Name: automated rat single-pellet reaching with 3-dimensional reconstruction of paw and digit trajectories
Uploaded: 2019-07-10T15:00:00
Description: Watch this Scientific Journal Video about Automated Rat Single-Pellet Reaching with 3-Dimensional Reconstruction of Paw and Digit Trajectories at JoVE.com

The authors would like to thank Karunesh Ganguly and his laboratory for advice on the skilled reaching task, and Alexander and Mackenzie Mathis for their help in adapting DeepLabCut. This work was supported by the National Institute of Neurological Disease and Stroke (grant number K08-NS072183) and the University of Michigan.

All methods involving animal use described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Michigan.
1. Setting up the reaching chamber
NOTE: See Ellens et al.14 for details and diagrams of the apparatus. Part numbers refer to Figure 1.
<ol>
	<li>Bond clear polycarbonate panels with acrylic cement to build the reaching chamber (15 cm wide ...

<ol>
	<li>Chen, 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=An+automated+behavioral+apparatus+to+combine+parameterized+reaching+and+grasping+movements+in+3D+space.">An automated behavioral apparatus to combine parameterized reaching and grasping movements in 3D space.</a> Journal of Neuroscience Methods. , (2018).</li><li>Sacrey, L. A. R. A., 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=Similar+hand+shaping+in+reaching-for-food+(skilled+reaching)+in+rats+and+humans+provides+evidence+of+homology+in+release,+collection,+and+manipulation+movements.">Similar hand shaping in reaching-for-food (skilled reaching) in rats and humans provides evidence of homology in release, collection, and manipulation movements.</a> Behavioural Brain Research. 204, 153-161 (2009).</li><li>Whishaw, I. Q., Kolb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decortication+abolishes+place+but+not+cue+learning+in+rats.">Decortication abolishes place but not cue learning in rats.</a> Behavioural Brain Research. 11, 123-134 (1984).</li><li>Klein, A., Dunnett, S. B. <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+Skilled+Forelimb+Movement+in+Rats:+The+Single+Pellet+Reaching+Test+and+Staircase+Test.">Analysis of Skilled Forelimb Movement in Rats: The Single Pellet Reaching Test and Staircase Test.</a> Current Protocols in Neuroscience. 58, 8.28.1-8.28.15 (2012).</li><li>Whishaw, I. Q., Pellis, S. 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+structure+of+skilled+forelimb+reaching+in+the+rat:+a+proximally+driven+movement+with+a+single+distal+rotatory+component.">The structure of skilled forelimb reaching in the rat: a proximally driven movement with a single distal rotatory component.</a> Behavioural Brain Research. 41, 49-59 (1990).</li><li>Zeiler, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medial+premotor+cortex+shows+a+reduction+in+inhibitory+markers+and+mediates+recovery+in+a+mouse+model+of+focal+stroke.">Medial premotor cortex shows a reduction in inhibitory markers and mediates recovery in a mouse model of focal stroke.</a> Stroke. 44, 483-489 (2013).</li><li>Fenrich, K. K., 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=Improved+single+pellet+grasping+using+automated+ad+libitum+full-time+training+robot.">Improved single pellet grasping using automated ad libitum full-time training robot.</a> Behavioural Brain Research. 281, 137-148 (2015).</li><li>Azim, E., Jiang, J., Alstermark, B., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skilled+reaching+relies+on+a+V2a+propriospinal+internal+copy+circuit.">Skilled reaching relies on a V2a propriospinal internal copy circuit.</a> Nature. , (2014).</li><li>Guo, J. Z. 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=Cortex+commands+the+performance+of+skilled+movement.">Cortex commands the performance of skilled movement.</a> Elife. 4, e10774 (2015).</li><li>Nica, I., Deprez, M., Nuttin, B., Aerts, 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=Automated+Assessment+of+Endpoint+and+Kinematic+Features+of+Skilled+Reaching+in+Rats.">Automated Assessment of Endpoint and Kinematic Features of Skilled Reaching in Rats.</a> Frontiers in Behavioral Neuroscience. 11, 255 (2017).</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> Journal of Neuroscience Methods. 246, 30-37 (2015).</li><li>Torres-Esp&#237;n, A., Forero, J., Schmidt, E. K. A., Fouad, K., Fenrich, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+motorized+pellet+dispenser+to+deliver+high+intensity+training+of+the+single+pellet+reaching+and+grasping+task+in+rats.">A motorized pellet dispenser to deliver high intensity training of the single pellet reaching and grasping task in rats.</a> Behavioural Brain Research. 336, 67-76 (2018).</li><li>Mathis, 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=DeepLabCut:+markerless+pose+estimation+of+user-defined+body+parts+with+deep+learning.">DeepLabCut: markerless pose estimation of user-defined body parts with deep learning.</a> Nature Neuroscience. 21, 1281-1289 (2018).</li><li>Ellens, D. 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=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> Journal of Neuroscience Methods. 271, 119-127 (2016).</li></ol>

Rodent skilled reaching has become a standard tool to study motor system physiology and pathophysiology. We have described how to implement an automated rat skilled reaching task that allows: training and testing with minimal supervision, 3-D paw and digit trajectory reconstruction (during reaching, grasping, and paw retraction), real-time identification of the paw during reaching, and synchronization with external electronics. It is well-suited to correlate forelimb kinematics with physiology or to perform precisely-tim...

Humans depend heavily on dexterous skill, defined as movements that require precisely coordinated multi-joint and digit movements. These skills are affected by a range of common central nervous system pathologies including structural lesions (e.g., stroke, tumor, demyelinating lesions), neurodegenerative disease (e.g., Parkinson's disease), and functional abnormalities of motor circuits (e.g., dystonia). Understanding how dexterous skills are learned and implemented by central motor circuits therefore has the potential to improve quality of life for a large population. Furthermore, such understanding is likely to improve motor performance in healthy people by optimizing training and rehabilitation strategies.
Dissecting the neural circuits underlying dexterous skill in humans is limited by technological and ethical considerations, necessitating the use of animal models. Nonhuman primates are commonly used to study dexterous limb movements given the similarity of their motor systems and behavioral repertoire to humans1. However, non-human primates are expensive with long generation times, limiting numbers of study subjects and genetic interventions. Furthermore, while the neuroscientific toolbox applicable to nonhuman primates is larger than for humans, many recent technological advances are either unavailable or significantly limited in primates.
Rodent skilled reaching is a complementary approach to studying dexterous motor control. Rats and mice can be trained to reach for, grasp, and retrieve a sugar pellet in a stereotyped sequence of movements homologous to human reaching patterns2. Due to their relatively short generation time and lower housing costs, as well as their ability to acquire skilled reaching over days to weeks, it is possible to study large numbers of subjects during both learning and skill consolidation phases. The use of rodents, especially mice, also facilitates the use of powerful modern neuroscientific tools (e.g., optogenetics, calcium imaging, genetic models of disease) to study dexterous skill.
Rodent skilled reaching has been used for decades to study normal motor control and how it is affected by specific pathologies like stroke and Parkinson's disease3. However, most versions of this task are labor and time-intensive, mitigating the benefits of studying rodents. Typical implementations involve placing rodents in a reaching chamber with a shelf in front of a narrow slot through which the rodent must reach. A researcher manually places sugar pellets on the shelf, waits for the animal to reach, and then places another one. Reaches are scored as successes or failures either in real time or by video review4. However, simply scoring reaches as successes or failures ignores rich kinematic data that can provide insight into how (as opposed to simply whether) reaching is impaired. This problem was addressed by implementing detailed review of reaching videos to identify and semi-quantitatively score reach submovements5. While this added some data regarding reach kinematics, it also significantly increased experimenter time and effort. Further, high levels of experimenter involvement can lead to inconsistencies in methodology and data analysis, even within the same lab.
More recently, several automated versions of skilled reaching have been developed. Some attach to the home cage6,7, eliminating the need to transfer animals. This both reduces stress on the animals and eliminates the need to acclimate them to a specialized reaching chamber. Other versions allow paw tracking so that kinematic changes under specific interventions can be studied8,9,10, or have mechanisms to automatically determine if pellets were knocked off the shelf11. Automated skilled reaching tasks are especially useful for high-intensity training , as may be required for rehabilitation after an injury12. Automated systems allow animals to perform large numbers of reaches over long periods of time without requiring intensive researcher involvement. Furthermore, systems which allow paw tracking and automated outcome scoring reduce researcher time spent performing data analysis.
We developed an automated rat skilled reaching system with several specialized features. First, by using a movable pedestal to bring the pellet into "reaching position" from below, we obtain a nearly unobstructed view of the forelimb. Second, a system of mirrors allows multiple simultaneous views of the reach with a single camera, allowing three-dimensional (3-D) reconstruction of reach trajectories using a high resolution, high-speed (300 fps) camera. With the recent development of robust machine learning algorithms for markerless motion tracking13, we now track not only the paw but individual knuckles to extract detailed reach and grasp kinematics. Third, a frame-grabber that performs simple video processing allows real-time identification of distinct reaching phases. This information is used to trigger video acquisition (continuous video acquisition is not practical due to file size), and can also be used to trigger interventions (e.g., optogenetics) at precise moments. Finally, individual video frames are triggered by transistor-transistor logic (TTL) pulses, allowing the video to be precisely synchronized with neural recordings (e.g., electrophysiology or photometry). Here, we describe how to build this system, train rats to perform the task, synchronize the apparatus with external systems, and reconstruct 3-D reach trajectories.

<table>
	<tbody>
		<tr>
			<td>clear polycarbonate panels</td>
			<td>TAP Plastics</td>
			<td></td>
			<td>cut to order (see box design)</td>
		</tr>
		<tr>
			<td>infrared source/detector</td>
			<td>Med Associates</td>
			<td>ENV-253SD</td>
			<td>30&#34; range</td>
		</tr>
		<tr>
			<td>camera</td>
			<td>Basler</td>
			<td>acA2000-340kc</td>
			<td>2046 x 1086 CMV2000 340 fps Color Camera Link</td>
		</tr>
		<tr>
			<td>camera lens</td>
			<td>Megapixel (computar)</td>
			<td>M0814-MP2</td>
			<td>2/3&#34; 8mm f1.4 w/ locking Iris &#38; Focus</td>
		</tr>
		<tr>
			<td>camera cables</td>
			<td>Basler</td>
			<td>#2000031083</td>
			<td>Cable PoCL Camera Link SDR/MDR Full, 5 m - Data Cables</td>
		</tr>
		<tr>
			<td>mirrors</td>
			<td>Amazon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>linear actuator</td>
			<td>Concentrics</td>
			<td>LACT6P</td>
			<td>Linear Actuator 6&#34; Stroke (nominal), 110 Lb Force, 12 VDC, with Potentiometer</td>
		</tr>
		<tr>
			<td>pellet reservoir/funnel</td>
			<td>Amico (Amazon)</td>
			<td>a12073000ux0890</td>
			<td>6&#34; funnel</td>
		</tr>
		<tr>
			<td>guide tube</td>
			<td>ePlastics</td>
			<td>ACREXT.500X.250</td>
			<td>1/2&#34; OD x 1/4&#34; ID Clear. Extruded Plexiglass Acrylic Tube x 6ft long</td>
		</tr>
		<tr>
			<td>pellet delivery rod</td>
			<td>ePlastics</td>
			<td>ACRCAR.250</td>
			<td>0.250&#34; DIA. Cast Acrylic Rod (2&#39; length)</td>
		</tr>
		<tr>
			<td>plastic T connector</td>
			<td>United States Plastic Corp</td>
			<td>#62065</td>
			<td>3/8&#34; x 3/8&#34; x 3/8&#34; Hose ID Black HDPE Tee</td>
		</tr>
		<tr>
			<td>LED lights</td>
			<td>Lighting EVER</td>
			<td>4100066-DW-F</td>
			<td>12V Flexible Waterproof LED Light Strip, LED Tape, Daylight White, Super Bright 300 Units 5050 LEDS, 16.4Ft 5 M Spool</td>
		</tr>
		<tr>
			<td>Light backing</td>
			<td>ePlastics</td>
			<td>ACTLNAT0.125X12X36</td>
			<td>0.125&#34; x 12&#34; x 36&#34; Natural Acetal Sheet</td>
		</tr>
		<tr>
			<td>Light diffuser films</td>
			<td>inventables</td>
			<td>23114-01</td>
			<td>.007x8.5x11&#34;, matte two sides</td>
		</tr>
		<tr>
			<td>cabinet and custom frame materials</td>
			<td>various (Home Depot, etc.)</td>
			<td></td>
			<td>3/4&#34; fiber board (see protocol for dimensions of each structure)</td>
		</tr>
		<tr>
			<td>acoustic foam</td>
			<td>Acoustic First</td>
			<td></td>
			<td>FireFlex Wedge Acoustical Foam (2&#34; Thick)</td>
		</tr>
		<tr>
			<td>ventilation fans</td>
			<td>Cooler Master (Amazon)</td>
			<td>B002R9RBO0</td>
			<td>Rifle Bearing 80mm Silent Cooling Fan for Computer Cases and CPU Coolers</td>
		</tr>
		<tr>
			<td>cabinet door hinges</td>
			<td>Everbilt (Home Depot</td>
			<td>#14609</td>
			<td>continuous steel hinge (1.4&#34; x 48&#34;)</td>
		</tr>
		<tr>
			<td>cabinet wheels</td>
			<td>Everbilt (Home Depot</td>
			<td>#49509</td>
			<td>Soft rubber swivel plate caster with 90 lb. load rating and side brake</td>
		</tr>
		<tr>
			<td>cabinet door handle</td>
			<td>Everbilt (Home Depot</td>
			<td>#15094</td>
			<td>White light duty door pull (4.5&#34;)</td>
		</tr>
		<tr>
			<td>computer</td>
			<td>Hewlett Packard</td>
			<td>Z620</td>
			<td>HP Z620 Desktop Workstation</td>
		</tr>
		<tr>
			<td>Camera Link Frame Grabber</td>
			<td>National Instruments</td>
			<td>#781585-01</td>
			<td>PCIe-1473 Virtex-5 LX50 Camera Link - Full</td>
		</tr>
		<tr>
			<td>Multifunction RIO Board</td>
			<td>National Instruments</td>
			<td>#781100-01</td>
			<td>PCIe-17841R</td>
		</tr>
		<tr>
			<td>Analog RIO Board Cable</td>
			<td>National Instruments</td>
			<td>SCH68M-68F-RMIO</td>
			<td>Multifunction Cable</td>
		</tr>
		<tr>
			<td>Digital RIO Board Cable</td>
			<td>National Instruments</td>
			<td>#191667-01</td>
			<td>SHC68-68-RDIO Digital Cable for R Series</td>
		</tr>
		<tr>
			<td>Analog Terminal Block</td>
			<td>National Instruments</td>
			<td>#782536-01</td>
			<td>SCB-68A Noise Rejecting, Shielded I/O Connector Block</td>
		</tr>
		<tr>
			<td>Digital Terminal Block</td>
			<td>National Instruments</td>
			<td>#782536-01</td>
			<td>SCB-68A Noise Rejecting, Shielded I/O Connector Block</td>
		</tr>
		<tr>
			<td>24 position relay rack</td>
			<td>Measurement Computing Corp.</td>
			<td>SSR-RACK24</td>
			<td>Solid state relay backplane (Gordos/OPTO-22 type relays), 24-channel</td>
		</tr>
		<tr>
			<td>DC switch</td>
			<td>Measurement Computing Corp.</td>
			<td>SSR-ODC-05</td>
			<td>Solid state relay module, single, DC switch, 3 to 60 VDC @ 3.5 A</td>
		</tr>
		<tr>
			<td>DC Sense</td>
			<td>Measurement Computing Corp.</td>
			<td>SSR-IDC-05</td>
			<td>solid state relay module, single, DC sense, 3 to 32 VDC</td>
		</tr>
		<tr>
			<td>DC Power Supply</td>
			<td>BK Precision</td>
			<td>1671A</td>
			<td>Triple-Output 30V, 5A Digital Display DC Power Supply</td>
		</tr>
		<tr>
			<td>sugar pellets</td>
			<td>Bio Serv</td>
			<td>F0023</td>
			<td>Dustless Precision Pellets, 45 mg, Sucrose (Unflavored)</td>
		</tr>
		<tr>
			<td>LabVIEW</td>
			<td>National Instruments</td>
			<td>LabVIEW 2014 SP1, 64 and 32-bit versions</td>
			<td>64-bit LabVIEW is required to access enough memory to stream videos, but FPGA coding must be performed in 32-bit LabVIEW</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td>Matlab R2019a</td>
			<td>box calibration and trajectory reconstruction software is written in Matlab and requires the Computer Vision toolbox</td>
		</tr>
	</tbody>
</table>

automated rat single-pellet reaching with 3-dimensional reconstruction of paw and digit trajectories

Rodent skilled reaching is commonly used to study dexterous skills, but requires significant time and effort to implement the task and analyze the behavior. Several automated versions of skilled reaching have been developed recently. Here, we describe a version that automatically presents pellets to rats while recording high-definition video from multiple angles at high frame rates (300 fps). The paw and individual digits are tracked with DeepLabCut, a machine learning algorithm for markerless pose estimation. This system can also be synchronized with physiological recordings, or be used to trigger physiologic interventions (e.g., electrical or optical stimulation).

Rats acquire the skilled reaching task quickly once acclimated to the apparatus, with performance plateauing in terms of both numbers of reaches and accuracy over 1&#8211;2 weeks (Figure 5). Figure 6 shows sample video frames indicating structures identified by DeepLabCut, and Figure 7 shows superimposed individual reach trajectories from a single session. Finally, in Figure 8, we illustrate what happen...

Watch this Scientific Journal Video about Automated Rat Single-Pellet Reaching with 3-Dimensional Reconstruction of Paw and Digit Trajectories at JoVE.com

Automated Rat Single-Pellet Reaching with 3-Dimensional Reconstruction of Paw and Digit Trajectories

Rodent skilled reaching is commonly used to study dexterous skills, but requires significant time and effort to implement the task and analyze the behavior. We describe an automated version of skilled reaching with motion tracking and three-dimensional reconstruction of reach trajectories.

Rodent skilled reaching is commonly used to study dexterous skills, but requires significant time and effort to implement the task and analyze the ...

This method builds upon established rat skilled reaching protocols to automate training and testing providing an efficient means to acquire large data sets relatively quickly. It minimizes experimenter effort while allowing three-dimensional reconstruction of forelimb kinematics. The kinematics can be used to evaluate the effects of precisely timed interventions or correlated with physiologic recordings.With some adjustments, this technique can also be applied in mice. Begin by turning on the lights and place the rat in a skilled reaching chamber. Use forceps to hold a pellet through the reaching slot at the front of the box.Allow the rat to eat three pellets from the forceps. Pull the pellet back the next time the rat tries to eat the pellet from the forceps and repeat until the rat reaches for the pellet with one paw the most out of 11 reaches indicating paw preference. Next, align the pellet delivery rod with the side of the reaching slot contralateral to the rat's preferred paw.Place a pellet on the delivery rod. Then use forceps to bait the rat with the pellet but direct the rat towards the delivery rod so that its paw hits the pellet on the rod. After the rat has attempted 10 reaches to the delivery rod without being baited, advance the rat to the next phase.Next, position the pellet delivery rod based on the rat's paw preference and set it to position two. Set the height position of the pellet delivery rod using the actuator remote. Once the delivery rod is at the correct height, hold down the desired number until the light blinks red to set.Then place the rat in the chamber and bait the rat to the back with a pellet. When the rat moves far enough to the back of the chamber that it would break the infrared beam if the automated version was running, move the pellet delivery rod to position three. Finally, wait for the rat to reach for the pellet and then move the pellet delivery rod back to position two.Place a new pellet on the delivery rod if knocked off. Repeat these steps gradually baiting the rat less and less until the rat begins to one, move to the back to request a pellet without being baited, and two, immediately move to the front after requesting a pellet in the back. After the rat has done this 10 times, begin training on the automated task.To set up the automated system, turn on the lights in the chamber and refill the pellet reservoir. Position the pellet delivery rod according to the rat's paw preference. Check that the actuator positions are set correctly.Next, turn on the computer and open the skilled reaching program. Enter the rat ID number under subject and select the paw preference from the hand dropdown menu. Specify the save path for the videos.Then set the session time and the number of max videos. Set the pellet lift duration as well and enable the early reach penalty. Next, to take calibration images, place the helping hand inside the reaching chamber and poke the alligator clip through the reaching slot.Hold the cube in front of the reaching slot with the alligator clip. Then position the cube so that the red side appears in the top mirror, the green side in the left mirror, and the blue side in the right mirror. In the behavioral program, ensure the ROI threshold is set to a very large value.Click the run button and once the camera initialized button turns green, press start to begin video acquisition. Click Cal Mode then take an image by clicking Take Cal Image. Move the cube slightly and take another image.Repeat again for a total of three images. Finally, stop the program by clicking stop and then click the stop sign button. Remove the helping hand and cube from the box.Be careful not to bump anything in the behavioral chamber after calibration images have been taken that day. First, place the rat in the skilled reaching chamber. Click on the white arrow to run the program.Then set the position of the ROI for paw detection by adjusting x offset, y offset, ROI width, and ROI height. Position the ROI in the side mirror that shows the dorsum of the paw directly in front of the reaching slot and click start to begin the program. While the rat is not reaching, adjust the low ROI threshold value until the live ROI trigger value is oscillating between zero and one.Then set the ROI threshold to be significantly greater than the live ROI trigger value during nose pokes and lower than the live ROI trigger value when the rat reaches. Adjust until videos are consistently triggered when the rat reaches but not when it pokes its nose through the slot. Monitor the first few trials to ensure that everything is working correctly.When a rat reaches before requesting a pellet, observe the early reaches number increase. When a rat reaches after requesting a pellet, observe the videos number increase while a copy of the video is saved as a bin file. Finally, after the session time or max number of videos is reached, press the stop sign button.Results indicate that after 20 days, rats acquire the skilled reaching task. Here, the reach trajectories from a single session are shown. This represents only initial paw advancement for ease of presentation.Further, if the paw detection trigger is not accurately set, there is significant variability in the frame at which the paw breaches the reaching slot which may lead to variability in when interventions are triggered during reaching movements. Other than careful handling of the animals, no precautions will be taken in this protocol. It is most important to set the ROI threshold correctly so that videos or other interventions are triggered at the appropriate moments.This method can be paired with several other techniques such as optogenetics, calcium imaging, and genetic models which can address questions ranging from basic motor control to movement disorder pathophysiology. This technique permits correlation of detailed reach and grasp kinematics with physiologic recordings or interventions allowing researchers to answer not just if but how movements change with neural circuitry changes.

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Behavior

Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the &#34;cloud&#34; and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in&#160;cabinet; two such cabinets (16 environments) may be controlled by one computer.

Fully automated system for measuring physiologically meaningful properties of the mechanisms mediating spatial localization, temporal localization, duration, rate&#160;and probability estimation, risk assessment, impulsivity, and the accuracy and precision of memory, in order to assess the effects of genetic and pharmacological manipulations on foundational mechanisms of cognition in mice.

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and ...

Study Motor Skill Learning by Single-pellet Reaching Tasks in Mice

Reaching for and retrieving objects require precise and coordinated motor movements in the forelimb. When mice are repeatedly trained to grasp and retrieve food rewards positioned at a specific location, their motor performance (defined as accuracy and speed) improves progressively over time, and plateaus after persistent training. Once such reaching skill is mastered, its further maintenance does not require constant practice. Here we introduce a single-pellet reaching task to study the acquisition and maintenance of skilled forelimb movements in mice. In this video, we first describe the behaviors of mice that are commonly encountered in this learning and memory paradigm, and then discuss how to categorize these behaviors and quantify the observed results. Combined with mouse genetics, this paradigm can be utilized as a behavioral platform to explore the anatomical underpinnings, physiological properties, and molecular mechanisms of learning and memory.

Persistent practice improves the precision of coordinated movements.&#160;Here we introduce a single-pellet reaching task, which is designed to assess the learning and memory of forelimb skill in mice.&#160;

Reaching for and retrieving objects require precise and coordinated motor movements in the forelimb. When mice are repeatedly trained to grasp and ...

Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the test mouse is placed in a standardized apparatus and relevant behaviors are assessed in three different sessions (called session I, II, and III).
The apparatus has three compartments (see Figure 1), the left and right compartments contain an inverted cup which can house a mouse (called &#34;stimulus mouse&#34;).
In session I, the test mouse is placed in the cage and its mobility is characterized by the number of transitions made between compartments. In session II, a stimulus mouse is placed under one of the inverted cups and the sociability of the test mouse is quantified by the amounts of time it spends near the cup containing the enclosed stimulus mouse vs. the empty inverted cup. In session III, the inverted cups are removed and both mice interact freely. The sociability of the test mouse in session III is quantified by the number of social approaches it makes toward the stimulus mouse and by the number of times it avoids a social approach by the stimulus mouse.
The automated evaluation of the movie detects the nose of the test mouse, which allows the determination of all described sociability measures in session I and II (in session III, approaches are identified automatically but classified manually). To find the nose, the image of an empty cage is digitally subtracted from each frame of the movie and the resulting image is binarized to identify the mouse pixels. The mouse tail is automatically removed and the two most distant points of the remaining mouse are determined; these are close to nose and base of tail. By analyzing the motion of the mouse and using continuity arguments, the nose is identified.
<img alt="Figure 1" src="/files/ftp_upload/52508/52508fig1.jpg" /> 
Figure 1. Assessment of Sociability During 3 sessions. Session I (top): Acclimation of test mouse to the cage. Session II (middle): Test mouse moving freely in the cage while the stimulus mouse is enclosed in an inverted cup. Session III (bottom): Both test mouse and stimulus mouse are allowed to move freely and interact with each other.

This protocol describes a method to quantify mouse sociability. Mice are videotaped as they move and interact in a special cage. Movie processing allows for the automated quantification of sociability with excellent accuracy and reliability.

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the ...

Paw-Dragging: a Novel, Sensitive Analysis of the Mouse Cylinder Test

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore by rearing and touching the walls of the cylinder with their forelimb paws for postural support. Following ischemic injury to the forelimb sensorimotor cortex, rats rely more heavily on their unaffected forelimb paw for postural support resulting in fewer touches with their affected paw which is termed forelimb asymmetry. In contrast, focal ischemic damage in the mouse brain fails to result in comparable consistent deficits in forelimb asymmetry. While forelimb asymmetry deficits are infrequently observed, mice do demonstrate a novel behaviour post stroke termed &#8220;paw-dragging&#8221;. Paw-dragging is the tendency for a mouse to drag its affected paw along the cylinder wall rather than directly push off from the wall when dismounting from a rear to a four-legged stance. We have previously demonstrated that paw-dragging behaviour is highly sensitive to small cortical ischemic injuries to the forelimb motor cortex. Here we provide a detailed protocol for paw-dragging analysis. We define what a paw-drag is and demonstrate how to quantify paw-dragging behaviour. The cylinder test is a simple and inexpensive test to administer and does not require pre-training or food deprivation strategies. In using paw-dragging analysis with the cylinder test, it fills a niche for predicting cortical ischemic injuries such as photothrombosis and Endothelin-1 (ET-1)-induced ischemia &#8211; two models that are ever-increasing in popularity and produce smaller focal injuries than middle cerebral artery occlusion. Finally, measuring paw-dragging behaviour in the cylinder test will allow studies of functional recovery after cortical injury using a wide cohort of transgenic mouse strains where previous forelimb asymmetry analysis has failed to detect consistent deficits.

Classical forelimb asymmetry analysis of the cylinder test is routinely used to assess behavioural deficits in rats following brain injury or stroke; however, it fails to detect consistent deficits in mice. This study demonstrates that quantifying paw-dragging behaviour is a more sensitive analysis of brain injury in mice.

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore ...

Assessment of Social Cognition in Non-human Primates Using a Network of Computerized Automated Learning Device (ALDM) Test Systems

Fagot &#38; Paleressompoulle1 and Fagot &#38; Bonte2 have published an automated learning device (ALDM) for the study of cognitive abilities of monkeys maintained in semi-free ranging conditions. Data accumulated during the last five years have consistently demonstrated the efficiency of this protocol to investigate individual/physical cognition in monkeys, and have further shown that this procedure reduces stress level during animal testing3. This paper demonstrates that networks of ALDM can also be used to investigate different facets of social cognition and in-group expressed behaviors in monkeys, and describes three illustrative protocols developed for that purpose. The first study demonstrates how ethological assessments of social behavior and computerized assessments of cognitive performance could be integrated to investigate the effects of socially exhibited moods on the cognitive performance of individuals. The second study shows that batteries of ALDM running in parallel can provide unique information on the influence of the presence of others on task performance. Finally, the last study shows that networks of ALDM test units can also be used to study issues related to social transmission and cultural evolution. Combined together, these three studies demonstrate clearly that ALDM testing is a highly promising experimental tool for bridging the gap in the animal literature between research on individual cognition and research on social cognition.

Assessment of Social Cognition in Non-human Primates Using a Network of Computerized Automated Learning Device ALDM Test Systems

Fagot &#38; Paleressompoulle1 have published an automated learning device (ALDM) aimed at testing individual cognitive abilities in semi-free ranging monkeys. The main goal of our protocol is to use a network of ALDM test units to study social cognition in non-human primates.

Fagot &#38; Paleressompoulle1 and Fagot &#38; Bonte2 have published an automated learning device (ALDM) for the study of cognitive abilities of monkeys ...

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 ...

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

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.

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 ...

Automated Measurements of Sleep and Locomotor Activity in Mexican Cavefish

Across phyla, sleep is characterized by highly conserved behavioral characteristics that include elevated arousal threshold, rebound following sleep deprivation, and consolidated periods of behavioral immobility. The Mexican cavefish, Astyanax mexicanus (A. mexicanus), is a model for studying trait evolution in response to environmental perturbation. A. mexicanus exist as in eyed surface-dwelling forms and multiple blind cave-dwelling populations that have robust morphological and behavioral differences. Sleep loss has occurred in multiple, independently-evolved cavefish populations. This protocol describes a methodology for quantifying sleep and locomotor activity in A. mexicanus cave and surface fish. A cost-effective video monitoring system allows for behavioral imaging of individually-housed larval or adult fish for periods of a week or longer. The system can be applied to fish aged 4 days post fertilization through adulthood. The approach can also be adapted for measuring the effects of social interactions on sleep by recording multiple fish in a single arena. Following behavioral recordings, data is analyzed using automated tracking software and sleep analysis is processed using customized scripts that quantify multiple sleep variables including duration, bout length, and bout number. This system can be applied to measure sleep, circadian behavior, and locomotor activity in almost any fish species including zebrafish and sticklebacks.

This protocol details the methodology for quantifying locomotor behavior and sleep in the Mexican cavefish. Previous analyses are extended to measure these behaviors in socially-housed fish. This system can be widely applied to study sleep and activity in other fish species.

Across phyla, sleep is characterized by highly conserved behavioral characteristics that include elevated arousal threshold, rebound following sleep ...

Paw-Print Analysis of Contrast-Enhanced Recordings (PrAnCER): A Low-Cost, Open-Access Automated Gait Analysis System for Assessing Motor Deficits

Gait analysis is used to quantify changes in motor function in many rodent models of disease. Despite the importance of assessing gait and motor function in many areas of research, the available commercial options have several limitations such as high cost and lack of accessible, open code. To address these issues, we developed PrAnCER, Paw-Print Analysis of Contrast-Enhanced Recordings, for automated quantification of gait. The contrast-enhanced recordings are produced by using a translucent floor that obscures objects not in contact with the surface, effectively isolating the rat&#8217;s paw prints as it walks. Using these videos, our simple software program reliably measures a variety of spatiotemporal gait parameters. To demonstrate that PrAnCER can accurately detect changes in motor function, we employed a haloperidol model of Parkinson&#8217;s disease (PD). We tested rats at two doses of haloperidol: high dose (0.30 mg/kg) and low dose (0.15 mg/kg). Haloperidol significantly increased stance duration and hind paw contact area in the low dose condition, as might be expected in a PD model. In the high dose condition, we found a similar increase in contact area but also an unexpected increase in stride length. With further research, we found that this increased stride length is consistent with the bracing-escape phenomenon commonly observed at higher doses of haloperidol. Thus, PrAnCER was able to detect both expected and unexpected changes in rodent gait patterns. Additionally, we confirmed that PrAnCER is consistent and accurate when compared with manual scoring of gait parameters.

Paw-Print Analysis of Contrast-Enhanced Recordings PrAnCER: A Low-Cost, Open-Access Automated Gait Analysis System for Assessing Motor Deficits

We describe a novel gait analysis system, Paw-Print Analysis of Contrast-Enhanced Recordings (PrAnCER), an open-access automated system for the quantification of gait characteristics in rats that utilizes a novel semitransparent floor to automatically quantify gait. This system was validated using the haloperidol model of Parkinson&#8217;s Disease.

Gait analysis is used to quantify changes in motor function in many rodent models of disease. Despite the importance of assessing gait and motor function ...