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 border="0" cellpadding="0" cellspacing="0" width="1007">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:361px;">MATLAB&#174; R2013a</td>
			<td style="width:107px;">MathWorks</td>
			<td style="width:119px;"></td>
			<td style="width:420px;">Design platform for custom videoa video annotation software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sony HDR-CX380/B High Definition Handycam</td>
			<td style="width:107px;">Sony</td>
			<td>27-HDRCX330/B</td>
			<td>Video acquisition device.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Jif Creamy Peanut Butter - Gluten Free 454 g</td>
			<td style="width:107px;">J.M. Smucker Company</td>
			<td>NA</td>
			<td>Food reward stimulus.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:361px;">Sucrose Tablet - Chocolate 1800 g</td>
			<td style="width:107px;">TestDiet</td>
			<td>1811256</td>
			<td>Food reward stimulus.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:361px;">Manzanita Wood Gnawing Sticks</td>
			<td style="width:107px;">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

8.7K 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

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of chronic CNS disorders, there is an increasing demand for murine models with high validity at the behavioral level. However, multiple pathogenic mechanisms and complex functional deficits often impose challenges to reliably measure and interpret behavior of chronically sick mice. Therefore, the assessment of peripheral pathology and a behavioral profile at several time points using a battery of tests are required. Video-tracking, behavioral spectroscopy, and remote acquisition of physiological measures are emerging technologies that allow for comprehensive, accurate, and unbiased behavioral analysis in a home-base-like setting. This report describes a refined phenotyping protocol, which includes a custom-made monitoring apparatus (Integrated Behavioral Station, INBEST) that focuses on prolonged measurements of basic functional outputs, such as spontaneous activity, food/water intake and motivated behavior in a relatively stress-free environment. Technical and conceptual improvements in INBEST design may further promote reproducibility and standardization of behavioral studies.

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station INBEST

Prolonged and comprehensive monitoring of mice in a home-cage environment provides a deeper understanding of aberrant behavior in murine models of brain diseases. This paper describes the Integrated Behavioral Station (INBEST) as the key component of contemporary behavioral analysis.

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

Performing Behavioral Tasks in Subjects with Intracranial Electrodes

Patients having stereo-electroencephalography (SEEG) electrode, subdural grid or depth electrode implants have a multitude of electrodes implanted in different areas of their brain for the localization of their seizure focus and eloquent areas. After implantation, the patient must remain in the hospital until the pathological area of brain is found and possibly resected. During this time, these patients offer a unique opportunity to the research community because any number of behavioral paradigms can be performed to uncover the neural correlates that guide behavior. Here we present a method for recording brain activity from intracranial implants as subjects perform a behavioral task designed to assess decision-making and reward encoding. All electrophysiological data from the intracranial electrodes are recorded during the behavioral task, allowing for the examination of the many brain areas involved in a single function at time scales relevant to behavior. Moreover, and unlike animal studies, human patients can learn a wide variety of behavioral tasks quickly, allowing for the ability to perform more than one task in the same subject or for performing controls. Despite the many advantages of this technique for understanding human brain function, there are also methodological limitations that we discuss, including environmental factors, analgesic effects, time constraints and recordings from diseased tissue. This method may be easily implemented by any institution that performs intracranial assessments; providing the opportunity to directly examine human brain function during behavior.

Patients implanted with intracranial electrodes provide a unique opportunity to record neurological data from multiple areas of the brain while the patient performs behavioral tasks. Here, we present a method of recording from implanted patients that can be reproducible at other institutions with access to this patient population.

Patients having stereo-electroencephalography (SEEG) electrode, subdural grid or depth electrode implants have a multitude of electrodes implanted in ...

Combined Shuttle-Box Training with Electrophysiological Cortex Recording and Stimulation as a Tool to Study Perception and Learning

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the necessary equipment is now available by several commercial companies. This protocol provides a detailed description of a two-way shuttle-box avoidance learning paradigm in rodents (here Mongolian gerbils; Meriones unguiculatus) in combination with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical electrophysiological in vivo recordings. The detailed protocol is applicable to study multiple aspects of learning behavior and perception in different rodent species.
Site-specific ICMS of auditory cortical circuits as conditioned stimuli here is used as a tool to test the perceptual relevance of specific afferent, efferent and intracortical connections. Distinct activation patterns can be evoked by using different stimulation electrode arrays for local, layer-dependent ICMS or distant ICMS sites. Utilizing behavioral signal detection analysis it can be determined which stimulation strategy is most effective for eliciting a behaviorally detectable and salient signal. Further, parallel multichannel-recordings using different electrode designs (surface electrodes, depth electrodes, etc.) allow for investigating neuronal observables over the time course of such learning processes. It will be discussed how changes of the behavioral design can increase the cognitive complexity (e.g. detection, discrimination, reversal learning).

Shuttle-box avoidance learning is well-established in behavioral neuroscience. This protocol describes how shuttle-box learning in rodents can be combined with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical in vivo recordings as a tool to study multiple aspects of learning and perception.

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the ...

Novel Object Exploration as a Potential Assay for Higher Order Repetitive Behaviors in Mice

Restricted, repetitive behaviors (RRBs) are a core feature of autism spectrum disorder (ASD) and disrupt the lives of affected individuals. RRBs are commonly split into lower-order and higher-order components, with lower order RRBs consisting of motor stereotypies and higher order RRBs consisting of perseverative and sequencing behaviors. Higher order RRBs are challenging to model in mice. Current assays for RRBs in mice focus primarily on the lower order components, making basic biomedical research into potential treatments or interventions for higher-order RRBs difficult. Here we describe a new assay, novel object exploration. This assay uses a basic open-field arena with four novel objects placed around the perimeter. The test mouse is allowed to freely explore the arena and the order in which the mouse investigates the novel objects is recorded. From these data, patterned sequences of exploration can be identified, as can the most preferred object for each mouse. The representative data shared here and past results using the novel object exploration assay illustrate that inbred mouse strains do demonstrate different behavior in this assay and that strains with elevated lower order RRBs also show elevated patterned behavior. As such, the novel object exploration assay appears to possess good face validity for higher order RRBs in humans and may be a valuable assay for future studies investigating novel therapeutics for ASD.

Higher order restricted, repetitive behaviors (RRBs) disrupt the lives of affected individuals. These behaviors are challenging to model in rodents, making basic biomedical research into potential treatments or interventions for RRBs difficult. Here we describe novel object exploration as a potential assay for higher order RRBs in mice.

Restricted, repetitive behaviors (RRBs) are a core feature of autism spectrum disorder (ASD) and disrupt the lives of affected individuals.  RRBs are ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

Battery of Behavioral Tests Assessing General Locomotion, Muscular Strength, and Coordination in Mice

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the animal&#39;s wellbeing and health. There are numerous behavioral tests that may be applied. We selected a test for general locomotion, the open field test (OFT); a test for muscular strength, the mesh test (MT); and a test for coordination, the rotarod test (RR). Testing can be accomplished on a weekly or monthly basis. As a test for general locomotion, the OFT works by objectively monitoring movement parameters while the mouse is in an open field apparatus. The field is generally a 2&#39; x 2&#39; box, and the movements are recorded through laser sensing or through video capture. The mouse is placed in the center of the open field and allowed to move freely for the test. The MT uses the latency for a mouse to fall off an inverted screen as a measure of muscular strength. A mouse is placed on a screen, which is inverted over a clear box, and is timed for their latency to fall. Three trials are performed, with the best of the three trials scored for that day. A score of 60 s is the maximum time a mouse is left inverted. Mice are given a 5-min rest period between mesh test trials. Lastly, an accelerated protocol on the RR assesses motor coordination and endurance. During a trial, a mouse walks on a rotating rod as it increases in speed from 4 rpm to 40 rpm over 5 min. The trial ends when the mouse touches the magnetized pressure sensor upon falling. Each mouse undergoes three trials, and the best trial is scored for that day. This combined behavioral data allows for the global assessment of mobility, coordination, strength, and movement of the test animals. At least two out of the three behavioral testing measures must show improvement for an animal to qualify as having overall improved motor function.

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects of a disease or treatment on an animal's wellbeing. In order to globally assess motor functioning, we selected tests for general locomotion, muscular strength, and coordination: the open field test, the mesh test, and the rotarod test, respectively.

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the ...

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer scale. However, current methods mostly rely on tethered cable recordings or only use unidirectional systems, allowing either recording or stimulation of neural activity, but not at the same time or same target. Here, a new wireless, bidirectional device for simultaneous multichannel recording and stimulation of neural activity in freely behaving rats is described. The system operates through a single portable head stage that both transmits recorded activity and can be targeted in real-time for brain stimulation using a telemetry-based multichannel software. The head stage is equipped with a preamplifier and a rechargeable battery, allowing stable long-term recordings or stimulation for up to 1 h. Importantly, the head stage is compact, weighs 12 g (including battery) and thus has minimal impact on the animal&#180;s behavioral repertoire, making the method applicable to a broad set of behavioral tasks. Moreover, the method has the major advantage that the effect of brain stimulation on neural activity and behavior can be measured simultaneously, providing a tool to assess the causal relationships between specific brain activation patterns and behavior. This feature makes the method particularly valuable for the field of deep brain stimulation, allowing precise assessment, monitoring, and adjustment of stimulation parameters during long-term behavioral experiments. The applicability of the system has been validated using the inferior colliculus as a model structure.

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

A wireless, bidirectional system for multi-channel neural recordings and stimulation in freely behaving rats is introduced. The system is light and compact, thus having minimal impact on the animal&#180;s behavioral repertoire. Moreover, this bidirectional system provides a sophisticated tool to assess causal relationships between brain activation patterns and behavior.

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer ...

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often stereotyped actions elicited in response to innately aversive sensory stimuli, but their success requires enough flexibility for adapting to different spatial environments, which can change rapidly. Here, we describe a behavioral assay to evaluate the influence of learned spatial knowledge on defensive behaviors in mice. We have adapted the widely used Barnes maze spatial memory assay to investigate how mice navigate to a shelter during escape responses to innately aversive sensory stimuli in a novel environment, and how they adapt to acute changes in the environment. This new assay is an ethological paradigm that does not require training and exploits the natural exploration patterns and navigation strategies in mice. We propose that the set of protocols described here are a powerful means of studying goal-directed behaviors and stimulus-triggered navigation, which should be of interest to both the fields of instinctive behaviors and spatial memory.

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

Automated, Long-term Behavioral Assay for Cognitive Functions in Multiple Genetic Models of Alzheimer's Disease, Using IntelliCage

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive decline, such as Alzheimer&#39;s disease (AD), have become a promising tool to elucidate the underlying mechanisms and promote the therapeutic advances. An important step is the validation and characterization of expected behavioral abnormality in the models, in the case of AD, cognitive decline. The long-term behavioral investigations of laboratory animals to study the effect of aging demand substantial efforts from researchers. The IntelliCage system is a high-throughput and cost-effective test battery for mice that eliminates the need for daily human handling. Here, we describe how the system is utilized in the long-term phenotyping of a genetic Alzheimer&#39;s disease model, specifically focusing on the cognitive functions. The experiment employs repeated battery of tests that assess spatial learning and executive functions. This cost-effective age-dependent phenotyping allows us to identify the transient and/or permanent effects of genes on various cognitive aspects.

This paper describes a protocol for cognitive assessments for genetic models of the Alzheimer&#39;s disease using the IntelliCage system, which is a high throughput automated behavioral monitoring system with operant conditioning.

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive ...

Asymmetric Walkway: A Novel Behavioral Assay for Studying Asymmetric Locomotion

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Performing Behavioral Tasks in Subjects with Intracranial Electrodes

Combined Shuttle-Box Training with Electrophysiological Cortex Recording and Stimulation as a Tool to Study Perception and Learning

Novel Object Exploration as a Potential Assay for Higher Order Repetitive Behaviors in Mice

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

Battery of Behavioral Tests Assessing General Locomotion, Muscular Strength, and Coordination in Mice

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Automated, Long-term Behavioral Assay for Cognitive Functions in Multiple Genetic Models of Alzheimer's Disease, Using IntelliCage