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&#8217;hara &#38; 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&#8217;hara &#38; 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&#8217;hara &#38; 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 dishe (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

18.1K 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

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This video shows the resident-intruder paradigm in rats. This test is a standardized method to measure offensive aggression, defensive behavior and violence in a semi-natural setting. The use of the paradigm for social stress experiments is explained as well.

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Here we present the unpredictable chronic mild stress protocol in mice. This protocol induces a long-term depressive-like phenotype and enables to assess the efficacy of putative antidepressants in reversing the behavioral and neuromolecular depressive-like deficits.

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by ...

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, ...

Paw-Print Analysis of Contrast-Enhanced Recordings (PrAnCER): A Low-Cost, Open-Access Automated Gait Analysis System for Assessing Motor Deficits

Gait analysis is used to quantify changes in motor function in many rodent models of disease. Despite the importance of assessing gait and motor function in many areas of research, the available commercial options have several limitations such as high cost and lack of accessible, open code. To address these issues, we developed PrAnCER, Paw-Print Analysis of Contrast-Enhanced Recordings, for automated quantification of gait. The contrast-enhanced recordings are produced by using a translucent floor that obscures objects not in contact with the surface, effectively isolating the rat&#8217;s paw prints as it walks. Using these videos, our simple software program reliably measures a variety of spatiotemporal gait parameters. To demonstrate that PrAnCER can accurately detect changes in motor function, we employed a haloperidol model of Parkinson&#8217;s disease (PD). We tested rats at two doses of haloperidol: high dose (0.30 mg/kg) and low dose (0.15 mg/kg). Haloperidol significantly increased stance duration and hind paw contact area in the low dose condition, as might be expected in a PD model. In the high dose condition, we found a similar increase in contact area but also an unexpected increase in stride length. With further research, we found that this increased stride length is consistent with the bracing-escape phenomenon commonly observed at higher doses of haloperidol. Thus, PrAnCER was able to detect both expected and unexpected changes in rodent gait patterns. Additionally, we confirmed that PrAnCER is consistent and accurate when compared with manual scoring of gait parameters.

Paw-Print Analysis of Contrast-Enhanced Recordings PrAnCER: A Low-Cost, Open-Access Automated Gait Analysis System for Assessing Motor Deficits

We describe a novel gait analysis system, Paw-Print Analysis of Contrast-Enhanced Recordings (PrAnCER), an open-access automated system for the quantification of gait characteristics in rats that utilizes a novel semitransparent floor to automatically quantify gait. This system was validated using the haloperidol model of Parkinson&#8217;s Disease.

Gait analysis is used to quantify changes in motor function in many rodent models of disease. Despite the importance of assessing gait and motor function ...