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...

<ol>
	<li>Warner, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+disorders.">Movement disorders.</a> Practical Guide to Neurogenetics. , (2008).</li><li>Brashear, A., DeLeon, D., Bressman, S. B., Thyagarajan, D., Farlow, M. R., Dobyns, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid-onset+dystonia-parkinsonism+in+a+second+family.">Rapid-onset dystonia-parkinsonism in a second family.</a> Neurology. 48 (4), 1066-1069 (1997).</li><li>Linazasoro, G., Indakoetxea, B., Ruiz, J., Van Blercom, N., Lasa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+sporadic+rapid-onset+dystonia-parkinsonism.">Possible sporadic rapid-onset dystonia-parkinsonism.</a> Movement Disorders. 17 (3), 608-609 (2002).</li><li>Svetel, M., Ozelius, L. J., 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=Rapid-onset+dystonia-parkinsonism:+case+report.">Rapid-onset dystonia-parkinsonism: case report.</a> Journal of Neurology. 257 (3), 472-474 (2010).</li><li>Vrinten, D. H., Hamers, F. F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="CatWalk"+automated+quantitative+gait+analysis+as+a+novel+method+to+assess+mechanical+allodynia+in+the+rat;+a+comparison+with+von+Frey+testing.">"CatWalk" automated quantitative gait analysis as a novel method to assess mechanical allodynia in the rat; a comparison with von Frey testing.</a> PAIN. 102 (1), 203-209 (2003).</li><li>Berryman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DigigaitTM+quantitation+of+gait+dynamics+in+rat+rheumatoid+arthritis+model.">DigigaitTM quantitation of gait dynamics in rat rheumatoid arthritis model.</a> Journal of Musculoskeletal and Neuronal Interactions. 9 (2), 89-98 (2009).</li><li>Beare, J. E., Morehouse, J. R., 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=Gait+analysis+in+normal+and+spinal+contused+mice+using+the+TreadScan+system.">Gait analysis in normal and spinal contused mice using the TreadScan system.</a> Journal of Neurotrauma. 26 (11), 2045-2056 (2009).</li><li>Rushton, R., Steinberg, H., Tinson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+a+single+experience+on+subsequent+reactions+to+drugs.">Effects of a single experience on subsequent reactions to drugs.</a> British Journal of Pharmacology and Chemotherapy. 20, 99-105 (1963).</li><li>Lee, C. C., Peters, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxicity+and+behavioral+effects+of+thiram+in+rats.">Neurotoxicity and behavioral effects of thiram in rats.</a> Environmental health perspectives. 17, 35-43 (1976).</li><li>van der Zee, C. E., Schuurman, T., Traber, J., Gispen, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+administration+of+nimodipine+accelerates+functional+recovery+following+peripheral+nerve+damage+in+the+rat.">Oral administration of nimodipine accelerates functional recovery following peripheral nerve damage in the rat.</a> Neuroscience Letters. 83 (1-2), 143-148 (1987).</li><li>Leroy, T., Stroobants, S., Aerts, J. -. M., D'Hooge, R., Berckmans, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+analysis+of+altered+gait+in+arylsulphatase+A-deficient+mice+in+the+open+field.">Automatic analysis of altered gait in arylsulphatase A-deficient mice in the open field.</a> Behavior Research Methods. 41 (3), 787-794 (2009).</li><li>Sango, K., McDonald, M. P., 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=Mice+lacking+both+subunits+of+lysosomal+beta-hexosaminidase+display+gangliosidosis+and+mucopolysaccharidosis.">Mice lacking both subunits of lysosomal beta-hexosaminidase display gangliosidosis and mucopolysaccharidosis.</a> Nature Genetics. 14 (3), 348-352 (1996).</li><li>Deacon, R. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+Strength+of+Mice.">Measuring the Strength of Mice.</a> Journal of Visualized Experiments. (76), e2610 (2013).</li><li>Djamshidian, A., Lees, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+stress+trigger+Parkinson's+disease?.">Can stress trigger Parkinson's disease?.</a> Journal of Neurology, Neurosurgey, and Psychiatry. 85 (8), 879-882 (2014).</li><li>Brashear, A., Dobyns, W. B., 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=The+phenotypic+spectrum+of+rapid-onset+dystonia-parkinsonism+(RDP)+and+mutations+in+the+ATP1A3.">The phenotypic spectrum of rapid-onset dystonia-parkinsonism (RDP) and mutations in the ATP1A3.</a> Brain. 130 (Pt 3), 828-835 (2007).</li><li>Kirshenbaum, G. S., Saltzman, K., Rose, B., Petersen, J., Vilsen, B., Roder, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+neuronal+Na+,K+-ATPase+activity+in+Atp1a3+heterozygous+mice+increases+susceptibility+to+depression-like+endophenotypes+by+chronic+variable+stress.">Decreased neuronal Na+,K+-ATPase activity in Atp1a3 heterozygous mice increases susceptibility to depression-like endophenotypes by chronic variable stress.</a> Genes, Brain and Behavior. 10 (5), 542-550 (2011).</li><li>DeAndrade, M. P., Yokoi, F., van Groen, T., Lingrel, J. B., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Atp1a3+mutant+mice+as+a+model+of+rapid-onset+dystonia+with+parkinsonism.">Characterization of Atp1a3 mutant mice as a model of rapid-onset dystonia with parkinsonism.</a> Behavioral Brain Research. 216 (2), 659-665 (2011).</li><li>Sugimoto, H., Ikeda, K., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterozygous+mice+deficient+in+Atp1a3+exhibit+motor+deficits+by+chronic+restraint+stress.">Heterozygous mice deficient in Atp1a3 exhibit motor deficits by chronic restraint stress.</a> Behavioral Brain Research. 272, 100-110 (2014).</li><li>Zimprich, A., Garrett, 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=A+robust+and+reliable+non-invasive+test+for+stress+responsivity+in+mice.">A robust and reliable non-invasive test for stress responsivity in mice.</a> Frontiers in Behavioral Neuroscience. 8, 125 (2014).</li><li>Buynitsky, T., Mostofsky, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restraint+stress+in+biobehavioral+research:+recent+developments.">Restraint stress in biobehavioral research: recent developments.</a> Neuroscience and Biobehavioral Reviews. 33 (7), 1089-1098 (2009).</li><li>Ikeda, K., Satake, S., 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=Enhanced+inhibitory+neurotransmission+in+the+cerebellar+cortex+of+Atp1a3-deficient+heterozygous+mice.">Enhanced inhibitory neurotransmission in the cerebellar cortex of Atp1a3-deficient heterozygous mice.</a> The Journal of Physiology. 591 (13), 3433-3449 (2013).</li><li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+functions.">Motor functions.</a> What's Wrong with My Mouse?. , (2007).</li><li>. R: A language and environment for statistical computing Available from: <a target="_blank" href="https://www.R-project.org/">https://www.R-project.org/</a> (2014)</li><li>Wils, H., Kleinberger, G., 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=TDP-43+transgenic+mice+develop+spastic+paralysis+and+neuronal+inclusions+characteristic+of+ALS+and+frontotemporal+lobar+degeneration.">TDP-43 transgenic mice develop spastic paralysis and neuronal inclusions characteristic of ALS and frontotemporal lobar degeneration.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (8), 3858-3863 (2010).</li><li>Eilam, R., Peter, Y., 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=Selective+loss+of+dopaminergic+nigro-striatal+neurons+in+brains+of+Atm-deficient+mice.">Selective loss of dopaminergic nigro-striatal neurons in brains of Atm-deficient mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 95 (21), 12653-12656 (1998).</li><li>Lin, C. -. H., Tallaksen-Greene, S., 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>Dang, M. T., Yokoi, F., 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=Generation+and+characterization+of+Dyt1+&#916;GAG+knock-in+mouse+as+a+model+for+early-onset+dystonia.">Generation and characterization of Dyt1 &#916;GAG knock-in mouse as a model for early-onset dystonia.</a> Experimental Neurology. 196 (2), 452-463 (2005).</li><li>Glynn, D., Drew, C. J., Reim, K., Brose, N., Morton, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profound+ataxia+in+complexin+I+knockout+mice+masks+a+complex+phenotype+that+includes+exploratory+and+habituation+deficits.">Profound ataxia in complexin I knockout mice masks a complex phenotype that includes exploratory and habituation deficits.</a> Human Molecular Genetics. 14 (16), 2369-2385 (2005).</li><li>Becker, E. B. E., Oliver, P. 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=A+point+mutation+in+TRPC3+causes+abnormal+Purkinje+cell+development+and+cerebellar+ataxia+in+moonwalker+mice.">A point mutation in TRPC3 causes abnormal Purkinje cell development and cerebellar ataxia in moonwalker mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (16), 6706-6711 (2009).</li><li>Heck, D. H., Zhao, Y., Roy, S., LeDoux, M. S., Reiter, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cerebellar+function+in+Ube3a-deficient+mice+reveals+novel+genotype-specific+behaviors.">Analysis of cerebellar function in Ube3a-deficient mice reveals novel genotype-specific behaviors.</a> Human Molecular Genetics. 17 (14), 2181-2189 (2008).</li><li>Kirshenbaum, G. S., Dawson, N., 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=Alternating+hemiplegia+of+childhood-related+neural+and+behavioural+phenotypes+in+Na+,K+-ATPase+&#945;3+missense+mutant+mice.">Alternating hemiplegia of childhood-related neural and behavioural phenotypes in Na+,K+-ATPase &#945;3 missense mutant mice.</a> PLoS ONE. 8 (3), e60141 (2013).</li><li>Klein, A., Wessolleck, J., Papazoglou, A., Metz, G. A., Nikkhah, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Walking+pattern+analysis+after+unilateral+6-OHDA+lesion+and+transplantation+of+foetal+dopaminergic+progenitor+cells+in+rats.">Walking pattern analysis after unilateral 6-OHDA lesion and transplantation of foetal dopaminergic progenitor cells in rats.</a> Behavioral Brain Research. 199 (2), 317-325 (2009).</li><li>Geldenhuys, W. J., Guseman, T. L., Pienaar, I. S., Dluzen, D. E., Young, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+biomechanical+analysis+of+gait+changes+in+the+MPTP+mouse+model+of+Parkinson's+disease.">A novel biomechanical analysis of gait changes in the MPTP mouse model of Parkinson's disease.</a> PeerJ. 3 (Pt 7), e1175 (2015).</li><li>Cecchi, M., Khoshbouei, H., Morilak, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulatory+effects+of+norepinephrine,+acting+on+alpha1+receptors+in+the+central+nucleus+of+the+amygdala,+on+behavioral+and+neuroendocrine+responses+to+acute+immobilization+stress.">Modulatory effects of norepinephrine, acting on alpha1 receptors in the central nucleus of the amygdala, on behavioral and neuroendocrine responses to acute immobilization stress.</a> Neuropharmacology. 43 (7), 1139-1147 (2002).</li><li>Chu, X., Zhou, Y., 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=24-hour-restraint+stress+induces+long-term+depressive-likephenotypes+in+mice.">24-hour-restraint stress induces long-term depressive-likephenotypes in mice.</a> Scientific Reports. 6, 32935 (2016).</li><li>Freeman, M. L., Sheridan, B. S., Bonneau, R. H., Hendricks, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychological+Stress+Compromises+CD8++T+cell+control+of+latent+herpes+simplex+virus+type+1+infections.">Psychological Stress Compromises CD8+ T cell control of latent herpes simplex virus type 1 infections.</a> The Journal of Immunology. 179 (1), 322-328 (2007).</li><li>Lauretti, E., Di Meco, A., Merali, S., Pratic&#242;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+behavioral+stress+exaggerates+motor+deficit+and+neuroinflammation+in+the+MPTP+mouse+model+of+Parkinson's+disease.">Chronic behavioral stress exaggerates motor deficit and neuroinflammation in the MPTP mouse model of Parkinson's disease.</a> Translational Psychiatry. 6, e733 (2016).</li><li>Quartermain, D., Stone, E. A., Charbonneau, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+stress+disrupts+risk+assessment+behavior+in+mice.">Acute stress disrupts risk assessment behavior in mice.</a> Physiology and Behavior. 59 (4-5), 937-940 (1996).</li><li>Bannon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Behavioural effects of stress and aluminum toxicity on a mouse model of amyotrophic lateral sclerosis Parkinsonism-dementia complex. , 1-186 (2015).</li></ol>

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 dystonia<sup class="xref...

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; At...

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 ...

Our protocol using gait analysis is very useful in determining whether mouse motors of movement disorders mimic the symptom of patient with gait disturbance. So, gait analysis in the stress loading test can be performed easily, and without expensive equipment, to detect certain motor phenotypes in mice, with no spontaneous effects. Before initiating the behavioral test, record the weight of each mouse, and use a marking pen to label each tail.When all of the animals have been weighed and marked, place the mice in the experimental room for at least 30 minutes, and set a clear, 25 by 25 by 40 centimeter hanging box, with a rotatable mesh lid, onto the lab bench. When the mice have acclimated, place one mouse into the center of the mesh lid, and carefully turn the lid upside-down. Then, measure the fall latency of the mouse from the mesh lid, before returning the mouse to it's home cage.When all of the mice have been tested, place a 9.9 by 42 centimeter piece of white paper onto the lab bench, and place a dark goal box at the distal end of the paper. Place other boxes, approximately the same length as the paper, on both sides of the runway to prevent escape. And add black and red ink to separate 35 milliliter Petri dishes.To train the mice for the analysis, place a mouse at the proximal end of the paper, facing the goal box, and let the mouse walk from the proximal end of the paper to the goal box. When the mouse reaches the box, gently scruff the animal, tucking the back and tail between the ball of the thumb and the other fingers, to limit the movement of the hind limbs. And immerse the bottoms of the forelimbs in red ink, and the bottom of the hind limbs in black ink.Then, immediately place the mouse back onto the proximal end of the paper, facing the goal box, and let the mouse walk from the proximal end to the goal box. After air-drying the foot printed paper, use a ruler to obtain three measurements of the stride length, by measuring the distances between the same parts of the paw prints. Of the front base-width, by drawing a line between consecutive, same-side, front footprints, and measuring the length of the vertical line from the pad of the front footprint, to the line drawn between the footprints.And of the hind base-width, by drawing a line between consecutive right hind footprints, and measuring the length of the vertical line from the pad of the left hind footprint, to the line drawn between the other right footprints. For overlap, measure the distance between the pads of the front and hind footprints. To prepare a restraint tube, use a square drill to make 16 two millimeter holes in a 50 milliliter tube along the scale marks, and the backside of each scale mark.Cut off the tip of the tube to make a five millimeter hole, for breathing, and make a four millimeter hole in the tube cap, for the tail. For the stress-loading test, scruff a mouse, and place the animal into the restraining tube head first, with the forelimbs gently confined. Pass the tail through the hole in the cap before capping the tube.And place the mouse on the bench top for two hours at room temperature. At the end of the stress-loading period, remove the mouse from the tube, and return the animal to its home cage. As observed in these representative figures, heterozygous rapid-onset dystonia Parkinsonism model mice, demonstrate significantly shorter fore and hindlimb stride lengths, than do their wild-type litter mates at four weeks of age.Stress heterozygous rapid-onset dystonia Parkinsonism model mice, also exhibit significantly shorter stride lengths of both limbs than those of stressed wild-type litter mates at eight weeks of age. Asymmetries of the stride lengths of both limbs are not significantly different, in any group of mice, at any age. The front base and overlaps of both limbs are also similar, in all groups, at all ages.The hind base is significantly wider in these stressed heterozygous rapid-onset dystonia Parkinsonism model mice, than that observed in the stress wild-type animals at 10 weeks of age, indicating that stress causes motor deficits in heterozygous rapid-onset dystonia Parkinsonism model mice. No significant differences in hanging time, between wild-type and mutant, are observed in four to 10 week old animals. At 12 weeks of age, however, the hanging time of stress wild-type mice is significantly longer than that of the other animals, suggesting that the motor deficit in heterozygous rapid-onset dystonia Parkinsonism model mice, is distinguishable from wild-type mice by restraint test in older animals.Place the mouse onto the paper immediately after the mouse hangs the bottoms of limbs in the ink to avoid the ink drying before the walking test. Additional tests for emotional characteristics, such as open field, elevated plus-maze, and whole swim test, can be performed to answer whether the gait abnormality result from only motor deficit. Our protocol can be used to explore the susceptibility to stress, by changing the number and the duration of the stress loading, and by comparing motor phenotypes between various loading conditions.Our protocol can be used to explore the susceptibility to stress by changing the number and duration of the stress loadings, and by comparing motor phenotypes between various loading conditions.

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

Research

JoVE Journal

Behavior

17.5K 次观看.  Jichi Medical University. 我们的协议使用步态分析是非常有用的，以确定小鼠运动障碍的电机是否模仿有步态障碍的患者的症状。因此，在压力负荷测试中，步态分析可以方便地进行，无需昂贵的设备，即可检测小鼠的某些运动表型，无自发效应。在开始行为测试之前，记录每只鼠标的重量，并使用标记笔标记每只尾巴。当所有动物都称重和标记后，将小鼠放在实验室至少30分钟，并在实验室?...