia limits the frequency of repeated recording/imaging episodes in longitudinal studies. Hence, new methods that would allow stable recordings from non-anesthetized behaving mice are expected to advance the fields of cellular and cognitive neurosciences. Existing solutions range from mere physical restraint to more sophisticated approaches, such as linear and spherical treadmills used in combination with computer-generated virtual reality. Here, a novel method is described where a head-fixed mouse can move around an air-lifted mobile homecage and explore its environment under stress-free conditions. This method allows researchers to perform behavioral tests (e.g., learning, habituation or novel object recognition) simultaneously with two-photon microscopic imaging and/or patch-clamp recordings, all combined in a single experiment. This video-article describes the use of the awake animal head fixation device (mobile homecage), demonstrates the procedures of animal habituation, and exemplifies a number of possible applications of the method.

This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal&#8217;s head.

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a ...

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal experimentation whenever possible. When there is no alternative to animal experimentation, the total number of laboratory animals used should be the minimum needed to obtain valuable data. Moreover, appropriate refinement measures should be applied to minimize pain, suffering, and distress accompanying the experimental procedure. The categories used to classify the degree of pain, suffering, and distress are non-recovery, mild, moderate, or severe (EU Directive 2010/63). To determine which categories apply in individual cases, it is crucial to use scientifically sound tools.
The well-being-assessment protocol presented here is designed for procedures during which general anesthesia is used. The protocol focuses on home cage activity, the Mouse Grimace Scale, and luxury behaviors such as burrowing and nest building behavior as indicators of well-being. It also uses the free exploratory paradigm for trait anxiety-related behavior. Fecal corticosterone metabolites as indicators of acute stress are measured over the 24-h post-anesthetic period.
The protocol provides scientifically solid information on the well-being of mice following general anesthesia. Due to its simplicity, the protocol can easily be adapted and integrated in a planned study. Although it does not provide a scale to classify distress in categories according to the EU Directive 2010/63, it can help researchers estimate the degree of severity of a procedure using scientifically sound data. It provides a way to improve the assessment of well-being in a scientific, animal-centered manner.

We developed a protocol to assess well-being in mice during procedures using general anesthesia. A series of behavioral parameters indicating levels of well-being as well as glucocorticoid metabolites were analyzed. The protocol can serve as a general aid to estimate the degree of severity in a scientific, animal-centered manner.

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

The Tail Suspension Test

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

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

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

Neuroscience

Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. The Barnes maze can also assess other mouse behaviors, such as general motivation to escape from the maze platform and exploratory behavior. The Barnes maze can measure whether a genetic mutation or environmental variable can impact the acquisition and retention of spatial memories, as well as provide information about the search strategy employed by the mice. Here we use the Barnes maze to detect a memory deficit in adult mice following a single developmental ethanol exposure event. The newly described Damsel-in-Distress paradigm exposes a male mouse to a female mouse trapped in a chamber in the open center field of the arena. It provides an opportunity for the mouse to socially respond to the trapped female and exhibit prosocial behavior. The Damsel-in-Distress paradigm can also be used to examine mouse behavior in a novel arena and measure locomotor activity. Both the Barnes Maze and the Damsel-in-Distress protocols require minimal financial investment and most aspects of the tests can be constructed from common lab supplies. These flexible and accessible tools can also be used to detect behavioral changes over the course of development.

This protocol measures spatial learning and memory using the Barnes maze. A novel Damsel-in-Distress paradigm is used to assess locomotor activity and prosocial behavior in mice.

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. ...

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

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

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

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

A Chronic Immobilization Stress Protocol for Inducing Depression-Like Behavior in Mice

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, therapeutic treatments for depression or research on depression is scarce. Thus, in the present paper, we propose a mouse model of depression induced by movement restriction. Chronic mild stress (CMS) is a well-known technique to induce depressive-like behavior. However, it necessitates a complex procedure consisting of a combination of various mild stresses. In contrast, chronic immobilization stress (CIS) is a readily accessible chronic stress model, modified from a restraint model that induces depressive behavior by restricting movement using a restrainer for a certain period. To evaluate the depressive-like behaviors, the sucrose preference test (SPT), the tail suspension test (TST), and the ELISA assay to measure stress marker corticosterone levels are combined in the present experiment. The described protocols illustrate the induction of CIS and evaluation of the changes in behavior and physiological factors for the validation of depression.

This article provides a simplified and standardized protocol for induction of depressive-like behavior in chronically immobilized mice by using a restrainer. In addition, behavior and physiological techniques to verify induction of depression are explained.

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, ...

Handling Techniques to Reduce Stress in Mice

Laboratory animals are subjected to multiple manipulations by scientists or animal care providers. The stress this causes can have profound effects on&#160;animal well-being and can also be a confounding factor for experimental variables such as anxiety measures. Over the years, handling techniques that minimize handling-related stress have been developed with a particular focus on rats, and little attention to mice. However, it has been shown that mice can be habituated to manipulations using handling techniques. Habituating mice to handling reduces stress, facilitates routine handling, improves animal wellbeing, decreases data variability, and improves experimental reliability. Despite beneficial effects of handling, the tail-pick up approach, which is particularly stressful, is still widely used. This paper provides a detailed description and demonstration of a newly developed mouse-handling technique intended to minimize the stress experienced by the animal during human interaction. This manual technique is performed over 3 days (3D-handling technique) and focuses on the animal&#39;s capacity to habituate to the experimenter. This study also shows the effect of previously established tunnel handling techniques (using a polycarbonate tunnel) and the tail-pick up technique. Specifically studied are their effects on anxiety-like behaviors, using behavioral tests (Elevated-Plus Maze and Novelty Suppressed Feeding), voluntary interaction with experimenters and physiological measurement (corticosterone levels). The 3D-handling technique and the tunnel handling technique reduced anxiety-like phenotypes. In the first experiment, using 6-month-old male mice, the 3D-handling technique significantly improved experimenter interaction. In the second experiment, using 2.5-month-old female, it reduced corticosterone levels. As such, the 3D-handling is a useful approach in scenarios where interaction with the experimenter is required or preferred, or where tunnel handling may not be possible during the experiment.

This paper describes a handling technique in mice, the 3D-handling technique, which facilitates routine handling by reducing anxiety-like behaviors and presents details on two existing related techniques (tunnel and tail handling).

Laboratory animals are subjected to multiple manipulations by scientists or animal care providers. The stress this causes can have profound effects ...

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using ...

Impact of Restraint Stress on Physiology and Neurobiology in Rodent Models

Restraint to Induce Stress in Mice and Rats

Across all animal species, exposure to stressful conditions induces stress responses. One method to study the effects of stress using rodent models is the restraint stress procedure. Restraint stress has been used for decades to investigate changes in physiology, genetics, neurobiology, immunology, and other systems impacted by stress. Due to the ease of performing the procedure, low cost, and numerous modifications to scale for the intensity and duration of stress exposure, a vast literature of studies has used restraint stress in mice and rats. As one example, this study presents previously published data showing the impact of restraint stress in transgenic mice on plasma corticosterone levels and optogenetically-induced norepinephrine release. Acute restraint stress increased plasma corticosterone levels, yet this effect was blunted in mice following repeat restraint stress. However, stimulated norepinephrine release in the bed nucleus of the stria terminalis was increased only in the repeat restraint stress group. These data highlight important considerations of restraint parameters on dependent measures. Additional descriptions of restraint stress in rats are also included for comparison. Finally, the influence of the parameters of the restraint (e.g., acute vs. chronic) and characteristics of the animal subjects (strain, sex, age) are discussed.

This article covers the procedures to induce stress responses using physical restraint stress in mice and rats. Additional considerations that should be observed when selecting and using restraint stress in rodent models are discussed.

Across all animal species, exposure to stressful conditions induces stress responses. One method to study the effects of stress using rodent models is the ...

Assessing Gait by Footprint Analysis: A Method to Test Motor Coordination in Mice

Source: Sugimoto, H. and Kawakami, K. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/59027/low-cost-protocol-footprint-analysis-hanging-box-test-for-mice">Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress.</a>&#160;J. Vis. Exp. (2019).
In this video, we describe the footprint testing procedure and related analysis to assess gait and motor coordination in mice.

Source: Sugimoto, H. and Kawakami, K. Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress.&#160;J. ...