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.

<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>

The authors have nothing to disclose.

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 ...

automated-rat-single-pellet-reaching-with-3-dimensional

Research

JoVE Journal

Behavior

13.8K Views.  University of Michigan. 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.

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

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 ...

A Fully Automated Rodent Conditioning Protocol for Sensorimotor Integration and Cognitive Control Experiments

Rodents have been traditionally used as a standard animal model in laboratory experiments involving a myriad of sensory, cognitive, and motor tasks. Higher cognitive functions that require precise control over sensorimotor responses such as decision-making and attentional modulation, however, are typically assessed in nonhuman primates. Despite the richness of primate behavior that allows multiple variants of these functions to be studied, the rodent model remains an attractive, cost-effective alternative to primate models. Furthermore, the ability to fully automate operant conditioning in rodents adds unique advantages over the labor intensive training of nonhuman primates while studying a broad range of these complex functions.
Here, we introduce a protocol for operantly conditioning rats on performing working memory tasks. During critical epochs of the task, the protocol ensures that the animal's overt movement is minimized by requiring the animal to 'fixate' until a Go cue is delivered, akin to nonhuman primate experimental design. A simple two alternative forced choice task is implemented to demonstrate the performance. We discuss the application of this paradigm to other tasks.

A fully automated protocol for rodent operant conditioning is proposed. The protocol relies on precise temporal control of behavioral events to investigate the extent to which this control influences neural activity underlying sensorimotor integration and cognitive control experiments.

Rodents have been traditionally used as a standard animal model in laboratory experiments involving a myriad of sensory, cognitive, and motor tasks. ...

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 ...

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 ...

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific responses to changing temperatures according to their physiological tolerance and adaptability. Drosophila flies also possess a temperature sensing system that has become fundamental to understanding the neural basis of temperature processing in ectotherms. We present here a temperature-controlled arena that permits fast and precise temperature changes with temporal and spatial control to explore the response of individual flies to changing temperatures. Individual flies are placed in the arena and exposed to pre-programmed temperature challenges, such as uniform gradual increases in temperature to determine reaction norms or spatially distributed temperatures at the same time to determine preferences. Individuals are automatically tracked, allowing the quantification of speed or location preference. This method can be used to rapidly quantify the response over a large range of temperatures to determine temperature performance curves in Drosophila or other insects of similar size. In addition, it can be used for genetic studies to quantify temperature preferences and reactions of mutants or wild-type flies. This method can help uncover the basis of thermal speciation and adaptation, as well as the neural mechanisms behind temperature processing.

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Here we present a protocol to automatically determine the locomotor performance of Drosophila at changing temperatures using a programmable temperature-controlled arena that produces fast and accurate temperature changes in time and space.

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific ...

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 ...