This study was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI (no. 18H03129, 21K19709, 21H03302, 15K10441) and the Japan Agency for Medical Research and Development (AMED) (no. 15bk0104037h0002).

The present study was approved by the Kyoto University Animal Experimental Committee (Med Kyo 14033) and performed in compliance with National Institute of Health guidelines (Guide for the Care and Use of Laboratory Animals, 8th Edition). 7-week-old male Wistar rats were used for the present study. A schematic representing the sequence of procedures is provided in Supplementary File 1.
1.&#160;Familiarizing rats with treadmill walking
NOTE: Please see the previously published report13 for details regarding the procedure.
<ol>
	<li>Place the rat on a treadmill designed for rodents (see Table of Materials). In the first session, allow the animal to explore the treadmill to become accustomed to the environment. 
	NOTE: This process takes approximately 5 min.</li>
	<li>Gradually increase the belt&#39;s velocity to the desired level (20 cm/s) and walk the rat. Use an electric shock at the end of the treadmill if needed14. 
	NOTE: One walking session lasts approximately 10-20 min.</li>
	<li>Repeat this process every other day for 1 week or more frequently if needed15,16,17. 
	NOTE: Start the familiarization period 1 week prior to step 2.</li>
	<li>Keep the rats in groups in cages (2-3 rats in each cage) with a 12 h light-dark cycle. Provide food and water ad libitum.</li>
</ol>
2. Application of hindlimb unloading to the rats and setting up of joint markers
NOTE: Elevate the hindlimbs of the rat using thread and adhesive tape attached to the tail as described in previous reports18,19,20. Make sure the thread and tape are attached at the base of the tail to prevent slippage of the tail skin. Monitor the animals thoroughly and adjust the unloading height or tightness of the tape if needed.
<ol>
	<li>Under 2-5% isoflurane inhalation with an anesthetic mask, wrap the first half of a 30 cm long strip of adhesive tape around the proximal portion of the rat&#39;s tail.</li>
	<li>Fold a 1 m long cotton thread (cotton kitchen twine, approximately 1 mm diameter) into half. Make a loop by tying a knot at the folded 50 cm midpoint. The knot must be approximately 5 cm from the tip to leave a 10 cm circumference loop.</li>
	<li>Allow the remaining 15 cm of the adhesive tape to pass once through the thread loop to secure the tape. Wrap the remaining tape around the distal portion of the tail.</li>
	<li>Secure the other tip of the thread on the overhead platform of the cage. Keep the animals in a cage that is high enough to elevate their hindlimbs by their tails. Other than the unloading, provide the same environment as those for the Ctrl group, such as food, water, and floor bedding.</li>
	<li>Set up the joint markers and software (see Table of Materials) following the steps below. 
	NOTE: For details regarding this step, please see Wang et al.13.
	<ol>
		<li>Under 2-5% isoflurane inhalation, attach colored semispherical markers (3 mm diameter) to the shaved skin corresponding to bony landmarks. Keep the isoflurane level as low as possible to prevent very deep anesthesia.</li>
		<li>Ensure that the landmarks are the anterior superior iliac spine (ASIS), trochanter major (hip joint), knee joint (knee), lateral malleolus (ankle), and fifth metatarsophalangeal joint (MTP)21. 
		NOTE: Paint the tip of the toe if the angle of the toe is needed. Use an oil-based paint marker (see Table of Materials). Liquid glue is preferable for adhesive since the liquid form dries faster.</li>
	</ol>
	</li>
</ol>
3. Marker tracking using captured videos
<ol>
	<li>Open the MotionRecorder app (see Table of Materials) and turn on the treadmill. Place the rat on the treadmill belt. 
	NOTE: The four cameras for video capture (see Table of Materials) are laid out along the long edges of the treadmill: two cameras on each edge, approximately 50 cm x 50 cm apart, facing the center of the treadmill belt area.</li>
	<li>Increase the belt speed up to 20 cm/s. As the rat begins walking normally at the desired speed, click on the Record icon to start the video capture. Once enough steps (5 consecutive steps, preferably 10 steps) are obtained, stop the capture by clicking on the Record icon again. 
	NOTE: Capture data on multiple animals in one experiment. Try up to five times for each rat. If a rat does not walk, capture a different one and try the first one later. The capturing rate of the camera was 120 frames/s.</li>
	<li>Open the 3DCalculator app (see Table of Materials) and the video file to be analyzed.</li>
	<li>Crop the video by adjusting the horizontal slider bar on the top to contain enough numbers of consecutive steps. Captured image changes by dragging the yellow slide bar&#39;s end tip icon(s).</li>
	<li>To capture the markers, select the marker legends by clicking on the marker legends on the stick picture model, dragging them to the corresponding marker on the captured video, and releasing the button. This process allocates the color of the marker to the marker legend in the stick picture. Repeat this process for every marker to be tracked.</li>
	<li>Click on the Automatic trace icon. If the system does not accurately track markers or the tracking process halts due to marker loss, switch to manual mode. 
	NOTE: This automatic process does not halt unless the markers are missed. If halts happen more often than every few frames, consider repositioning the lost markers.</li>
	<li>If manual mode is needed, click the Manual icon to switch. Click the missing marker legend on the stick picture and the corresponding marker on the video. The video proceeds with one frame for each click in the manual mode. 
	&#8203;NOTE: Utilize freely available apps that enable auto click to prevent fatigue of those who track (digitize) the markers (see Table of Materials).</li>
</ol>
4. Computation of desired parameters
<ol>
	<li>Open the KineAnalyzer app (see Table of Materials) and load the file.</li>
	<li>Go to the View &#62; Edit Marker Master menu.It opens the &#34;Marker master edit&#34; window. 
	NOTE: Captured markers have simple numbers until they are labeled.</li>
	<li>Click on the desired label (landmark) on the marker tab, then click on the desired color. This process designates each marker to a specific landmark.</li>
	<li>Go to the link tab. Create lines by clicking on two markers consecutively. This process creates lines that correspond to each limb using labeled markers.</li>
	<li>Assign colors to the created lines by selecting the desired color from the Color column.</li>
	<li>Define angles by assigning reference/moving lines and directions of the angles. Go to the angle tab. After naming the angle, assign Vector A (reference line) and Vector B (moving line) by clicking on the markers corresponding to each landmark. Then, define the direction of the angle with a value in the operate section in the same tab. 
	NOTE: For the present study, the parameters mainly focused on were at the middle of the stance phase (midstance): KSt (knee angle), ASt (ankle angle), MHD (metatarso hip distance: equivalent to the height of the hip, see the next section). Knee angle and ankle angle were defined as the angle between the femur and tibia and the tibia and fifth metatarsal bone, respectively. A 0&#176; angle means that the joint was fully flexed.</li>
	<li>On the distance tab, define the distance parameter (MHD). Select two corresponding markers in the Distance Setting section. Joint trajectories as a function of normalized step cycle will also be available. 
	NOTE: Defining angles/parameters needs to be performed only one time. The settings of the parameters will be available for later evaluations once this defining process is completed.</li>
</ol>

<ol>
	<li>Bloomfield, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+musculoskeletal+structure+and+function+with+prolonged+bed+rest.">Changes in musculoskeletal structure and function with prolonged bed rest.</a> Medicine and Science in Sports and Exercise. 29 (2), 197-206 (1997).</li><li>Booth, F. W., Roberts, C. K., Laye, 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=Lack+of+exercise+is+a+major+cause+of+chronic+diseases.">Lack of exercise is a major cause of chronic diseases.</a> Comprehensive Physiology. 2 (2), 1143-1211 (2012).</li><li>Walton, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+development+under+conditions+of+simulated+weightlessness+and+space+flight.">Postnatal development under conditions of simulated weightlessness and space flight.</a> Brain Research Reviews. 28 (1-2), 25-34 (1998).</li><li>Canu, M. H., Falempin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+hindlimb+unloading+on+locomotor+strategy+during+treadmill+locomotion+in+the+rat.">Effect of hindlimb unloading on locomotor strategy during treadmill locomotion in the rat.</a> European Journal of Applied Physiology and Occupational Physiology. 74 (4), 297-304 (1996).</li><li>Shpakov, A. V., Voronov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+effects+of+simulated+weightlessness+and+lunar+gravitation+on+the+biomechanical+parameters+of+gait+in+humans.">Studies of the effects of simulated weightlessness and lunar gravitation on the biomechanical parameters of gait in humans.</a> Neuroscience and Behavioral Physiology. 48 (2), 199-206 (2018).</li><li>Kawano, 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=Tension-+and+afferent+input-associated+responses+of+neuromuscular+system+of+rats+to+hindlimb+unloading+and/or+tenotomy.">Tension- and afferent input-associated responses of neuromuscular system of rats to hindlimb unloading and/or tenotomy.</a> American Journal of Physiology - Regulatory, Integrative and Comparative Physiology. 287 (1), 76-86 (2004).</li><li>Canu, M. H., Falempin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+hindlimb+unloading+on+interlimb+coordination+during+treadmill+locomotion+in+the+rat.">Effect of hindlimb unloading on interlimb coordination during treadmill locomotion in the rat.</a> European Journal of Applied Physiology and Occupational Physiology. 78 (6), 509-515 (1998).</li><li>Canu, M. H., Falempin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+hindlimb+unloading+on+two+hindlimb+muscles+during+treadmill+locomotion+in+rats.">Effect of hindlimb unloading on two hindlimb muscles during treadmill locomotion in rats.</a> European Journal of Applied Physiology and Occupational Physiology. 75 (4), 283-288 (1997).</li><li>Walker, J. L., Evans, J. M., Meade, P., Resig, P., Sisken, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gait-stance+duration+as+a+measure+of+injury+and+recovery+in+the+rat+sciatic+nerve+model.">Gait-stance duration as a measure of injury and recovery in the rat sciatic nerve model.</a> Journal of Neuroscience Methods. 52 (1), 47-52 (1994).</li><li>Rui, 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=Gait+cycle+analysis+parameters+sensitive+for+functional+evaluation+of+peripheral+nerve+recovery+in+rat+hind+limbs.">Gait cycle analysis parameters sensitive for functional evaluation of peripheral nerve recovery in rat hind limbs.</a> Annals of Plastic Surgery. 73 (4), 405-411 (2014).</li><li>Ueno, M., Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinematic+analyses+reveal+impaired+locomotion+following+injury+of+the+motor+cortex+in+mice.">Kinematic analyses reveal impaired locomotion following injury of the motor cortex in mice.</a> Experimental Neurology. 230 (2), 280-290 (2011).</li><li>Z&#246;rner, 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=Profiling+locomotor+recovery:+Comprehensive+quantification+of+impairments+after+CNS+damage+in+rodents.">Profiling locomotor recovery: Comprehensive quantification of impairments after CNS damage in rodents.</a> Nature Methods. 7 (9), 701-711 (2010).</li><li>Wang, T., Ito, A., Tajino, J., Kuroki, H., Aoyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+kinematic+analysis+for+the+functional+evaluation+in+the+rat+model+of+sciatic+nerve+crush+injury.">3D kinematic analysis for the functional evaluation in the rat model of sciatic nerve crush injury.</a> Journal of Visualized Experiments. (156), e60267 (2020).</li><li>Canu, M. H., Garnier, C., Lepoutre, F. X., Falempin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+3D+analysis+of+hindlimb+motion+during+treadmill+locomotion+in+rats+after+a+14-day+episode+of+simulated+microgravity.">A 3D analysis of hindlimb motion during treadmill locomotion in rats after a 14-day episode of simulated microgravity.</a> Behavioural Brain Research. 157 (2), 309-321 (2005).</li><li>Gruner, J. A., Altman, J., Spivack, N. <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+arrested+cerebellar+development+on+locomotion+in+the+rat:+Cinematographic+and+electromyographic+analysis.">Effects of arrested cerebellar development on locomotion in the rat: Cinematographic and electromyographic analysis.</a> Experimental Brain Research. 40 (4), 361-373 (1980).</li><li>Bou&#235;t, V., Borel, L., Harlay, F., Gah&#233;ry, Y., Lacour, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinematics+of+treadmill+locomotion+in+rats+conceived,+born,+and+reared+in+a+hypergravity+field+(2+g):+Adaptation+to+1+g.">Kinematics of treadmill locomotion in rats conceived, born, and reared in a hypergravity field (2 g): Adaptation to 1 g.</a> Behavioural Brain Research. 150 (1-2), 207-216 (2004).</li><li>Bojados, M., Herbin, M., Jamon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinematics+of+treadmill+locomotion+in+mice+raised+in+hypergravity.">Kinematics of treadmill locomotion in mice raised in hypergravity.</a> Behavioural Brain Research. 244, 48-57 (2013).</li><li>Morey-Holton, E. R., Globus, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hindlimb+unloading+rodent+model:+Technical+aspects.">Hindlimb unloading rodent model: Technical aspects.</a> Journal of Applied Physiology. 92 (4), 1367-1377 (2002).</li><li>Tajino, 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=Discordance+in+recovery+between+altered+locomotion+and+muscle+atrophy+induced+by+simulated+microgravity+in+rats.">Discordance in recovery between altered locomotion and muscle atrophy induced by simulated microgravity in rats.</a> Journal of Motor Behavior. 47 (5), 397-406 (2015).</li><li>Liu, x., Gao, X., Tong, J., Yu, L., Xu, M., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+Osteoporosis+in+Rats+With+Hind-Limb+Unloading+Treated+With+Pulsed+Electromagnetic+Field+and+Whole-Body+Vibration.">Improvement of Osteoporosis in Rats With Hind-Limb Unloading Treated With Pulsed Electromagnetic Field and Whole-Body Vibration.</a> Physical Therapy &#38; Rehabilitation Journal. , (2022).</li><li>Thota, A. K., Watson, S. C., Knapp, E., Thompson, B., Jung, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromechanical+control+of+locomotion+in+the+rat.">Neuromechanical control of locomotion in the rat.</a> Journal of Neurotrauma. 22 (4), 442-465 (2005).</li><li>Canu, M. H., Langlet, C., Dupont, E., Falempin, M. <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+hypodynamia-hypokinesia+on+somatosensory+evoked+potentials+in+the+rat.">Effects of hypodynamia-hypokinesia on somatosensory evoked potentials in the rat.</a> Brain Research. 978 (1-2), 162-168 (2003).</li><li>Dupont, E., Canu, M. H., Falempin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+14-day+period+of+hindpaw+sensory+deprivation+enhances+the+responsiveness+of+rat+cortical+neurons.">A 14-day period of hindpaw sensory deprivation enhances the responsiveness of rat cortical neurons.</a> Neuroscience. 121 (2), 433-439 (2003).</li><li>Langlet, C., Bastide, B., Canu, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hindlimb+unloading+affects+cortical+motor+maps+and+decreases+corticospinal+excitability.">Hindlimb unloading affects cortical motor maps and decreases corticospinal excitability.</a> Experimental Neurology. 237 (1), 211-217 (2012).</li><li>Trinel, D., Picquet, F., Bastide, B., Canu, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+spine+remodeling+induced+by+hindlimb+unloading+in+adult+rat+sensorimotor+cortex.">Dendritic spine remodeling induced by hindlimb unloading in adult rat sensorimotor cortex.</a> Behavioural Brain Research. 249, 1-7 (2013).</li><li>Alkner, B. A., Norrbrand, L., Tesch, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromuscular+adaptations+following+90+days+bed+rest+with+or+without+resistance+exercise.">Neuromuscular adaptations following 90 days bed rest with or without resistance exercise.</a> Aerospace Medicine and Human Performance. 87 (7), 610-617 (2016).</li><li>English, K. L., Bloomberg, J. J., Mulavara, A. P., Ploutz-Snyder, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+countermeasures+to+neuromuscular+deconditioning+in+spaceflight.">Exercise countermeasures to neuromuscular deconditioning in spaceflight.</a> Comprehensive Physiology. 10 (1), 171-196 (2020).</li><li>Parks, M. T., Wang, Z., Siu, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+low-cost+video-based+motion+analysis+options+for+clinical+rehabilitation:+A+systematic+review.">Current low-cost video-based motion analysis options for clinical rehabilitation: A systematic review.</a> Physical Therapy. 99 (10), 1405-1425 (2019).</li></ol>

The authors declare that there is no conflict of interest.

Alteration of environments leads to fluctuating functional aspects and musculoskeletal components of locomotor systems26,27. Aberrations in contractile structures or environments may affect functional abilities, persisting even after resolving mechanical/environmental distortions19. Objective motion analysis helps to measure those functional abilities quantitatively. As shown above, video analysis is a powerful methodology for acquiring su...

Lack of physical activity or disuse leads to the deterioration of locomotor effectors, such as muscle atrophy and bone loss1 and whole-body deconditioning2. Moreover, it has recently been noticed that inactivity affects not only structural aspects of musculoskeletal components but also qualitative aspects of the movement. For example, the limb positions of rats exposed to a simulated microgravity environment were different from those of intact animals even 1 month after the intervention ended3,4. Nevertheless, little has been reported on motion deficits caused by inactivity. Also, comprehensive motion characteristics of the deteriorations have not been fully determined.
The current protocol demonstrates and discusses the application of kinematic evaluation to visualize motion alterations by referring to gait motion deficits evoked through disuse in rats subjected to hindlimb unloading.
It has been shown that hyperextensions of limbs in walking after a simulated microgravity environment are observed both in humans5 and animals4,6,7,8. Therefore, for universality, we focused on general parameters in this study: angles of the knee and ankle joints and vertical distance between the metatarsophalangeal joint and hip (roughly equivalent to the height of the hip) at the middle point of the stance phase (midstance). Further, potential applications of video kinematic evaluation are suggested in the discussion.
A series of kinematic analyses may be an effective measure to assess functional aspects of neural control. However, although motion analyses have been developed from footprint observation or simple measuring on captured video9,10 to multiple camera systems11,12, universal methods and parameters are yet to be established. The method in this study is intended to provide this joint motion analysis with comprehensive parameters.
In the previous work13, we tried illustrating gait alterations in nerve lesion model rats using comprehensive video analysis. However, in general, the potential outcomes of motion analyses are often restricted to predetermined variables provided in the analysis frameworks. For this reason, the present study further detailed how to incorporate user-defined parameters that are broadly applicable. Kinematic evaluations using video analyses may be of further use if proper parameters are implemented.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adhesive Tape</td>
			<td>NICHIBAN CO.,LTD.</td>
			<td>SEHA25F</td>
			<td>Adhesive tape to secure thread on tails of rats for hindlimb unloading</td>
		</tr>
		<tr>
			<td>Anesthetic Apparatus for Small Animals</td>
			<td>SHINANO MFG CO.,LTD.</td>
			<td>SN-487-0T</td>
			<td></td>
		</tr>
		<tr>
			<td>Auto clicker</td>
			<td>N.A.</td>
			<td>N.A.</td>
			<td>free software available to download to PC (https://www.google.com/search?client=firefox-b-1-d&#38;q=auto+clicker)</td>
		</tr>
		<tr>
			<td>CCD Camera</td>
			<td>Teledyne FLIR LLC</td>
			<td>GRAS-03K2C-C</td>
			<td>CCD (Charge-Coupled Device) cameras for video capture</td>
		</tr>
		<tr>
			<td>Cotton Thread</td>
			<td>N.A.</td>
			<td>N.A.</td>
			<td>Thread to hang tails of rats from the ceiling of cage</td>
		</tr>
		<tr>
			<td>ISOFLURANE Inhalation Solution</td>
			<td>Pfizer Japan Inc.</td>
			<td>(01)14987114133400</td>
			<td></td>
		</tr>
		<tr>
			<td>Joint marker</td>
			<td>TOKYO MARUI Co., Ltd</td>
			<td>0.12g BB</td>
			<td>6 mm airsoft pellets that were used as semispherical markers with modification</td>
		</tr>
		<tr>
			<td>Kine Analyzer</td>
			<td>KISSEI COMTEC CO.,LTD.</td>
			<td>N.A.</td>
			<td>Software for analysis</td>
		</tr>
		<tr>
			<td>Konishi Aron Alpha</td>
			<td>TOAGOSEI CO.,LTD.</td>
			<td>#31204</td>
			<td>Super glue to attach spherical markers on randmarks of rats</td>
		</tr>
		<tr>
			<td>Motion Recorder</td>
			<td>KISSEI COMTEC CO.,LTD.</td>
			<td>N.A.</td>
			<td>Software for video recording</td>
		</tr>
		<tr>
			<td>Paint Marker</td>
			<td>MITSUBISHI PENCIL CO., LTD</td>
			<td>PX-21.13</td>
			<td>Oil based paint marker to mark toes of animals</td>
		</tr>
		<tr>
			<td>Three-dimensional motion capture apparatus (KinemaTracer for small animals)</td>
			<td>KISSEI COMTEC CO.,LTD.</td>
			<td>N.A.</td>
			<td>3D motion analysis system that consists of four cameras (https://www.kicnet.co.jp/solutions/biosignal/animals/kinematracer-for-animal/ or https://micekc.com/en/)</td>
		</tr>
		<tr>
			<td>Three-dimensional(3D) Calculator</td>
			<td>KISSEI COMTEC CO.,LTD.</td>
			<td>N.A.</td>
			<td>Software fo marker tracking</td>
		</tr>
		<tr>
			<td>Treadmill</td>
			<td>MUROMACHI KIKAI CO.,LTD</td>
			<td>MK-685</td>
			<td>Treadmill equipped with transparent housing, electrical shocker, and speed control unit</td>
		</tr>
		<tr>
			<td>Wistar Rats (male, 7-week old)</td>
			<td>N.A.</td>
			<td>N.A.</td>
			<td>Commercially available at experimental animal sources</td>
		</tr>
	</tbody>
</table>

comprehensive understanding of inactivity-induced gait alteration in rodents

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics is still unclear. The present study describes a motion analysis approach that utilizes three-dimensional (3D) reconstruction from video captures. Using this technology, disuse-evoked alterations of walking performances were observed in rodents exposed to a simulated microgravity environment by unloading their hindlimb by their tail. After 2 weeks of unloading, the rats walked on a treadmill, and their gait motions were captured with four charge-coupled device (CCD) cameras. 3D motion profiles were reconstructed and compared to those of control subjects using the image processing software. The reconstructed outcome measures successfully portrayed distinct aspects of distorted gait motion: hyperextension of the knee and ankle joints and higher position of the hip joints during the stance phase. Motion analysis is useful for several reasons. First, it enables quantitative behavioral evaluations instead of subjective observations (e.g., pass/fail in certain tasks). Second, multiple parameters can be extracted to fit specific needs once the fundamental datasets are obtained. Despite hurdles for broader application, the disadvantages of this method, including labor intensity and cost, may be alleviated by determining comprehensive measurements and experimental procedures.

12 animals were randomly assigned to one of two groups: the unloading group (UL, n = 6) or the control group (Ctrl, n = 6). For the UL group, the hindlimbs of the animals were unloaded by the tail for 2 weeks (UL period), whereas the Ctrl group animals were left free. 2 weeks after unloading, the UL group showed a distinct gait pattern compared with the Ctrl group. Figure 1 shows normalized joint trajectories of representative subjects. During the stance phase, the UL group exhibited further...

Watch this Scientific Journal Video about Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents at JoVE.com

Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents

The present protocol describes three-dimensional motion tracking/evaluation to depict gait motion alteration of rats after exposure to a simulated disuse environment.

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics ...

comprehensive-understanding-inactivity-induced-gait-alteration

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

2.3K Views.  Kyoto University. The present protocol describes three-dimensional motion tracking/evaluation to depict gait motion alteration of rats after exposure to a simulated disuse environment. 

Video: Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents

Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the sensorimotor cortex and the underneath white matter (undercut) so that a specific region of the cerebral cortex is largely isolated from the neighboring cortex and subcortical regions1-3. After a latency of two or more weeks following the surgery, epileptiform discharges can be recorded in brain slices from rodents1; and electrical or behavior seizures can be observed in vivo from other species such as cat and monkey4-6. This well established animal model is efficient to generate and mimics several important characteristics of traumatic brain injury. However, it is technically challenging attempting to make precise cortical lesions in the small rodent brain with a free hand. Based on the procedure initially established in Dr. David Prince's lab at the Stanford University1, here we present an improved technique to perform a surgery for the preparation of this model in mice and rats. We demonstrate how to make a simple surgical device and use it to gain a better control of cutting depth and angle to generate more precise and consistent results. The device is easy to make, and the procedure is quick to learn. The generation of this animal model provides an efficient system for study on the mechanisms of posttraumatic epileptogenesis.

Partially isolated cortex (&#x201C;undercut&#x201D;) is an efficient animal model of posttraumatic epileptogenesis. Here we demonstrate how to make a novel surgical device and use it to make more precise and consistent lesions to generate this model.

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the ...

Habituation and Prepulse Inhibition of Acoustic Startle in Rodents

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated within a few milliseconds, leading to a whole body flinch in rodents1. Although startle responses are reflexive responses that can be reliably elicited, they are not stereotypic. They can be modulated by emotions such as fear (fear potentiated startle) and joy (joy attenuated startle), by non-associative learning processes such as habituation and sensitization, and by other sensory stimuli through sensory gating processes (prepulse inhibition), turning startle responses into an excellent tool for assessing emotions, learning, and sensory gating, for review see 2, 3. The primary pathway mediating startle responses is very short and well described, qualifying startle also as an excellent model for studying the underlying mechanisms for behavioural plasticity on a cellular/molecular level3.
We here describe a method for assessing short-term habituation, long-term habituation and prepulse inhibition of acoustic startle responses in rodents. Habituation describes the decrease of the startle response magnitude upon repeated presentation of the same stimulus. Habituation within a testing session is called short-term habituation (STH) and is reversible upon a period of several minutes without stimulation. Habituation between testing sessions is called long-term habituation (LTH)4. Habituation is stimulus specific5. Prepulse inhibition is the attenuation of a startle response by a preceding non-startling sensory stimulus6. The interval between prepulse and startle stimulus can vary from 6 to up to 2000 ms. The prepulse can be any modality, however, acoustic prepulses are the most commonly used.
Habituation is a form of non-associative learning. It can also be viewed as a form of sensory filtering, since it reduces the organisms' response to a non-threatening stimulus. Prepulse inhibition (PPI) was originally developed in human neuropsychiatric research as an operational measure for sensory gating7. PPI deficits may represent the interface of "psychosis and cognition" as they seem to predict cognitive impairment8-10. Both habituation and PPI are disrupted in patients suffering from schizophrenia11, and PPI disruptions have shown to be, at least in some cases, amenable to treatment with mostly atypical antipsychotics12, 13. However, other mental and neurodegenerative diseases are also accompanied by disruption in habituation and/or PPI, such as autism spectrum disorders (slower habituation), obsessive compulsive disorder, Tourette's syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's Disease (PPI)11, 14, 15 Dopamine induced PPI deficits are a commonly used animal model for the screening of antipsychotic drugs16, but PPI deficits can also be induced by many other psychomimetic drugs, environmental modifications and surgical procedures.

Habituation and prepulse inhibition of startle are operational measures of sensory gating. Sensory gating is disrupted in schizophrenia, and some other mental disorders and neurodegenerative diseases. We here describe a standard protocol to assess short-term and long-term habituation as well as prepulse inhibition of acoustic startle responses in rats and mice.

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

A Low Cost Setup for Behavioral Audiometry in Rodents

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the experiments. Such parameters may be physiological response characteristics of the auditory pathway, e.g. via brainstem audiometry (BERA). But these methods allow only indirect and uncertain extrapolations about the auditory percept that corresponds to these physiological parameters. To assess the perceptual level of hearing, behavioral methods have to be used. A potential problem with the use of behavioral methods for the description of perception in animal models is the fact that most of these methods involve some kind of learning paradigm before the subjects can be behaviorally tested, e.g. animals may have to learn to press a lever in response to a sound. As these learning paradigms change perception itself 1,2 they consequently will influence any result about perception obtained with these methods and therefore have to be interpreted with caution. Exceptions are paradigms that make use of reflex responses, because here no learning paradigms have to be carried out prior to perceptual testing. One such reflex response is the acoustic startle response (ASR) that can highly reproducibly be elicited with unexpected loud sounds in na&iuml;ve animals. This ASR in turn can be influenced by preceding sounds depending on the perceptibility of this preceding stimulus: Sounds well above hearing threshold will completely inhibit the amplitude of the ASR; sounds close to threshold will only slightly inhibit the ASR. This phenomenon is called pre-pulse inhibition (PPI) 3,4, and the amount of PPI on the ASR gradually depends on the perceptibility of the pre-pulse. PPI of the ASR is therefore well suited to determine behavioral audiograms in na&iuml;ve, non-trained animals, to determine hearing impairments or even to detect possible subjective tinnitus percepts in these animals. In this paper we demonstrate the use of this method in a rodent model (cf. also ref. 5), the Mongolian gerbil (Meriones unguiculatus), which is a well know model species for startle response research within the normal human hearing range (e.g. 6).

A fast and inexpensive method for the behavioral determination of hearing parameters like hearing thresholds, hearing impairments or phantom perceptions (subjective tinnitus) is described. It uses pre-pulse inhibition of the acoustic startle response and can be easily implemented in a personal computer using a programmable AD/DA-converter and a piezo sensor.

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Acrylic Resin Molding Based Head Fixation Technique in Rodents

Head fixation is a technique of immobilizing animal's head by attaching a head-post on the skull for rigid clamping. Traditional head fixation requires surgical attachment of metallic frames on the skull. The attached frames are then clamped to a stationary platform resulting in immobilization of the head. However, metallic frames for head fixation have been technically difficult to design and implement in general laboratory environment. In this study, we provide a novel head fixation method. Using a custom-made head fixation bar, head mounter is constructed during implantation surgery. After the application of acrylic resin for affixing implants such as electrodes and cannula on the skull, additional resins applied on top of that to build a mold matching to the port of the fixation bar. The molded head mounter serves as a guide rails, investigators conveniently fixate the animal&#8217;s head by inserting the head mounter into the port of the fixation bar. This method could be easily applicable if implantation surgery using dental acrylics is necessary and might be useful for laboratories that cannot easily fabricate CNC machined metal head-posts.

Traditional head fixation techniques have used metallic frames attached on the skull as an interface for head fixation apparatus. This protocol demonstrates a frameless, acrylic resin molding based head fixation technique for behavioral tasks in rodents.

Head fixation is a technique of immobilizing animal's head by attaching a head-post on the skull for rigid clamping. Traditional head fixation requires ...

Transcranial Electrical Brain Stimulation in Alert Rodents

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in humans is transcranial direct current stimulation (tDCS). Less frequently, transcranial alternating current stimulation (tACS) or transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range, is used. The increase of noninvasive electrical brain stimulation research in humans, both for experimental and clinical purposes, has yielded an increased need for basic, mechanistic, safety studies in animals. This article describes a model for transcranial electrical brain stimulation (tES) through the intact skull targeting the motor system in alert rodents. The protocol provides step-by-step instructions for the surgical set-up of a permanent epicranial electrode socket combined with an implanted counter electrode on the chest. By placing a stimulation electrode into the epicranial socket, different electrical stimulation types, comparable to tDCS, tACS, and tRNS in humans, can be delivered. Moreover, the practical steps for tES in alert rodents are introduced. The applied current density, stimulation duration, and stimulation type may be chosen depending on the experimental needs. The caveats, advantages, and disadvantages of this set-up are discussed, as well as safety and tolerability aspects.

This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact skull.

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...