Kriss Franklin, Amanda Pollard and Justine Shaffer assisted in animal training and data collection. Sarah Freeman and Alisa Ivanova contributed to data analysis. This study is supported by WVU School of Medicine Start-Up, NIH/NIGMS U54GM104942, and NIH CoBRE P20GM109098.

The following training paradigm employs the analysis of phase adjustments of the average adult Sprague-Dawley rat. Please ensure that the protocol described herein is in accordance with your institutional animal care guidelines. All procedures in this study were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) and Office for Laboratory Animal Welfare (OLAW) at West Virginia University School of Medicine and abides by the National Institutes of Health guidelines for the use of experimental animals.
1. Equipment Setup
<ol>
	<li>Construct the asymmetric walkway as an open-top plastic box braced with aluminum supports at each corner measuring 155 cm x 104 cm (Figure 1). Brace the top edges of the box with aluminum bars grooved on both sides to allow for alternate peg placement, along the perimeter of the box, so that each consecutive peg on the same side defines the stride length.</li>
	<li>Place a 20 cm x 20 cm platform on each corner (four total) separating the conditions represented on each side. This distance should be sufficient for the inclusion of the distance traversed by a single rat step cycle.
	<ol>
		<li>Use pegs made of aluminum with dimensions of 20 cm x 1 cm x 0.5 cm. Bend the top of each peg 2.5 cm from the tip to produce a foot placement platform.</li>
		<li>Secure the pegs to the grooved bars using sliding inside brackets through machined holes at the same distance to ensure level horizontal placement. Adjust positions using a screwdriver and a ruler. Use a 1 cm peg width that corresponds approximately to the average rat paw size; thinner or wider pegs are either uncomfortable or increase the foot placement variability.</li>
	</ol>
	</li>
	<li>Manipulate the peg placement on each side to produce one of three precise stepping challenge conditions.
	<ol>
		<li>Produce a symmetric locomotor task with a 15 cm stride length (SL15) by setting the left inter-stride length (lISL) and right inter-stride length (rISL) to the half of stride length (7.5 cm).</li>
		<li>Impose an additional symmetric condition (SL12) by changing lISL and rISL lengths to 6.0 cm.</li>
		<li>Produce the asymmetric tasks by changing the distance between pegs on the left and right sides, termed the inter-stride length. To challenge the motor system asymmetrically, change the lISL and rISL by 20% to impose short inter-stride lengths either on the left (L6R9 condition) or on the right (L9R6) side. The 1.5 cm perturbations impose an lISL of 6 cm and rISL of 9 cm for the L6R9 condition, or an lISL of 9 cm and a rISL of 6 cm for the L9R6 condition</li>
	</ol>
	</li>
	<li>For rats, keep the stride length for all conditions except for SL12 at a preferred 15 cm.</li>
	<li>For convenience, assign each long side of the walkway an asymmetric condition favoring either the left or the right side of the subject, while reserving the two short sides for the symmetric control condition.</li>
	<li>Setup a high definition camera with a sampling rate of at least 60 Hz so that the placement of limbs on pegs is unobstructed with camera pointing perpendicularly to the walkway with the field of view covering about 7 steps. The first and last steps in proximity to platforms are ignored.</li>
</ol>
2. Training on Apparatus
<ol>
	<li>Please use standard training resources, e.g., NIH Training in Basic Biomethodology for Laboratory Rats, to familiarize with general behavioral training of rodents.</li>
	<li>In the beginning of training, acclimate subjects by placing and rewarding them on the 20 x 20 cm platform for at least 5 min. Then, guide the animals across a peg arrangement with a 1 cm inter-stride length to the next platform by the presentation of a food reward. Reward animals verbally and with petting for reaching the platform.</li>
	<li>After 5 training runs, space the pegs an extra 1 - 2 cm apart and perform the next 5 training runs. The number of repetitions listed herein is sufficient to produce statistically appropriate sample size (20 - 35 steps).
	<ol>
		<li>If the animal acquires the task more slowly as judged by consistency of stepping (no stopping) and posture (arched back), then focus training on the strengthening of these skills at the short stride lengths (S12) before resuming training on the long strides (S15) eventually approaching the desired stride length.</li>
		<li>If the new spacing induces anxiety or discomfort with the task, readjust the pegs to the previous setting and repeat the training paradigm.</li>
		<li>Proceed with this training until the appropriate inter-stride lengths are achieved for the four conditions and locomotor standards are met. In our experience, the rats respond well to vocal encouragement as cues for initiating a trial. The testing can be done on the same day as training provided the subjects are motivated to perform the task. 
		Note: The locomotor standards are as follows: walking is consistent and does not involve stops or missteps; head-bobbing is minimal; the back is arched and the tail is raised during locomotion; each limb is clearly visible from an orthogonal view of the walkway at the onset and offset of the stance phase. This selection process is essential as the present study focuses only on walking rather than other gaiting behavior.</li>
	</ol>
	</li>
</ol>
3. Testing and Data Analysis
<ol>
	<li>Test animals on S12, S15, L9R6, and L6R9 tasks (described in section 1.3) using randomized session design. Use breaks to avoid adaptation within a task.</li>
	<li>Record sessions with high definition camera with a sampling rate of at least 60 Hz. Import video recordings without re-sampling into video editing software and select only the walking bouts for further analysis.</li>
	<li>Mark onsets and offsets of kinematic phases in video recordings from each subject.</li>
	<li>Here, use the custom software called videoa written in Matlab to manually identify the time of stance onset and offset for each limb on a frame-by-frame basis, where stance onset is indicated by the loss of motion blur associated with the limb placement on a peg, and stance offset, occurring at the onset of limb lift-off, is indicated by the first evidence of motion blur.</li>
	<li>Calculate the duration of swing phase as the time remaining between two consecutive kinematic stance onsets. Exclude any behavior not consistent with overground quadrupedal walking, e.g., when gait contains a double swing phase (both forelimbs or hindlimbs off the ground), from proceeding analyses.</li>
	<li>Plot the duration of each phase as a function of the corresponding step cycle duration. Capture the relationship with the linear regression model (Tphase = B1+B2*Tc) obtained for each limb, where Tc is cycle duration, Tphase is either Te extensor-related stance or Tf, which is the flexor-related swing, and B1 and B2 are empirical constants (offset and slope) of the regression model. 
	Note: The slope (B2) represents the amount of change in phase duration with the change in speed of locomotion.</li>
	<li>Use Equations 1 and 2 (Figure 2C) for each limb to calculate asymmetry index (AI). Both equations have the same form of a simple ratio that normalizes the difference of two values to their sum.
	<ol>
		<li>Using Equation 1, calculate the horizontal difference (AIh) that uses the difference between slopes of stance modulation left (l) and right (r) limbs. Similarly, calculate the vertical asymmetry (AIv) using the slopes from front/anterior (a) and back / posterior (p) limbs. The result of applying these two equations is the dataset of 4 x-y points corresponding to 1) forelimb asymmetry, aAIh ; 2) hindlimb asymmetry, pAIh ; 3) left forelimb-hindlimb asymmetry, lAIv ; 4) right forelimb-hindlimb asymmetry, rAIv .</li>
		<li>Plot these values as a patch (Figure 2B) for the visual representation of asymmetry across all limbs.</li>
	</ol>
	</li>
	<li>Calculate diagonality indices (DI) to assess diagonal coupling between parameters of a forelimb and its contralateral hindlimb (Equation 3, Figure 2C).</li>
	<li>Test the DI, as well as the difference of four AIs between conditions of opposing asymmetry (&#916;AI = |AIL9R6 - AIL6R9| ) for statistical significance using a one-way ANOVA with the post-hoc comparison of means analysis19</li>
</ol>

<ol>
	<li>Curzon, P., Zhang, M., Radek, R. J., Fox, G. B. <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 Behavioral Assessment of Sensorimotor Processes in the Mouse: Acoustic Startle, Sensory Gating, Locomotor Activity, Rotarod, and Beam Walking. Methods of Behavior Analysis in Neuroscience.. , (2009).</li><li>Basso, D. M., Beattie, M. S., Bresnahan, 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=A+sensitive+and+reliable+locomotor+rating+scale+for+open+field+testing+in+rats.">A sensitive and reliable locomotor rating scale for open field testing in rats.</a> Journal of Neurotrauma. 12 (1), 1-21 (1995).</li><li>Basso, D. M., Beattie, M. S., Bresnahan, 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=Graded+histological+and+locomotor+outcomes+after+spinal+cord+contusion+using+the+NYU+weight-drop+device+versus+transection.">Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection.</a> Experimental Neurology. 139 (2), 244-256 (1996).</li><li>Li, S., Shi, Z., 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=Assessing+gait+impairment+after+permanent+middle+cerebral+artery+occlusion+in+rats+using+an+automated+computer-aided+control+system.">Assessing gait impairment after permanent middle cerebral artery occlusion in rats using an automated computer-aided control system.</a> Behavioural Brain Research. 250, 174-191 (2013).</li><li>Vandeputte, C., Taymans, J. -. M., 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=Automated+quantitative+gait+analysis+in+animal+models+of+movement+disorders.">Automated quantitative gait analysis in animal models of movement disorders.</a> BMC Neuroscience. 11, 92 (2010).</li><li>Yakovenko, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+10+-+A+hierarchical+perspective+on+rhythm+generation+for+locomotor+control.">Chapter 10 - A hierarchical perspective on rhythm generation for locomotor control.</a> Progress in Brain Research. 188, 151-166 (2011).</li><li>Giszter, S. F., Hockensmith, G., Ramakrishnan, A., Udoekwere, U. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+spinalized+rats+can+walk:+biomechanics,+cortex+and+hindlimb+muscle+scaling+-+implications+for+rehabilitation.">How spinalized rats can walk: biomechanics, cortex and hindlimb muscle scaling - implications for rehabilitation.</a> Annals of the New York Academy of Sciences. 1198, 279-293 (2010).</li><li>Smith, J. L., Edgerton, V. R., Eldred, E., Zernicke, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chronic+spinalized+cat:+a+model+for+neuromuscular+plasticity.">The chronic spinalized cat: a model for neuromuscular plasticity.</a> Birth Defects Original Article Series. 19 (4), 357-373 (1983).</li><li>Yakovenko, S., Drew, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+motor+cortical+contribution+to+the+anticipatory+postural+adjustments+that+precede+reaching+in+the+cat.">A motor cortical contribution to the anticipatory postural adjustments that precede reaching in the cat.</a> Journal of Neurophysiology. 102 (2), 853-874 (2009).</li><li>Yakovenko, S., Krouchev, N., Drew, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+Activation+of+Motor+Cortical+Neurons+Contributes+to+Intralimb+Coordination+During+Reaching+in+the+Cat+by+Modulating+Muscle+Synergies.">Sequential Activation of Motor Cortical Neurons Contributes to Intralimb Coordination During Reaching in the Cat by Modulating Muscle Synergies.</a> Journal of Neurophysiology. 105, 388-409 (2011).</li><li>Pizzi, A., Carlucci, G., Falsini, C., Lunghi, F., Verdesca, S., Grippo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gait+in+hemiplegia:+Evaluation+of+clinical+features+with+the+Wisconsin+Gait+Scale.">Gait in hemiplegia: Evaluation of clinical features with the Wisconsin Gait Scale.</a> Journal of Rehabilitation Medicine. 39 (9), 170-174 (2007).</li><li>Bohannon, R. W., Horton, M. G., Wikholm, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+four+variables+of+walking+to+patients+with+stroke.">Importance of four variables of walking to patients with stroke.</a> International Journal of Rehabilitation Research. 14 (3), 246-250 (1991).</li><li>Richards, C., Malouin, F., Dumas, F., Tardif, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gait+velocity+as+an+outcome+measure+of+locomotor+recovery+after+stroke.">Gait velocity as an outcome measure of locomotor recovery after stroke.</a> Gait Analysis. Theory and Application. , 355-364 (1995).</li><li>Thaut, M. H., McIntosh, G. C., Rice, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhythmic+facilitation+of+gait+training+in+hemiparetic+stroke+rehabilitation.">Rhythmic facilitation of gait training in hemiparetic stroke rehabilitation.</a> Journal of the Neurological Sciences. 151, 207-212 (1997).</li><li>Hsu, A. -. L., Tang, P. -. F., Jan, 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=Analysis+of+impairments+influencing+gait+velocity+and+asymmetry+of+hemiplegic+patients+after+mild+to+moderate+stroke.">Analysis of impairments influencing gait velocity and asymmetry of hemiplegic patients after mild to moderate stroke.</a> Archives of Physical Medicine and Rehabilitation. 84 (8), 1185-1193 (2003).</li><li>Jansen, K., De Groote, F., Duysens, J., Jonkers, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+contributions+to+center+of+mass+acceleration+adapt+to+asymmetric+walking+in+healthy+subjects.">Muscle contributions to center of mass acceleration adapt to asymmetric walking in healthy subjects.</a> Gait &amp; Posture. 38 (4), 739-744 (2013).</li><li>Halbertsma, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stride+cycle+of+the+cat:+the+modelling+of+locomotion+by+computerized+analysis+of+automatic+recordings.">The stride cycle of the cat: the modelling of locomotion by computerized analysis of automatic recordings.</a> Acta physiologica Scandinavica. 521, 1-75 (1983).</li><li>Metz, G. A., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ladder+rung+walking+task:+a+scoring+system+and+its+practical+application.">The ladder rung walking task: a scoring system and its practical application.</a> Journal of Visualized Experiments : JoVE. (28), 4-7 (2009).</li><li>Hogg, R. V., Ledolter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+Statistics.">Engineering Statistics.</a> , (1987).</li><li>Brown, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intrinsic+factors+in+the+act+of+progression+in+the+mammal.">The intrinsic factors in the act of progression in the mammal.</a> Proceedings of the Royal Society of London. Series B, Containing Papers of a Biological Character. 84 (572), 308-319 (1911).</li><li>Kiehn, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locomotor+circuits+in+the+mammalian+spinal+cord.">Locomotor circuits in the mammalian spinal cord.</a> Annual Review of Neuroscience. 29, 279-306 (2006).</li><li>Blitz, D. M., Nusbaum, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State-dependent+presynaptic+inhibition+regulates+central+pattern+generator+feedback+to+descending+inputs.">State-dependent presynaptic inhibition regulates central pattern generator feedback to descending inputs.</a> The Journal of Neuroscience. 28 (38), 9564-9574 (2008).</li><li>Martin, J. H., Ghez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+nucleus+and+motor+cortex:+parallel+motor+systems+for+the+initiation+and+control+of+skilled+movement.">Red nucleus and motor cortex: parallel motor systems for the initiation and control of skilled movement.</a> Behavioural Brain Research. 28 (1-2), 271-223 (1998).</li><li>Drew, T., Jiang, W., Kably, B., Lavoie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+motor+cortex+in+the+control+of+visually+triggered+gait+modifications.">Role of the motor cortex in the control of visually triggered gait modifications.</a> Canadian Journal of Physiology and Pharmacology. 74 (4), 426-442 (1996).</li><li>Drew, T., Andujar, J. -. E., Lajoie, K., Yakovenko, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+mechanisms+involved+in+visuomotor+coordination+during+precision+walking.">Cortical mechanisms involved in visuomotor coordination during precision walking.</a> Brain Research Reviews. 57 (1), 199-211 (2008).</li><li>Longa, E. Z., Weinstein, P. R., Carlson, S., Cummins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+middle+cerebral+artery+occlusion+without+craniectomy+in+rats.">Reversible middle cerebral artery occlusion without craniectomy in rats.</a> Stroke. 20 (1), 84-91 (1989).</li><li>Ulu&#231;, K., Miranpuri, A., Kujoth, G. C., Akt&#252;re, E., Ba&#351;kaya, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+Cerebral+Ischemia+Model+by+Endovascular+Suture+Occlusion+of+the+Middle+Cerebral+Artery+in+the+Rat.">Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat.</a> Journal of Visualized Experiments : JoVE. 48, e1978 (2011).</li><li>Hackney, D. B., Finkelstein, S. D., Hand, C. M., Markowitz, R. S., Black, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmortem+Magnetic+Resonance+Imaging+of+Experimental+Spinal+Cord+Injury+Magnetic+Resonance+Findings+versus+In+Vivo+Functional+Deficit.">Postmortem Magnetic Resonance Imaging of Experimental Spinal Cord Injury Magnetic Resonance Findings versus In Vivo Functional Deficit.</a> Neurosurgery. 35 (6), 1104-1111 (1994).</li><li>Kjaerulff, O., Kiehn, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+Networks+Generating+and+Coordinating+Locomotor+Activity+in+the+Neonatal+Rat+Spinal+Cord+In+Vitro:+A+Lesion+Study.">Distribution of Networks Generating and Coordinating Locomotor Activity in the Neonatal Rat Spinal Cord In Vitro: A Lesion Study.</a> The Journal of Neuroscience. 16 (18), 5777-5794 (1996).</li><li>Liddell, E. G. T., Phillips, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Striatal+and+pyramidal+lesions+in+the+cat.">Striatal and pyramidal lesions in the cat.</a> Brain. 69 (4), 264-279 (1946).</li><li>Beloozerova, I. N., Sirota, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+the+Motor+Cortex+in+the+Control+of+Accuracy+of+Locomotor+Movements+in+the+Cat.">The Role of the Motor Cortex in the Control of Accuracy of Locomotor Movements in the Cat.</a> Journal of Physiology. 461, 1-25 (1993).</li><li>Hill, K. D., Goldie, P. A., Baker, P. A., Greenwood, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retest+reliability+of+the+temporal+and+distance+characteristics+of+hemiplegic+gait+using+a+footswitch+system.">Retest reliability of the temporal and distance characteristics of hemiplegic gait using a footswitch system.</a> Archives of Physical Medicine and Rehabilitation. 75 (5), 577-583 (1994).</li><li>Hillyer, J. E., Joynes, 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=A+new+measure+of+hindlimb+stepping+ability+in+neonatally+spinalized+rats.">A new measure of hindlimb stepping ability in neonatally spinalized rats.</a> Behavioural Brain Research. 202 (2), 291-302 (2009).</li></ol>

The authors have nothing to disclose.

The rationale for this study was to develop a behavioral task that quantitatively assesses the changes in precise control of asymmetric locomotor behaviors. The existence of the spinal CPG has been functionally demonstrated for some time20, but the anatomical and functional characteristics that describe its mechanism as well as its modulatory inputs from descending or sensory feedback pathways have not been characterized until the past decade6,21,22. The current consensus is that the intrinsic spina...

Post-stroke morbidity in the surviving population includes gross motor impairments that pose a challenge for quantitative evaluation in both humans post stroke and animal models of neurologic impairment1. In the clinical setting, these motor impairments are measured using subjective criteria which are more sensitive to severe rather than moderate impairment exhibited by the majority of patients. Similarly, such subjective assessments of post-injury motor behavior in animals are common, e.g., the Basso, Beattie, and Bresnahan (BBB) locomotor scale method2,3. While these subjective evaluation methods are helping translation between gait rehabilitation studies in quadruped animal models and humans, the details of motor deficits associated with activity of separate muscle groups are not assessed. Moreover, the assessment of motor cortical contribution to locomotion, as the putative culprit of motor deficit in cerebrovascular accident, can only be obtained indirectly even using the most novel automated quantitative methods4,5, as they rely on open-field or linear walking tasks. These tasks do not require cortical contribution and can be performed by the neural mechanisms of the spinal cord, i.e., the central pattern generator (CPG) network which is spared in most animal models of neural damage, e.g., spinalized animals6-8. Essential cortical contribution to these spinal mechanisms has been experimentally implicated in tasks that require anticipated postural adjustments9 and reaching10, as well as precise stepping10.
Moreover, most neurological damage is asymmetric; for example, stroke causes hemiparesis, i.e., weakness on one side of the body, which results in an asymmetric gait11-14. The asymmetry of hemiplegic gait is produced by asymmetric spatiotemporal muscle activation most significantly manifested in the shortening of the extensor-associated stance phase and the lengthening of the flexor-associated swing phase of the step cycle on the paretic side15,16. This trend has not yet been explored across a range of locomotor speeds in healthy or paretic animals. In the current study, we employed the analysis of phase duration characteristics17 that describes the relationship between the duration of swing or stance phases as a function of cycle duration in each step. The obtained linear regression model was then further described with an analysis of asymmetry across all limbs.
We report a novel low-cost method for assessing the activity of descending cortical inputs in the motor system of quadruped animals based on a precise stepping locomotor task. This task is designed to challenge the motor cortex by imposing demands on foot placement over a natural range of walking speeds. In addition, foot-placement requirements are manipulated to preferentially challenge the left or right side of the motor system. In a similar locomotor task, Metz &#38; Whishaw (2009) examined the rates of failure, the number of missed steps on irregular rung walkway, in rats. Our method is complimentary to this previous study, and it details the quality of phase control in &#34;successful&#34; steps18.

<table><tbody><tr><td>MATLAB&amp;reg; R2013a</td><td>MathWorks</td><td></td><td>Design platform for custom videoa video annotation software</td></tr><tr><td>Sony HDR-CX380/B High Definition Handycam</td><td>Sony</td><td>27-HDRCX330/B</td><td>Video acquisition device.</td></tr><tr><td>Jif Creamy Peanut Butter - Gluten Free 454 g</td><td>J.M. Smucker Company</td><td></td><td>Food reward stimulus.</td></tr><tr><td>Sucrose Tablet - Chocolate 1800 g</td><td>TestDiet</td><td>1811256</td><td>Food reward stimulus.</td></tr><tr><td>Manzanita Wood Gnawing Sticks</td><td>BioServe</td><td>W0016</td><td>For presentation of food reward stimulus.</td></tr></tbody></table>

asymmetric walkway: a novel behavioral assay for studying asymmetric locomotion

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for quantifying locomotor deficits in rodents is to measure minute disturbances of unconstrained gait overground (e.g., manual BBB score or automated CatWalk). However, cortical inputs are not required for the generation of basic locomotion produced by the spinal central pattern generator (CPG). Thus, unconstrained walking tasks test locomotor deficits due to motor cortical impairment only indirectly. In this study, we propose a novel, precise foot-placement locomotor task that evaluates cortical inputs to the spinal CPG. An instrumented peg-way was used to impose symmetrical and asymmetrical locomotor tasks mimicking lateralized movement deficits. We demonstrate that shifts from equidistant inter-stride lengths of 20% produce changes in the forelimb stance phase characteristics during locomotion with preferred stride length. Furthermore, we propose that the asymmetric walkway allows for measurements of behavioral outcomes produced by cortical control signals. These measures are relevant for the assessment of impairment after cortical damage.

Figure 2 shows the analysis of asymmetry during the locomotor tasks for a single representative subject. The values were calculated for all conditions using Equation 1 and 2 from all subjects individually (Figure 2) and from composite data of 8 female Sprague-Dawley rats (250 - 400 g, Figure 3). Generally, the modulation of the forelimb stance phase was lesser for the side to which the locomotion condition was favored (short ISL), consist...

Watch this Scientific Journal Video about Asymmetric Walkway: A Novel Behavioral Assay for Studying Asymmetric Locomotion at JoVE.com

Asymmetric Walkway: A Novel Behavioral Assay for Studying Asymmetric Locomotion

Here, we present a protocol to quantify precise stepping in rodents. Cortical and the spinal central pattern generator signals are required for precise foot-placement during obstructed locomotion. We report here the novel constrained walking task that directly examines precise stepping behavior.

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for ...

asymmetric-walkway-novel-behavioral-assay-for-studying-asymmetric

Research

JoVE Journal

Behavior

8.8K Views.  West Virginia University School of Medicine. Here, we present a protocol to quantify precise stepping in rodents. Cortical and the spinal central pattern generator signals are required for precise foot-placement during obstructed locomotion. We report here the novel constrained walking task that directly examines precise stepping behavior.

Video: Asymmetric Walkway: A Novel Behavioral Assay for Studying Asymmetric Locomotion

Studying the Neural Basis of Adaptive Locomotor Behavior in Insects

Studying the neural basis of walking behavior, one often faces the problem that it is hard to separate the neuronally produced stepping output from those leg movements that result from passive forces and interactions with other legs through the common contact with the substrate. If we want to understand, which part of a given movement is produced by nervous system motor output, kinematic analysis of stepping movements, therefore, needs to be complemented with electrophysiological recordings of motor activity. The recording of neuronal or muscular activity in a behaving animal is often limited by the electrophysiological equipment which can constrain the animal in its ability to move with as many degrees of freedom as possible. This can either be avoided by using implantable electrodes and then having the animal move on a long tether (i.e. Clarac et al., 1987; Duch &amp; Pfl&uuml;ger, 1995; B&ouml;hm et al., 1997; Gruhn &amp; Rathmayer, 2002) or by transmitting the data using telemetric devices (Kutsch et al, 1993; Fischer et al., 1996; Tsuchida et al. 2004; Hama et al., 2007; Wang et al., 2008). Both of these elegant methods, which are successfully used in larger arthropods, often prove difficult to apply in smaller walking insects which either easily get entangled in the long tether or are hindered by the weight of the telemetric device and its batteries. In addition, in all these cases, it is still impossible to distinguish between the purely neuronal basis of locomotion and the effects exerted by mechanical coupling between the walking legs through the substrate. One solution for this problem is to conduct the experiments in a tethered animal that is free to walk in place and that is locally suspended, for example over a slippery surface, which effectively removes most ground contact mechanics. This has been used to study escape responses (Camhi and Nolen, 1981; Camhi and Levy, 1988), turning (Tryba and Ritzman, 2000a,b; Gruhn et al., 2009a), backward walking (Graham and Epstein, 1985) or changes in velocity (Gruhn et al., 2009b) and it allows the experimenter easily to combine intra- and extracellular physiology with kinematic analyses (Gruhn et al., 2006).
We use a slippery surface setup to investigate the timing of leg muscles in the behaving stick insect with respect to touch-down and lift-off under different behavioral paradigms such as straight forward and curved walking in intact and reduced preparations.

We describe a method to record motor activity, timed to the electrically recorded tarsal contact signal in a tethered insect, walking on a slippery surface. This is used to study the neural basis of adaptive behavior under reduced influence of mechanical interaction between legs through the substrate.

Studying the neural basis of walking behavior, one often faces the problem that it is hard to separate the neuronally produced stepping output from those ...

Neuroscience

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation in mouse models is a non-invasive and reproducible activity assay that is directly analogous to measures of patient ambulation such as the 6-minute walk test and related mobility scores. Many common methods for testing mouse ambulation speed and distance are based on the open field test, where an animal&#39;s free movement within an arena is measured over time. One major downside to this approach is that commercial software and equipment for high-resolution motion tracking is expensive and may require transferring mice to specialized facilities for testing. Here, we describe a low-cost, video-based system for measuring mouse ambulation that utilizes free and open-source software. Using this protocol, we demonstrate that voluntary ambulation in the dystrophin-null mdx mouse model for Duchenne muscular dystrophy (DMD) is decreased relative to wild-type mouse activity. In mdx mice expressing the utrophin transgene, these activity deficits are not observed and the total distance traveled is indistinguishable from wild-type mice. This method is effective for measuring changes in voluntary ambulation associated with dystrophic pathology, and provides a versatile platform that can be readily adapted to diverse research settings.

This protocol describes a flexible, low-cost system for measuring mouse ambulation in an open field activity assay. We show that a 6-minute ambulation assay based on this system detects a decrease in voluntary movement in mdx mice, and accurately distinguishes improvement in a muscle-specific rescue of these animals.

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation ...

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Biology

3D Kinematic Gait Analysis for Preclinical Studies in Rodents

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of complex algorithms and mathematical modeling that accompany 3D data collection and analysis procedures. This work provides a simple, user-friendly, step-by-step detailed methodology for 3D kinematic gait analysis during treadmill locomotion in healthy and neurotraumatic rats using a six-camera motion capture system. Also provided are details on 1) calibration of the system in an experimental set-up customized for quadrupedal locomotion, 2) data collection for treadmill locomotion in adult rats using markers positioned on all four limbs, 3) options available for video tracking and processing, and 4) basic 3D kinematic data generation and visualization and quantification of data using the built-in data collection software. Finally, it is suggested that the utility of this motion capture system be expanded to studying a variety of motor behaviors before and after neurotrauma.

Presented here is a protocol to collect and analyze three-dimensional kinematics of quadrupedal locomotion in rodents for preclinical studies.

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of ...

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. Specifically, gait analysis allows recapturing disease relevant phenotypes that are observed in human patients, especially in neurodegenerative diseases that affect motor abilities such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), and others. In early studies along this line, the measurement of gait parameters was laborious and depended on factors that were hard to control (e.g., running speed, continuous running). The development of ventral plane imaging (VPI) systems made it feasible to perform gait analysis at a large scale, making this method a useful tool for the assessment of motor behavior in rodents. Here, we present an in-depth protocol of how to use kinematic gait analysis to examine the age-dependent progression of motor deficits in mouse models of neurodegeneration; mouse lines with decreased levels of endophilin, in which neurodegenerative damage progressively increases with age, are used as an example.

In this study, we demonstrate the use of kinematic gait analysis based on ventral plane imaging to monitor the subtle changes in motor coordination as well as the progression of neurodegeneration with advancing age in mouse models (e.g., endophilin mutant mouse lines).

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. ...

Developmental Biology

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

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method of gait analysis demonstrated here is a simple but effective measure of gait abnormalities in murine models. Footprints are analyzed by painting a mouse&#8217;s feet with non-toxic washable paint and allowing the subject to walk through a tunnel on a sheet of paper. The design of the testing tunnel takes advantage of natural mouse behavior and their affinity for small dark places. The stride length, stride width, and toe spread of each mouse is easily measured using a ruler and a pencil. This is a well-established and reliable method, and it generates several metrics that are analogous to digital systems. This approach is sensitive enough to detect changes in stride early in phenotype presentation, and due to its non-invasive approach, it allows for testing of groups across life-span or phenotypic presentation.

Footprint analysis is a low-cost alternative to digitized gait analysis programs for researchers quantifying movement abnormalities in mice. Because of its speed, simplicity, and longitudinal potential, it is ideal for behavioral phenotyping of mouse models.

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method ...

Assessing Motor Performance in TH-Related Murine Models Using the Ladder Beam Test

A Versatile, Behavioral Method to Investigate Thyroid Hormone Effects on Cerebellar Function

Thyroid hormone (TH) action is essential during the development of the central nervous system, including the cerebellum. In case of TH deficiency in early life such as congenital hypothyroidism, patients display neurological disorders such as cognitive retardation and motor deficits. There are various studies using mouse models with tissue- or cell-specific TH deficiency to investigate the role of TH in the cerebellum. Compared to generalized congenital hypothyroid mice, cerebellar cell-specific TH-deficient mice display milder and subtler ataxic features, making the assessment of motor function difficult when using conventional tests such as the rotarod test. 
Due to the need for an alternative tool to assess motor function in TH-related animal models, we developed a versatile behavioral method called the "ladder beam test," in which we can design the various ladder tests depending on the severity of ataxia in model mice. We utilized transgenic mice expressing a dominant-negative TH receptor specifically in the cerebellar Purkinje cell, a sole output neuron in the cerebellar cortex modulating motor performance. The newly-built ladder beam test successfully detected robust impairments in motor performance in the transgenic mice at a greater level compared to the rotarod test. Disruption of motor learning was also detected in the ladder beam test but not in the rotarod test. The protocol with this novel behavioral apparatus can be applied to other animal models that may show mild ataxic phenotype to examine subtle changes in cerebellar function.

Author Spotlight: Accurately Assessing Thyroid Hormone-Driven Motor Alterations in Mouse

Here we present a protocol for a versatile behavior test developed recently, the ladder beam test. This test has the advantage of detecting subtle cerebellar ataxia caused by a defect of thyroid hormone action in the central nervous system over the conventional behavior tests assessing motor performance.

Thyroid hormone (TH) action is essential during the development of the central nervous system, including the cerebellum. In case of TH deficiency in early ...

Validating a Murine Model of Angelman Syndrome Using Behavioral Tests

Behavioral Characterization of an Angelman Syndrome Mouse Model

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of AS. We use the rotarod learning paradigm, detailed gait analysis, and nest building test to detect and characterize animal motor impairments. We test animal emotionality in the open field and elevated plus maze tests, as well as the affect in the tail suspension test. When AS mice are tested in the open field test, the results should be interpreted with care, since motor dysfunctions influence mouse behavior in the maze and alter activity scores.
The reproducibility and effectiveness of the presented behavioral tests has already been validated in several independent Uba3a mouse lines with different knockout variants, establishing this set of tests as an excellent validation tool in AS research. Models with the relevant construct and face validity will warrant further investigations to elucidate the pathophysiology of the disease and grant the development of causal treatments.

Author Spotlight: A Battery of Highly Reproducible Behavioral Tests to Validate an Angelman Syndrome Murine Model 

This manuscript presents a set of highly reproducible behavioral tests to validate an Angelman syndrome mouse model.

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...

Quantifying Locomotor Dysfunction in Spinal Cord Injury Mice Using MouseWalker

Using the MouseWalker to Quantify Locomotor Dysfunction in a Mouse Model of Spinal Cord Injury

The execution of complex and highly coordinated motor programs, such as walking and running, is dependent on the rhythmic activation of spinal and supra-spinal circuits. After a thoracic spinal cord injury, communication with upstream circuits is impaired. This, in turn, leads to a loss of coordination, with limited recovery potential. Hence, to better evaluate the degree of recovery after the administration of drugs or therapies, there is a necessity for new, more detailed, and accurate tools to quantify gait, limb coordination, and other fine aspects of locomotor behavior in animal models of spinal cord injury. Several assays have been developed over the years to quantitatively assess free-walking behavior in rodents; however, they usually lack direct measurements related to stepping gait strategies, footprint patterns, and coordination. To address these shortcomings, an updated version of the MouseWalker, which combines a frustrated total internal reflection (fTIR) walkway with tracking and quantification software, is provided. This open-source system has been adapted to extract several graphical outputs and kinematic parameters, and a set of post-quantification tools can be to analyze the output data provided. This manuscript also demonstrates how this method, allied with already established behavioral tests, quantitatively describes locomotor deficits following spinal cord injury.

Author Spotlight: Using the MouseWalker to Quantify Locomotor Dysfunction in a Mouse Model of Spinal Cord Injury  

An experimental pipeline to quantitatively describe the locomotor pattern of freely walking mice using the MouseWalker (MW) toolbox is provided, ranging from initial video recordings and tracking to post-quantification analysis. A spinal cord contusion injury model in mice is employed to demonstrate the usefulness of the MW system.