This research has been funded by NIH-NINDS R03 NS091737.

This protocol describes setting up the task and establishing living and feeding conditions, as well as training animals, testing animals after injury, and analyzing behavior data. The four steps of animal training are described as well: habituation, reward association, counterweight training, and training to criterion turn angle. All animal experiments were approved by the IACUC of Weill Cornell Medicine and University of Texas at Dallas.
1. Setting Up the Task
<ol>
	<li>Design a behavioral box made of clear plastic that measures 150 mm wide by 200 mm long by 250 mm tall. 
	NOTE: Here, the rectangular aperture is 14.2 mm wide by 25.4 mm high. The knob manipulandum of diameter 9.5 mm is made with methacrylate and has two stops that restrict supination to less than 100&#176;. Each of these parameters have been tested and optimized for the deficits described in the results section. The behavioral box and manipulandum are shown in Figure 1A.</li>
	<li>Connect the microcontroller box to the computer. Up to four microcontroller boxes can be connected to each computer.</li>
	<li>To begin the task, use the control software (Figure 1C).
	<ol>
		<li>Select the knob device for control. Input the subject name in the &#34;Subject&#34; field. Choose the stage for training under the &#34;Stage&#34; dropdown menu. 
		NOTE: The stages dictates three parameters for each trial: the &#34;Hit Window,&#34; &#34;Hit Threshold,&#34; and &#34;Initial Threshold.&#34; A hit is defined as reaching the criterion angle within a defined period of time. &#34;Hit Window&#34; refers to the time allotted for an animal to start a trial and reach the criterion angle. &#34;Hit Threshold&#34; is the criterion angle for a successful trial. &#34;Initial Threshold&#34; is the criterion angle to initiate a trial and hit window; this indicates the start of the turn. More information on Stages can be seen below under the section &#34;Training Animals.&#34;</li>
		<li>Click &#34;Start&#34; to begin the training session. Click &#34;Feed&#34; to manually feed the animal and &#34;Stop&#34; to stop the training session. 
		NOTE: Manual feeding should be used to maintain interest in the task and is described in detail in section 3. After stopping the session, session data will be saved to the C: drive.</li>
	</ol>
	</li>
</ol>
2. Living and Feeding Conditions
<ol>
	<li>Use adult female Sprague Dawley rats (175-200 g). House rats together in standard cages with reversed 12 h dark/12 h light cycle. Perform all training and testing during the dark cycle with the lights dimmed. 
	NOTE: Female rats are used since they are easier to train 21. Here Sprague-Dawley were used because it is a common strain used in motor training, and it is the predominant strain for transgenics. However, given the adjustable nature of the task (counterweight, turn angle criterion, distance to knob, and knob itself) these could easily be adjusted for different strains and larger (e.g. males) or smaller (e.g. Wistar) rats.</li>
	<li>For the first 5 days of the study, give rats a full trough in addition to nutritionally complete, chocolate flavored pellets for five days. After the fifth day, food-restrict rats to 85% of their normal weight curve.</li>
	<li>After food restriction has begun, feed the rats 7.5 g of chocolate pellets (adjust up or down to maintain 85% of age-adjusted weight) throughout the entirety of their training. If rats have not received 7.5 g of chocolate pellets in their training sessions, supplement withstandard chow after the final session. 
	NOTE: Weekend feeding is ad libitum with standard chow until Sunday evening, then food-deprive overnight. When performing surgeries or other invasive interventions, allow food ad libitum at least three days before and then for three days after the procedure (depending on recovery times) before putting rats on food restriction again.</li>
</ol>
3. Training Procedure
NOTE: An overview of the training procedure is shown in Figure 3A. Throughout the protocol, train the rats twice daily, once in the morning and once in the afternoon. Wait at least 3 h after the morning session before starting the afternoon session.
<ol>
	<li>Habituation 
	Note: The goal of habituation is to familiarize the rat with the testing box and with handling.
	<ol>
		<li>Retract the knob device fully by pressing the down arrow key on the auto positioner.</li>
		<li>Place the rat in the behavioral box for 15 min.</li>
		<li>After 15 min in the box, handle each rat in the hands for at least five min to familiarize the rat to the experimenter.</li>
		<li>Repeat this for five days.</li>
	</ol>
	</li>
	<li>Reward Association 
	NOTE: The goal is to train the rat to associate turning of the knob with a food reward. Expect to spend the full session length of 30 min on the task during the reward association stage of training.
	<ol>
		<li>Open the software and input the rat Name. Set the Stage to &#34;K1: Knob Shaping - No Pulley.&#34; 
		NOTE: This sets the manipulandum distance away from the aperture at 0.0 cm, the &#34;Init. Thresh.&#34; at 3&#176;, &#34;HIT Thresh.&#34; at 5 degrees, and &#34;HIT Window&#34; at 2 s. This allows the rat to be fed as long as the rat turns the knob past 3 degrees. The &#34;HIT Window&#34; will remain at 2 s throughout training.</li>
		<li>Put the rat in the behavioral box and press &#34;Start&#34; to begin the session.</li>
		<li>Familiarize the rat to the reward trough by dispensing 2 - 3 pellets at a time and tapping the side of the box where the reward trough is located. Dispense the pellets by using the manual feeder button.</li>
		<li>Once the rat knows the location of the reward, manually dispense 1 pellet when the rat is in front of the aperture.</li>
		<li>Once the rat moves to the aperture in anticipation of a pellet reward, condition the rat to interact with the manipulandum.
		<ol>
			<li>Prompt the rat to engage by placing a 45 mg food pellet near the manipulandum, or by applying pellet dust directly to the manipulandum, allowing it to reach out and grasp it. If the rat moves away from the aperture, tap on the box near the manipulandum to redirect its attention. 
			NOTE: If the rat has touched the knob with its reaching paw, the program will feed the rat. Any interactions that do not include the use of paws (i.e. biting, nosing the manipulandum) are incorrect and should not be rewarded.</li>
		</ol>
		</li>
		<li>Once the rat begins touching the knob with the desired paw and getting rewarded 10 consecutive times at 0.0 cm, use the down arrow key on the auto-positioner (Figure 1A) to retract the manipulandum by 0.25 cm. Repeat this in 0.25 cm increments until the manipulandum is 1.25 cm away from the aperture.</li>
		<li>Press &#34;Stop&#34; after 30 min to end the session.</li>
		<li>Continue reward association until the rat supinates the manipulandum with the desired paw at 1.25 cm away from the aperture (Figure 1B).</li>
		<li>Once the rat has completed 2 consecutive sessions supinating the manipulandum and retrieving the pellet reward, begin counterweight training.</li>
	</ol>
	</li>
	<li>Counterweight training to train the rats to supinate a 6 g counterweight.
	<ol>
		<li>Place a 3 g counterweight on the manipulandum by attaching the connector at the end of the counterweight to the L-shaped attachment point on the manipulandum. Feed the counterweight string through the pulley until the counterweight hangs freely.</li>
		<li>Open software and input the rat Name. Set the Stage to &#34;K2: Knob Shaping - Pulley.&#34; 
		NOTE: This sets the manipulandum distance away from the aperture at 1.25 cm, the &#34;Init. Thresh.&#34; at 3&#176;, and the &#34;HIT Thresh.&#34; at 5 degrees. The manipulandum distance will remain at 1.25 cm from this point forward.</li>
		<li>Put the rat in the behavioral box and press &#34;Start&#34; to begin the session.</li>
		<li>Press &#34;Stop&#34; after 100+ successful trials. Once the rat has completed 2 consecutive sessions of 100+ successful trials, increase the weight by 1 g after the end of a session. Increase the weight from 3 g to 6 g, over subsequent sessions.</li>
		<li>Proceed to step 3.4 after 2 consecutive sessions of supination at 6 g and 100+ successful trials. 
		NOTE: It takes an average of 6 sessions (3 days) to work up from 3 g to 6 g. Beginning in counterweight training, it is imperative that correct supination movement gets rewarded and incorrect supination movement gets shaped out. For a visual guide and explanation about correct and incorrect supination form, please refer to Figure 2.</li>
	</ol>
	</li>
	<li>Training to Baseline 
	NOTE: Remember to continue to shape correct supination movement. Again, refer to Figure 2 for a visual guide on correct/incorrect supination movement and how to ameliorate incorrect supination. Train the rats to supinate to the baseline criteria; here, this is 75 degrees with a 6 g counterweight at a success rate of 75% or higher.
	<ol>
		<li>Place a 6 g counterweight on the manipulandum.</li>
		<li>Open software and input the rat Name. Set the Stage to &#34;KSB4: Knob Training Median 75 Max.&#34; 
		NOTE: This sets the &#34;Init. Thresh.&#34; at 5&#176;, &#34;HIT Threshold Minimum&#34; at 15&#176;, and &#34;HIT Threshold Maximum&#34; at 75&#176;. This is called an &#34;adaptive&#34; training stage, meaning the threshold will increase as the rat&#39;s performance improves. For the &#34;KSB4&#34; stage, the threshold is first set to the previous session&#39;s final threshold. If no previous session was run on this stage, the threshold is set to the threshold minimum of 15 degrees. After the first 10 trials of the session, the threshold is calculated as the median of the peak angles in the previous 10 trials. Thus, the threshold is different for each trial and depends on the rat&#39;s performance in the previous trials.</li>
		<li>Stop the session after 30 min.</li>
		<li>Continue training using the adaptive stage until the average peak angle is 75 degrees or greater. Then, proceed to step 3.5. This generally occurs after about 10 days or 20 sessions.</li>
	</ol>
	</li>
	<li>Baseline assessment to record four consecutive baselines
	<ol>
		<li>Place a 6 g counterweight on the manipulandum.</li>
		<li>Open software and input the rat Name. Set the Stage to &#34;K27: 75 degrees.&#34; This sets the &#34;Init. Thresh.&#34; at 5&#176;and &#34;HIT Threshold at 75&#176;.</li>
		<li>Press &#34;Stop&#34; after 30 min or 100 trials, whichever comes first, to end the session.</li>
		<li>Continue until there are four consecutive baselines with a success rate of 75% or higher.</li>
	</ol>
	</li>
</ol>
4. Post-Injury Assessment
<ol>
	<li>Open software and input the rat name. Set the Stage to the same static stage as pre-injury baseline testing. This will retain the same parameters used for baseline.</li>
	<li>Press &#34;Start&#34; and run session until 30 min. 
	NOTE: Participation might be low after injury. This can be remedied by using the manual feed button or placing a reward pellet near the manipulandum to entice the rat to engage with the manipulandum.</li>
	<li>Repeat testing once a week in weekly intervals until the desired length of post-injury assessment has been reached. 
	NOTE: Here, we assess post-injury performance each week for six weeks.</li>
</ol>
5. Analyze Data
NOTE: Data are saved to a default location on the C: Drive of a PC. Data are captured at 100 Hz during the hit window and stored in binary format. Here, the data was analyzed using a custom program, called Dexterity. Please email Dr. Jason Carmel for access to this free software.
<ol>
	<li>Open Dexterity and click &#34;Standard.&#34;</li>
	<li>Locate the directory where rat data is and select the desired folder.</li>
	<li>After selecting the directory, select the rats for analysis.</li>
	<li>Click &#34;Keep&#34; or &#34;Discard&#34; to keep or discard files that are incomplete or contain no data. 
	NOTE: There are times when sessions are started using the wrong parameters and training stage and while they are immediately stopped, a file is still created. This file needs to be discarded during analysis. There are also instances, especially acutely after injury, when an animal performs no trials because of their injury or very little trials. For these sessions, a file must be kept because it is a representation of their performance. When a file with no successful trials is kept, a NaN is placed in the appropriate place for computed variables.</li>
	<li>Choose whether to annotate the experiment now or later; if &#34;Annotate Now&#34; is selected, a new window will open. Type in the &#34;Experiment Name,&#34; &#34;Group Names,&#34; &#34;Event Data,&#34; and &#34;Total Number of Weeks&#34; in the experiment.</li>
	<li>Assign subjects to a group.</li>
	<li>Input the number of data sessions in each week or time point. Separate the input sessions by a space and make sure not to put a space after the last number i.e. (1 1 1 1).</li>
	<li>Input the time point labels. Separate input labels by a space and make sure not to put a space after the last label i.e. (Pre Wk1 Wk2 Wk3).</li>
	<li>Select when the event occurred; an option to save the analysis session will appear. Click &#34;Yes&#34; to save the analysis session.</li>
	<li>An overview of the annotated and unannotated data will appear. This gives the option to plot data.</li>
	<li>For the unannotated data, click &#34;Graph Subject&#34; to plot an individual subject or &#34;Graph Subjects&#34; to plot all subjects on the same graph.</li>
	<li>For the annotated analysis, click &#34;Plot&#34; to plot the experiment.</li>
	<li>For both annotated and unannotated, click the dropdown arrow for a list of variables to be plotted. Click the variable to view the plot.</li>
	<li>Click export to export the graph and/or the data associated with the graph.</li>
</ol>

<ol>
	<li>Anderson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+recovery:+priorities+of+the+spinal+cord-injured+population.">Targeting recovery: priorities of the spinal cord-injured population.</a> J Neurotraum. 21 (10), 1371-1383 (2004).</li><li>Martin, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+neurobiology+of+restorative+neurology+and+future+directions+for+repair+of+the+damaged+motor+systems.">Systems neurobiology of restorative neurology and future directions for repair of the damaged motor systems.</a> Clin Neurol Neurosur. 114 (5), 515-523 (2012).</li><li>Lemon, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descending+pathways+in+motor+control.">Descending pathways in motor control.</a> Annu Rev Neurosci. 31, 195-218 (2008).</li><li>Feng, W., Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corticospinal+tract+lesion+load:+An+imaging+biomarker+for+stroke+motor+outcomes.">Corticospinal tract lesion load: An imaging biomarker for stroke motor outcomes.</a> Ann Neurol. 78 (6), 860-870 (2015).</li><li>Whishaw, I. Q., Pellis, S. M., Gorny, B. P., Pellis, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impairments+in+reaching+and+the+movements+of+compensation+in+rats+with+motor+cortex+lesions:+an+endpoint,+videorecording,+and+movement+notation+analysis.">The impairments in reaching and the movements of compensation in rats with motor cortex lesions: an endpoint, videorecording, and movement notation analysis.</a> Behav Brain Res. 42 (1), 77-91 (1991).</li><li>Irvine, K. A., Ferguson, A. R., Mitchell, K. D., Beattie, S. B., 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+novel+method+for+assessing+proximal+and+distal+forelimb+function+in+the+rat:+the+Irvine,+Beatties+and+Bresnahan+(IBB)+forelimb+scale.">A novel method for assessing proximal and distal forelimb function in the rat: the Irvine, Beatties and Bresnahan (IBB) forelimb scale.</a> J Vis Exp. (46), (2010).</li><li>Allred, R. P., Adkins, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vermicelli+handling+test:+a+simple+quantitative+measure+of+dexterous+forepaw+function+in+rats.">The vermicelli handling test: a simple quantitative measure of dexterous forepaw function in rats.</a> J Neurosci Meth. 170 (2), 229-244 (2008).</li><li>Sindhurakar, A., Butensky, S. D., 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=An+automated+test+of+rat+forelimb+supination+quantifies+motor+function+loss+and+recovery+after+corticospinal+injury.">An automated test of rat forelimb supination quantifies motor function loss and recovery after corticospinal injury.</a> Neurorehab Neural Re. 31 (2), 122-132 (2017).</li><li>Carmel, J. B., Kim, S., Brus-Ramer, M., Martin, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feed-forward+control+of+preshaping+in+the+rat+is+mediated+by+the+corticospinal+tract.">Feed-forward control of preshaping in the rat is mediated by the corticospinal tract.</a> Eur J Neurosci. 32 (10), 1678-1685 (2010).</li><li>Meyers, E., Sindhurakar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+supination+assessment+task:+An+automated+method+for+quantifying+forelimb+rotational+function+in+rats.">The supination assessment task: An automated method for quantifying forelimb rotational function in rats.</a> J Neurosci Meth. 266, 11-20 (2016).</li><li>Gomez-Marin, A., Paton, J. J., Kampff, A. R., Costa, R. M., Mainen, Z. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Big+behavioral+data:+psychology,+ethology+and+the+foundations+of+neuroscience.">Big behavioral data: psychology, ethology and the foundations of neuroscience.</a> Nat Neurosci. 17 (11), 1455-1462 (2014).</li><li>Wong, C. C., Ramanathan, D. S., Gulati, T., Won, S. J., Ganguly, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+behavioral+box+to+assess+forelimb+function+in+rats.">An automated behavioral box to assess forelimb function in rats.</a> J Neurosci Meth. 246, 30-37 (2015).</li><li>Alaverdashvili, M., 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=Motor+cortex+stroke+impairs+individual+digit+movement+in+skilled+reaching+by+the+rat.">Motor cortex stroke impairs individual digit movement in skilled reaching by the rat.</a> Eur J Neurosci. 28 (2), 311-322 (2008).</li><li>Ellens, D. J., Gaidica, 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=An+automated+rat+single+pellet+reaching+system+with+high-speed+video+capture.">An automated rat single pellet reaching system with high-speed video capture.</a> J Neurosci Meth. 271, 119-127 (2016).</li><li>Lai, S., Panarese, A., 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=Quantitative+kinematic+characterization+of+reaching+impairments+in+mice+after+a+stroke.">Quantitative kinematic characterization of reaching impairments in mice after a stroke.</a> Neurorehab Neural Re. 29 (4), 382-392 (2015).</li><li>Molina-Luna, K., Hertler, B., Buitrago, M. M., Luft, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+learning+transiently+changes+cortical+somatotopy.">Motor learning transiently changes cortical somatotopy.</a> Neuroimage. 40 (4), 1748-1754 (2008).</li><li>Ballermann, M., Tompkins, G., 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=Skilled+forelimb+reaching+for+pasta+guided+by+tactile+input+in+the+rat+as+measured+by+accuracy,+spatial+adjustments,+and+force.">Skilled forelimb reaching for pasta guided by tactile input in the rat as measured by accuracy, spatial adjustments, and force.</a> Behav Brain Res. 109 (1), 49-57 (2000).</li><li>Vigaru, B. C., Lambercy, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robotic+platform+to+assess,+guide+and+perturb+rat+forelimb+movements.">A robotic platform to assess, guide and perturb rat forelimb movements.</a> IEEE Trans Neural Syst Rehabil Eng. 21 (5), 796-805 (2013).</li><li>Martin, J. H., Choy, M., Pullman, S., Meng, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corticospinal+system+development+depends+on+motor+experience.">Corticospinal system development depends on motor experience.</a> J Neurosci. 24 (9), 2122-2132 (2004).</li><li>Murgia, A., Kyberd, P. J., Chappell, P. H., Light, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marker+placement+to+describe+the+wrist+movements+during+activities+of+daily+living+in+cyclical+tasks.">Marker placement to describe the wrist movements during activities of daily living in cyclical tasks.</a> Clin Biomech. 19 (3), 248-254 (2004).</li><li>Simpson, J., Kelly, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+of+whether+there+are+sex+differences+in+certain+behavioural+and+neurochemical+parameters+in+the+rat.">An investigation of whether there are sex differences in certain behavioural and neurochemical parameters in the rat.</a> Behav Brain Res. 229 (1), 289-300 (2012).</li><li>Hays, S. A., Khodaparast, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isometric+pull+task:+a+novel+automated+method+for+quantifying+forelimb+force+generation+in+rats.">The isometric pull task: a novel automated method for quantifying forelimb force generation in rats.</a> J Neurosci Meth. 212 (2), 329-337 (2013).</li></ol>

Dr. Rennaker and Dr. Sloan are the owners of Vulintus Inc., which manufactures the equipment in this publication. No conflicts of interest declared for the other authors.

The knob supination task evaluates forelimb supination in rats using quantitative and semi-automated methods. To achieve these endpoints, many of the parameters designed for the task, including knob alignment, manipulandum design, and training criteria, have been iterated over several years. For knob alignment, we experimented with three different alignments of the knob with regards to the aperture: left side of the knob aligned with the left side of the aperture, the knob centered in aperture, and the right side of the knob aligned with the right side of the aperture. We settled on the right side of the knob being aligned with the right side of the aperture, as this produced rats that were trained in the shortest amount of time and who supinated with minimal compensatory mechanisms, specifically, interference from the left paw.
As for manipulandum design, we altered several design features to maximize turning with the forelimb and minimize use of the body. In addition, we scaled the difficulty of the task to the severity of the predicted deficit. After pyramidotomy, supination is the movement that is most strongly affected, but the impairment is still relatively subtle. Thus, we trained the rats to a higher baseline criterion (75&#176;) to ensure that large deficits were observed after injury. For cortical lesions, which are more impairing, 60-degree threshold at 7.5 g was sufficient to demonstrate a significant deficit after injury. Additional parameters that were optimized through a trial-and-error approach include aperture size, knob distance from aperture, and time window to achieve a successful trial.
There are some critical points throughout the training protocol that require careful supervision. When training to baseline, the adaptive thresholding method has been used successfully to train rats to 75&#176; 10. However, rats can plateau at a peak angle less than 75&#176;; the performance remains the same after 4-5 sessions. To improve performance, a static threshold can be employed. A static threshold refers to the threshold remaining at a set degree, which is independent of rat performance, as opposed to an adaptive threshold that changes based on recent performance. If the rat plateaus during adaptive training, the experimenter should change to a static threshold. Static training stages range from 20 to 70 degrees in 10&#176; increments. (Stage K28 - K33). Choose the static stage based on the rat&#39;s average peak angle in the previous 2 sessions. For example, if the rat is averaging 45&#176;, select the static stage for 50 degrees (K31). All static stages set the &#34;Init. Thresh.&#34; at 5&#176;. During training, if the rat loses motivation, manually feed the rat if it supinates close to but not above the threshold.
In addition, during baseline assessment, approximately 5% of rats regress 5 - 10&#176; in their supination angle and 5 - 10% in success rate between sessions. If this happens, and the rat does not recover the 75&#176; average peak angle after 3 - 4 sessions, decrease the static stage to within 10 degrees of the rat&#39;s current average angle before returning to step 3.5. It is important to not reintroduce a rat to the adaptive stages once it has been placed on static training stages.
There are some limitations to the task. Once incorrect grasp position has been established, changing grasping behavior (Figure 2) can be difficult. Thus, early detection and correction is important. To correct a rat&#39;s grasp, the aperture can be altered by narrowing the size of the aperture in the horizontal and/or vertical direction; typically we tape a glass slide to the edge of the aperture that needs adjustment. For most rats, this improves their grasp form because it forces them to grasp the manipulandum in a specific manner. This, in turn, improves their ability to properly supinate.
In addition to this challenge, rats can develop compensatory mechanisms to supinate. These include the use of the head to aid the forelimb in supination; lowering the elbow and shoulder joint to turn the knob; using the left paw to help turn the knob or push the reaching paw down. All of these behaviors can be used to successfully complete the task. As mentioned above, behaviors relating to the grasp can be corrected by manipulating the aperture. Compensatory mechanisms outside of the grasp, however, require active participation by the experimenter to not reward compensatory behavior. After injury, we have observed rats taking several trials to place the paw in the proper position before supinating. Although we have not analyzed which components of the task might contribute to the loss of supination, these could include loss of accurate grip and impaired force modulation, among many possibilities.
The semi-automated supination task takes, on average, 20 &#177; 5 days to train rats to baseline, and 25% of animals are unable to be trained on the task. Contributing to the training time is the fact that we have not selected naturally right-preference rats but instead force all animals to use their right paws as is common in most reaching assays. We have not tried using left-preference rats, but it would be an interesting exploratory study to first identify paw preference and then train the dominant paw. To accommodate this, we would need to flip the orientation of the doors so that the aperture is reversed; this can easily be done.
Compared with traditional tasks like the IBB or single pellet reaching, the supination task quantitatively and objectively measures forelimb reaching. It shows sensitivity to severe injury (cortical lesion) and subtle injury (pyramidotomy), and the training procedure can be modified depending on the severity of the injury model. Because it is semi-automated, the task allows the experimenter to train multiple rats simultaneously, depending on training stage. This greatly improves the experimenter&#39;s productivity and rat throughput. The task is reliable and reproducible between rats. By creating a troubleshooting guide (Figure 2) for experimenters to refer to during the training protocol, we have standardized several incorrect behaviors as well as solutions to fix them. Lastly, the task offers an intuitive means to analyze large amounts of data and gives the experimenter the ability to delve deeper into the kinetics of supination.
In the future, we will be using the semi-automated supination task as a platform to evaluate type, dose, and timing of rehabilitation. Our lab is interested in the effects of stimulation on functional improvement after injury. In addition, we are interested in how therapies which stimulate neural repair or improve neural conduction and communication can affect rehabilitation. We also have interest in modifying the task to be compatible with electrophysiology so that we can study motor learning; rats with head caps routinely perform the task, and adding a commutator for recording or stimulation would be simple to do. The task, as described, is for rats, but there are also labs experimenting with using mice for the task. In general, this task can be used for evaluating forelimb function in rodents in a wide variety of injury models and disease states and in turn, for evaluating rehabilitative strategies. Moving forward, we will continue to improve the task, with refinements to help lessen incorrect behaviors and improve task acquisition rate and training time.

A loss of dexterity after nervous system injury or disease significantly diminishes independence and quality of life for affected individuals 1,2,3,4. Thus, dexterity is an important outcome measure for understanding the science of neural repair and rehabilitation as well the foundations of neural control of movement and motor learning. Traditionally, manual tasks like single pellet reaching, pasta manipulation, and Irvine, Beatties, and Bresnahan (IBB) Forelimb Scale have been used to evaluate dexterity in animals, specifically rodents 5,6,7. These tasks have become popularized due to their minimal task acquisition time. However, they are qualitative in nature, laborious for the experimenter, and, at times, insensitive to functional impairment after injury with subtle deficits 5,7,8,9. These limitations of traditional tasks have spurred the development of more quantitative measures of motor function in animals, specifically, forelimb reaching.
There are several advantages to automating tasks, namely objectivity, increased throughput, and decreased analysis time. New automated tasks provide a more sensitive measure of evaluating dexterity after injury than conventional tasks 8,10. In addition, they allow for adaptive training and testing which tailors the training and testing difficulty to an animal&#39;s performance. Lastly, automated tasks generate large amounts of data, which provide two advantages. Firstly, an increase in data both within a trial and in the number of trials increases the statistical power of a study. Secondly, it gives neuroscientists a larger data set from which to study motor learning, training, and compensation more robustly through analysis of kinetic and kinematic information 11.
Several groups have attempted to automate traditional tasks. High speed cameras can be used to gather kinematic data from tasks like the single pellet reaching task 12. Alaverdashvili and Wishaw have used high-speed cameras to capture reaching movements and analyze digit movements using frame-by-frame motion measurement software Peak Motus 13. However, this software does not identify digits using computer vision, but instead requires the experimenter to digitize moving points by cursor. Additionally, some tasks have been used in conjunction with feeders and cages to automate the training process 14,15,16.
Other groups have used force sensors as well as high speed cameras to evaluate spatial adjustments and force in skilled forelimb reaching using pasta manipulation, while others have designed tasks to capture more complex movements 17. One such task is a reach and pull task that uses a three-degree-of-freedom robotic device to capture planar and rotational movement of rat forelimb movements 18. This has advantages in being able to measure the kinetics of movements but with an increase in complexity and cost.
Here, we demonstrate a semi-automated forelimb task that measures supination in rats 8. Forelimb supination is the rotation of the paw from palm down to palm up. Supination is both an excellent marker of corticospinal tract function and a clinically relevant movement in humans that is required for daily living activities 8,19,20. In addition, supination is highly sensitive to injury and inactivation, especially when compared to single pellet reaching 8. The supination task, developed in a collaboration between the Burke Medical Research Institute and The University of Texas at Dallas, measures rotational movement in the horizontal plane 8,10. Rats are placed in a behavioral box (Figure 1A) and are trained to do three movements (Figure 1B): reach through a rectangular aperture; grasp a spherical manipulandum; supinate to a designated angle.
The behavior task is controlled by PC software (Figure 1C). The controlling software sends instructions to a microcontroller that&#39;s connected to the auto-positioner, optical encoder, speaker, and feeder. The microcontroller and its peripheral connections are referred to as the microcontroller box. Information flows from optical encoder, to the microcontroller, then the computer, and then back to the microcontroller. If the controlling software has signaled to the microcontroller that the trial was a success, the microcontroller triggers the feeder to dispense a pellet. At the beginning of each session, the controlling software relays the stage information to the microcontroller, which directs the auto-positioner to position the knob at the stage&#39;s defined distance from the aperture. The auto-positioner can also be operated manually by using the arrow keys located on the auto-positioner. The optical encoder records data at 100 Hz and measures changes in angle. All the data are stored in binary format.
The experimenter uses sequential training stages within the software to train the rat from habituation to supinating at a predetermined angle and success rate. During habituation, the knob manipulandum is placed inside the aperture window without any counterweight. After a week of highly supervised training, the rat associates the knob with a reward and begins turning the knob independently. Once the rat is able to turn independently, the knob is retracted to 1.25 cm in 0.25 cm increments until the rat can turn independently at 1.25 cm. Counterweight is then added in 1 g increments from 3 g to 6 g. Automated training stages train the animal to supinate the knob at 6 g up to 75 degrees. This stage of training is largely unsupervised; once rats adopt the task with proper form (discussed below), they continue to supinate properly. Training is complete when rats supinate 75 degrees at a success rate (hit rate) of 75% 8. Here, we describe a typical training protocol and present solutions to common issues we have encountered. We demonstrate the progression of representative successful and unsuccessful rats through the training protocol, and show that the task can be modified to show functional impairment with subtle or more severe deficits.

<table border="0" cellpadding="0" cellspacing="0" width="946">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Base Cage - Rat Model</td>
			<td style="width:167px;">Vulintus</td>
			<td style="width:147px;">MotoTrak Rat System</td>
			<td style="width:416px;">N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Controller</td>
			<td style="width:167px;">Vulintus</td>
			<td>MotoTrak Rat System</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Behavior Module</td>
			<td style="width:167px;">Vulintus</td>
			<td>MotoTrak Rat System</td>
			<td>Supination Task, Methacrylate Dual Stop Knobs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pellet Dispenser - 45mg</td>
			<td style="width:167px;">Vulintus</td>
			<td>MotoTrak Rat System</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Autopositioner</td>
			<td style="width:167px;">Vulintus</td>
			<td>MotoTrak Rat System</td>
			<td>N/A</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">45 mg, Chocolate Flavor, 50,000/Box</td>
			<td style="width:167px;">Bio-Serv</td>
			<td style="width:147px;">F0299</td>
			<td>N/A</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HP Z230 Tower WorkStation</td>
			<td style="width:167px;">HP</td>
			<td style="width:147px;">N/A</td>
			<td style="width:416px;">Intel Xeon CPU E3-1225 v3 @ 3.20 GHz, 16GB RAM, 1TB HDD. Min Requirements: 8GB RAM, Multi-Core Processor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dexterity</td>
			<td style="width:167px;">Burke Medical Research Institute</td>
			<td style="width:147px;"></td>
			<td>Matlab software for data analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Enviropak</td>
			<td style="width:167px;">WF Fisher and Son</td>
			<td style="width:147px;">N/A</td>
			<td>N/A</td>
		</tr>
	</tbody>
</table>

the knob supination task: a semi-automated method for assessing forelimb function in rats

Tasks that accurately measure dexterity in animal models are critical to understand hand function. Current rat behavioral tasks that measure dexterity largely use video analysis of reaching or food manipulation. While these tasks are easy to implement and are robust across disease models, they are subjective and laborious for the experimenter. Automating traditional tasks or creating new automated tasks can make the tasks more efficient, objective, and quantitative. Since rats are less dexterous than primates, central nervous system (CNS) injury produces more subtle deficits in dexterity, however, supination is highly affected in rodents and crucial to hand function in primates. Therefore, we designed a semi-automated task that measures forelimb supination in rats. Rats are trained to reach and grasp a knob-shaped manipulandum and turn the manipulandum in supination to receive a reward. Rats can acquire the skill within 20 &plusmn; 5 days. While the early part of training is highly supervised, much of the training is done without direct supervision. The task reliably and reproducibly captures subtle deficits after injury and shows functional recovery that accurately reflects clinical recovery curves. Analysis of data is performed by specialized software through a graphical user interface that is designed to be intuitive. We also give solutions to common problems encountered during training, and show that minor corrections to behavior early in training produce reliable acquisition of supination. Thus, the knob supination task provides efficient and quantitative evaluation of a critical movement for dexterity in rats.

Early in training, the experimenter spends more time on task shaping rat behavior. As the rats associate rat supination with reward, the hands on time decreases (Figure 3A). During habituation, reward association, and counterweight training, the full session length (30 min) is spent on the task. However, after a rat is supinating with a 6 g weight, the time on task gradually lessens to about 15 min as the rat&#39;s supination angle increases. Finally, when the rat reaches baseline, the time on task is at a minimum; the experimenter only needs to place the rat in the behavioral box and start the program. The maximum number of rats an experimenter can work with simultaneously is two rats during reward association, four rats during counterweight training and training to baseline, and as many rats as there are boxes during baseline assessment and post-injury testing. On average, 75% of rats (n = 56) acquire the task.
After the rat has associated supination with a reward, there is a positive progression in the rat&#39;s supination angle (Figure 3B). In Figure 3B, the rat progressed from 3 g to 6 g counterweight from day 3 to day 7. After counterweight training, there was a short period of adaptive training from day 7 to day 9, during which supination increased from 26 to 30 degrees. Because there was not much change, a static threshold was employed from day 9 to 18. During this period, the rat steadily increased from 30 degrees to 75 degrees in 8 days. There is day-to-day variability throughout training, in particular, days 12 and 14. But generally, there is an upward trend in supination angle. By the end of day 17 post-habituation, the rat had recorded its first baseline, and four sessions later, it finished baseline assessment. From habituation to recording of a fourth baseline, the training protocol takes an average of 20 &#177; 5 days.
While viewing an ideal progression through the training protocol is important, viewing an unsuccessful progression is equally important (Figure 4). In Figure 4A, the orange line shows a rat that successfully completes the protocol, the blue line shows an unsuccessful rat, and grey lines show another six successful rats. Successful rats reached baseline in 15 &#177; 0.6 days (n = 7). The representative successful rat uses a 1 o&#39;clock grasp, while the unsuccessful rat uses a 3 o&#39;clock grasp. Both rats associate the knob with a reward in 2 days. In addition, both rats show a similar supination angle (Figure 4A) progression in the first four days after the counterweight is added. However, after this point, the successful rat begins to break away from the unsuccessful rat. This is because the unsuccessful rat&#39;s grasp was unable to be corrected before this point (see Figure 2).
For the successful rat, there is a steep rise in supination angle that begins to plateau between 50 and 60 degrees but then resumes a steadier climb towards 75 degrees. However, for the unsuccessful rat, there is a more gradual increase in supination angle. As the rat plateaus around 20 degrees, the rat gets pushed to supinate more, but eventually, it loses interest in the task, even with manual feeding, and the supination angle decreases rapidly around Day 15 post-habituation. While there is a slight recovery after Day 17 post-habituation, the rat struggles to supinate more than 25 degrees. If a rat hasn&#39;t achieved baseline by day 20, we consider this rat unsuccessful and remove the rat from the study.
In addition to supination angle, one can perform a visual inspection of the supination waveforms (Figure 4B-D) for the successful and unsuccessful rat. When performing a visual inspection, we look for several waveform characteristics: slope of the line, latency, and number of peaks in the time window for the trial. The slope of the line is calculated as the derivative of the curve between the onset of the curve and the peak of the curve. The latency is calculated as the time between the initiation of the trial and the curve crossing the hit threshold. Lastly, peaks are calculated by using the derivative to find local maxima in the trial window. Previously, we found that slope of the line, or velocity, is a robust measure of supination kinetics and is sensitive to subtle deficit 8.
In the first third of training after beginning to supinate using 6 g (Figure 4B), the successful rat (Figure 4B1) shows a single waveform with a peak near 20 degrees, while the unsuccessful rat (Figure 4B2) shows a double turn, or two peaks, with the first peak near 10 degrees and the second peak near 5 degrees. In the middle third of training (Figure 4C), the successful rat (Figure 4C1) shows an increase in peak angle from 20 degrees to 50 degrees with a more defined, single peak curve. The unsuccessful rat (Figure 4C2), meanwhile, only shows a marginal increase in peak angle to 20 degrees but has improved in its form; it now only uses a single turn. By the final third of training (Figure 4D), the successful rat (Figure 4D1) shows a very pronounced single waveform with a peak around 65&#176;, versus the unsuccessful rat (Figure 4D2) with a peak angle of 20 degrees but now with an additional peak at 2 s of 15&#176;. This is another good indicator that with increasing training difficulty, the rat was unable to correct its 3 o&#39;clock grasp, and in turn, unable to supinate properly. Even if this rat wasn&#39;t excluded from the study and could eventually perform up to 75 degrees, questions would remain about whether it was true supination versus supination with compensation.
Lastly, the supination task detects functional impairment after multiple types of injury, including a cut lesion of the corticospinal tract, the main pathway for voluntary movement in people, and forelimb motor cortex lesion performed with endothelin injections (Figure 5) 8,10,22. Rats in the pyramidotomy group (purple, n = 8) were trained to supinate at least 75&#176; at 6 g at a success rate of 75% or above, while rats in the cortical lesion group (green, n = 10) were trained to supinate 60&#176; at 7.5 g at 75% or above. Rats in both groups showed a sharp decrease in success rate after injury (Figure 5A). Success rate for rats in the pyramidotomy group decreased from 90% &#177; 2% to 14% &#177; 8%. The success rate for rats with cortical lesion decreased from 76% &#177; 1% to 10% &#177; 3%. By week 6, both groups were still impaired: the pyramidotomy group was at 34% &#177; 11% while the cortical lesion group remained at 16% &#177; 7%. As for supination angle, both groups show a decrease from pre- to post-injury (Figure 5B). Due to the different criterion baseline supination angles, the pyramidotomy group had a higher pre-injury supination angle (85&#176; &#177; 2.9&#176;) than the cortical lesion group (67&#176; &#177; 0.52&#176;). The pyramidotomy group decreased to 38&#176; &#177; 10&#176; while the cortical lesion group decreased to 27&#176; &#177; 2.9&#176;.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56341/56341fig1.jpg" /> 
Figure 1: Supination Task Description. (A) The rat is placed in a Plexiglas box with an aperture through which it reaches and grasps a knob that must be turned in supination. The knob has two stops to prevent supination angles greater than 100&#176;. The knob also has a pulley with counterweight; this creates torque that the rat must overcome to supinate. The knob is connected to an optical encoder that measures angle with an accuracy of 0.25&#176;. This optical encoder is connected to a microcontroller, which in turn is connected to a computer which controls the task. The computer signals to the microcontroller when to trigger audio-feedback and dispense a pellet from the feeder if a success criterion is achieved. The microcontroller also controls the auto positioner whose position between 0 and 1.25 cm is dictated by the training stage set by the computer. (B) The rat performs the task in three successive movements: reaching through the aperture, grasping the knob with a power grasp located at 1 o&#39;clock, and supinating. (C) The knob supination task is controlled by control software. The experimenter inputs the subject&#39;s name and chooses the training stage, while the program sets the corresponding parameters. A waveform of a single successful supination trial is shown in blue, while the sequence of successful and failed trials are shown in green and red, respectively. A trial is marked successful by the control software if the supination angle is greater than the hit threshold within the defined time window, whereas a trial is marked unsuccessful if it does not. This program controls one box. Four programs can be run simultaneously per computer. This figure has been modified from Sindhurakar et al., 2017, Neurorehabilitation and Neural Repair8. <a href="https://cloudflare.jove.com/files/ftp_upload/56341/56341fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56341/56341fig2.jpg" /> 
Figure 2: Supination Movements. Diagrams and descriptions of common correct and incorrect supination movements encountered during the training protocol. Correct movements allow for true supination, while incorrect movements include compensatory mechanisms that may prevent true supination. Included are suggested solutions to correct improper movements. <a href="https://cloudflare.jove.com/files/ftp_upload/56341/56341fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56341/56341fig3.jpg" /> 
Figure 3: Training protocol. (A) Standard timeline. There are five periods of training lasting approximately 25 days in total: Habituation (5 d), Reward Association (1 - 3 d), Weight Training (3 - 4 d), Training to Baseline (8 - 12 d), and Baseline Assessment (2 - 4 d). The line pattern on the timeline designates the time required of the experimenter to spend on the task each session. As the training protocol proceeds, the time on task decreases. (B) General progression of a rat&#39;s ability to supinate from reward association to baseline assessment. Overall, there is a positive linear progression of the rat towards baseline, but as observed, there is variability in a rat&#39;s performance throughout the training protocol. After weight training, there is a period of adaptive training, where the supination angle threshold is changed to match the rat&#39;s performance. This adaptive training is followed by a static thresholding paradigm until the rat has achieved baseline. <a href="https://cloudflare.jove.com/files/ftp_upload/56341/56341fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56341/56341fig4.jpg" /> 
Figure 4: Successful and Unsuccessful Task Acquisition. (A) Progression of supination angle throughout training protocol for eight rats, seven successful and one unsuccessful. One representative rat that reaches baseline criterion (successful, orange) and one unsuccessful rat (blue) are further used as case studies. In the first seven days of the training after habituation, both the successful and unsuccessful rat showed similar progress in supination angle. By Day 11 post-habituation, the successful rat was supinating 55&#176; while the unsuccessful rat supinated 25&#176;. After Day 15 post-habituation, the successful rat showed a strong upward progression, while the unsuccessful rat declined in performance. In the final third of training post-habituation, the unsuccessful rat had plateaued at 30&#176; while the successful rat was supinating 80&#176;. (B) Average waveform (black line) with a 95% confidence interval (orange for successful, blue for unsuccessful) for the first third of training after 6 g of counterweight is added. (B1) Successful rat - single peak around 20&#176;. (B2) Unsuccessful rat - double peak with global maximum of 10&#176;. (C) Average waveform (black line) with a 95% confidence interval (orange for successful, blue for unsuccessful) for the second third of training after 6 g of counterweight is added. (C1) Successful rat - single peak at 45&#176;. (C2) Unsuccessful rat - improved form with single peak near 20&#176;. (D) Average waveform (black line) with a 95% confidence interval (orange for successful, blue for unsuccessful) for the final third of training after 6 g of counterweight is added. (D1) Successful rat - pronounced single peak at 65&#176;. (D2) Unsuccessful rat - double peak with global maximum at 20&#176;. <a href="https://cloudflare.jove.com/files/ftp_upload/56341/56341fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56341/56341fig5.jpg" /> 
Figure 5: Task Sensitivity to Different Injury Models. Rats in the pyramidotomy group (purple, n = 8) were trained to supinate 75&#176; at 6 g at a success rate of 75% or above, while rats in the cortical lesion group (green, n = 10) were trained to supinate 60&#176; at 6 g at 75% or above. Data shown are mean &#177; standard error. (A) Success rate for pyramidotomy lesion versus cortical lesion. Both injury models showed a sharp decrease in success rate from pre- to post-injury (week 1). Success rate for pyramidotomy decreased from 0.90 &#177; 0.02 to 0.14 &#177; 0.08, while success rate for cortical lesion decreased from 0.76 &#177; 0.01. (B) Supination angle for pyramidotomy versus cortical lesion. Both groups showed a decrease from pre- to post-injury: the pyramidotomy group decreased to 38.2&#176; &#177; 10.1&#176; while the cortical lesion group decreased to 27.1&#176; &#177; 2.9&#176;. <a href="https://cloudflare.jove.com/files/ftp_upload/56341/56341fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about The Knob Supination Task: A Semi-automated Method for Assessing Forelimb Function in Rats at JoVE.com

The Knob Supination Task: A Semi-automated Method for Assessing Forelimb Function in Rats

This manuscript describes a semi-automated task that quantifies supination in rats. Rats reach, grasp, and supinate a spherical manipulandum. The rat is rewarded with a pellet if the turn angle exceeds a criterion set by the user. This task increases throughput, sensitivity to injury, and objectivity compared to traditional tasks.

Tasks that accurately measure dexterity in animal models are critical to understand hand function. Current rat behavioral tasks that measure dexterity ...

the-knob-supination-task-semi-automated-method-for-assessing-forelimb

Research

JoVE Journal

Behavior

9.2K Views.  Burke Medical Research Institute. This manuscript describes a semi-automated task that quantifies supination in rats. Rats reach, grasp, and supinate a spherical manipulandum. The rat is rewarded with a pellet if the turn angle exceeds a criterion set by the user. This task increases throughput, sensitivity to injury, and objectivity compared to traditional tasks.

Video: The Knob Supination Task: A Semi-automated Method for Assessing Forelimb Function in Rats

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

Assessing Forelimb Function after Unilateral Cervical SCI using Novel Tasks: Limb Step-alternation, Postural Instability and Pasta Handling

Cervical spinal cord injury (cSCI) can cause devastating neurological deficits, including impairment or loss of upper limb and hand function. A majority of the spinal cord injuries in humans occur at the cervical levels. Therefore, developing cervical injury models and developing relevant and sensitive behavioral tests is of great importance. Here we describe the use of a newly developed forelimb step-alternation test after cervical spinal cord injury in rats. In addition, we describe two behavioral tests that have not been used after spinal cord injury: a postural instability test (PIT), and a pasta-handling test. All three behavioral tests are highly sensitive to injury and are easy to use. Therefore, we feel that these behavioral tests can be instrumental in investigating therapeutic strategies after cSCI.

Three new behavioral tests (forelimb step-alternation, postural instability test, pasta handling test) for evaluating forelimb function after cervical spinal cord injury in rodents are described.

Cervical spinal cord injury (cSCI) can cause devastating neurological deficits, including impairment or loss of upper limb and hand function. A majority ...

The 5-Choice Serial Reaction Time Task: A Task of Attention and Impulse Control for Rodents

This protocol describes the 5-choice serial reaction time task, which is an operant based task used to study attention and impulse control in rodents. Test day challenges, modifications to the standard task, can be used to systematically tax the neural systems controlling either attention or impulse control. Importantly, these challenges have consistent effects on behavior across laboratories in intact animals and can reveal either enhancements or deficits in cognitive function that are not apparent when rats are only tested on the standard task. The variety of behavioral measures that are collected can be used to determine if other factors (i.e., sedation, motivation deficits, locomotor impairments) are contributing to changes in performance. The versatility of the 5CSRTT is further enhanced because it is amenable to combination with pharmacological, molecular, and genetic techniques.

This protocol describes the 5-choice serial reaction time task, which is an operant based task used to study attention and impulse control in rodents. Test day challenges, which are modifications of the standard task, increase flexibility of the task and can be combined with other manipulations to more fully characterize behavior.

This protocol describes the 5-choice serial reaction time task, which is an operant based task used to study attention and impulse control in rodents. ...

Operant Procedures for Assessing Behavioral Flexibility in Rats

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these processes is the ability to adjust behavior in response to changes in one&#8217;s environment (i.e., behavioral flexibility). These processes are essential to normal human behavior, and may be disrupted in diverse neuropsychiatric conditions, including schizophrenia, alcoholism, depression, stroke, and Alzheimer&#8217;s disease. Understanding of the neurobiology of executive functions has been greatly advanced by the availability of animal tasks for assessing discrete components of behavioral flexibility, particularly strategy shifting and reversal learning. While several types of tasks have been developed, most are non-automated, labor intensive, and allow testing of only one animal at a time. The recent development of automated, operant-based tasks for assessing behavioral flexibility streamlines testing, standardizes stimulus presentation and data recording, and dramatically improves throughput. Here, we describe automated strategy shifting and reversal tasks, using operant chambers controlled by custom written software programs. Using these tasks, we have shown that the medial prefrontal cortex governs strategy shifting but not reversal learning in the rat, similar to the dissociation observed in humans. Moreover, animals with a neonatal hippocampal lesion, a neurodevelopmental model of schizophrenia, are selectively impaired on the strategy shifting task but not the reversal task. The strategy shifting task also allows the identification of separate types of performance errors, each of which is attributable to distinct neural substrates. The availability of these automated tasks, and the evidence supporting the dissociable contributions of separate prefrontal areas, makes them particularly well-suited assays for the investigation of basic neurobiological processes as well as drug discovery and screening in disease models.

The ability to assess executive functions such as behavioral flexibility in rats is useful for investigating the neurobiology of cognition in both intact animals and disease models. Here we describe automated tasks for assessing strategy shifting and reversal learning, which are particularly sensitive to disruptions in prefrontal cortical networks.

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these ...

Acquisition of a High-precision Skilled Forelimb Reaching Task in Rats

Movements are the main measurable output of central nervous system function. Developing behavioral paradigms that allow detailed analysis of motor learning and execution is of critical importance in order to understand the principles and processes that underlie motor function. Here we present a paradigm to study movement acquisition within a daily session of training (within-session) representing the fast learning component and primary acquisition as well as skilled motor learning over several training sessions (between-session) representing the slow learning component and consolidation of the learned task. This behavioral paradigm increases the degree of difficulty and complexity of the motor skill task due to two features: First, the animal realigns its body prior to each pellet retrieval forcing renewed orientation and preventing movement execution from the same angle. Second, pellets are grasped from a vertical post that matches the diameter of the pellet and is placed in front of the cage. This requires a precise grasp for successful pellet retrieval and thus prevents simple pulling of the pellet towards the animal. In combination with novel genetics, imaging and electrophysiological technologies, this behavioral method will aid to understand the morphological, anatomical and molecular underpinnings of motor learning and memory.

A paradigm is presented to analyze the acquisition of a high-precision skilled forelimb reaching task in rats.

Movements are the main measurable output of central nervous system function. Developing behavioral paradigms that allow detailed analysis of motor ...

The Emotional Stroop Task: Assessing Cognitive Performance under Exposure to Emotional Content

The emotional Stroop effect (ESE) is the result of longer naming latencies to ink colors of emotion words than to ink colors of neutral words. The difference shows that people are affected by the emotional content conveyed by the carrier words even though they are irrelevant to the color-naming task at hand. The ESE has been widely deployed with patient populations, as well as with non-selected populations, because the emotion words can be selected to match the tested pathology. The ESE is a powerful tool, yet it is vulnerable to various threats to its validity. This report refers to potential sources of confounding and includes a modal experiment that provides the means to control for them. The most prevalent threat to the validity of existing ESE studies is sustained effects and habituation wrought about by repeated exposure to emotion stimuli. Consequently, the order of exposure to emotion and neutral stimuli is of utmost importance. We show that in the standard design, only one specific order produces the ESE.

The emotional Stroop effect (ESE) is the result of longer naming latencies to ink colors of emotion words than those of neutral words. This report refers to potential sources of confounding and includes a modal experiment that provides the means to control for them.

The emotional Stroop effect (ESE) is the result of longer naming latencies to ink colors of emotion words than to ink colors of neutral words. The ...

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 ...

A New Method for Inducing a Depression-Like Behavior in Rats

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is no existing format for studying the mechanism of action, prevention, containment, and treatment of contagious depression. The purpose of this study, therefore, was to establish the first animal model of contagious depression.
Healthy rats can contract depressive behaviors if exposed to depressed rats. Depression is induced in rats by subjecting them to several manipulations of chronic unpredictable stress (CUS) over 5 weeks, as described in the protocol. A successful sucrose preference test confirmed the development of depression in the rats. The CUS-exposed rats were then caged with na&#239;ve rats from the contagion group (1 na&#239;ve rat/2 depressed rats in a cage) for an additional 5 weeks. 30 social groups were created from the combination of CUS-exposed rats and na&#239;ve rats.
This proposed depression-contagion protocol in animals consists mainly of cohabiting CUS-exposed and healthy rats for 5 weeks. To ensure that this method works, a series of tests are carried out - first, the sucrose preference test upon inducing depression to rats, then, the sucrose preference test, alongside the open field and forced-swim tests at the end of the cohabitation period. Throughout the experiment, rats are given tags and are always returned to their cages after each test.
A few limitations to this method are the weak differences recorded between the experimental and control groups in the sucrose preference test and the irreversible traumatic outcome of the forced swim test. These may be worth considering for suitability before any future application of the protocol. Nonetheless, following the experiment, na&#239;ve rats developed contagion depression after 5 weeks of sharing the same cage with the CUS-exposed rats.

This protocol describes a new model by which healthy rats could contract depression over a given time periodthrough contagion by exposure to chronic unpredictable stressed (CUS) rats.

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is ...

Three Laboratory Procedures for Assessing Different Manifestations of Impulsivity in Rats

The present article provides a guide for the conduction and analysis of three conditioning-based protocols to evaluate impulsivity in rats. Impulsivity is a meaningful concept because it is associated with psychiatric conditions in humans and with maladaptive behavior in non-human animals. It is believed that impulsivity is composed of separate factors. There are laboratory protocols devised to assess each of these factors using standardized automated equipment. Delay discounting is associated with the incapacity to be motivated by delayed outcomes. This factor is evaluated through intertemporal choice protocols, which consist&#160;of presenting the individual with a choice situation involving an immediate reward and a larger but delayed reward. Response inhibition deficit is associated with the incapacity to withhold prepotent responses. Differential reinforcement of low rates (DLR) and feature-negative discrimination protocols assess the response inhibition deficit factor of impulsivity. The former imposes a condition to a motivated individual in which most wait a minimum period of time for a response to be rewarded. The latter evaluates the capacity of individuals to refrain from food seeking responses when a signal of the absence of food is presented. The purpose of these protocols is to construct an objective quantitative measure of impulsivity, which serves to make cross-species comparisons, allowing the possibility of translational research. The advantages of these particular protocols include their easy set-up and application, which stems from the relatively small amount of equipment needed and the automated nature of these protocols.

We present three protocols that assess different forms of impulsivity in rats and other small mammals. Intertemporal choice procedures evaluate the tendency to discount the value of delayed outcomes. Differential reinforcement of low rates and feature-negative discrimination evaluate response inhibition capacity with and without punishment for inappropriate responses, respectively.

The present article provides a guide for the conduction and analysis of three conditioning-based protocols to evaluate impulsivity in rats. Impulsivity is ...

Assessing the Coherence of Parents' Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with parents. This paper focuses on such a method, namely Five-Minute Speech Sample-Coherence (FMSS-Coherence). In this method, a parent is asked to speak for five uninterrupted minutes about her/his child and their relationship. The resulted narrative is coded for coherence, namely the extent to which the parent provides&#160;in the narrative&#160;a clear, consistent, multidimensional and well-supported portrayal of the child. FMSS-Coherence is based on attachment research that shows that the coherence of parents&#8217; narratives is indicative of the quality of the parent-child relationship and child adjustment. It overcomes the limitations of attachment narrative measures which are typically labor intensive. FMSS-Coherence is less nuanced than extant attachment narrative measures. Yet, studies of families from different cultural backgrounds, across different child ages and in the context of typically developing children as well as children with special needs suggest that coherence can be reliably evaluated using the FMSS procedure. Furthermore, parents&#8217; FMSS-Coherence is associated with parenting quality and children&#8217;s socio-emotional adjustment. Thus, it holds promise for researchers and practitioners who seek a relatively time- and cost-effective method for assessing the coherence of parents&#8217; narratives regarding their child.

This paper introduces a method for assessing the coherence of parents' short narratives regarding their child and their relationship using the five-minute speech sample procedure.

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with ...