We acknowledge the audiovisual technician and video producer Rebeca De las Heras Ponce as well as the head veterinarian Gonzalo Moreno del Val, for the supervision of good practice during mouse experimentation.&#160;Work was funded by grants from the GVA Excellence Program (2022/8) and the Spanish Research Agency (PID2022143237OB-I00) to Isabel P&#233;rez-Ota&#241;o.

In the current study, adult (2-3 months old) C57BL/6J mice of both sexes were used. Animals were housed two to five per cage with ad libitum access to food and water in an animal unit under observation and maintained in a temperature-controlled environment on a 12 h dark/light cycle. All procedures were conducted in accordance with the European and Spanish regulations (2010/63/UE; RD 53/2013) and were approved by the Ethical Committee of the Generalitat Valenciana and the animal welfare committee of the Universidad Miguel Hern&#225;ndez.
1. Behavioral room setup
<ol>
	<li>Reserve the behavioral testing room every day at the same time and establish the list and order of mice to be used, as well as arrangements for their hosting.</li>
	<li>Keep the experimental mice outside the testing room so they do not hear the sounds of the air compressor and Erasmus Ladder tones when not being tested.</li>
	<li>Check that all components of the Erasmus Ladder system are in order and ready to use: the network router, the computer with the software (see Table of Materials), the air compressor, two goal boxes, and the ladder with the rungs properly positioned.</li>
	<li>Extensively clean the goal boxes, ladder, and rungs with water after each animal and with water and 70% ethanol at the end of each training day.</li>
</ol>
2. Behavioral test protocol
<ol>
	<li>Create an experiment and enter the protocol into the software (Supplementary Figure S1).
	<ol>
		<li>Turn on the software.</li>
		<li>To create an experiment, choose File | New experiment | New or Set up | Experiment protocol. 
		NOTE: The default protocol, used in this study, is named EMC and was designed at Erasmus University Medical Center, Rotterdam.</li>
		<li>Give the experiment a name and click OK.</li>
		<li>Check that the default EMC protocol selected consists of 4 days of undisturbed sessions (42 undisturbed trials per day) and 4 days of challenge sessions (42 daily mixed trials: undisturbed, CS-only (tone), US-only (obstacle), Paired (obstacle announced by tone) (see Figure 1B). In the right side panel, also check the light cue (3 s maximum duration), air cue (45 s maximum duration), and tailwind (Yes in all the trial types), used to encourage the mouse to cross the ladder, and the tone (250 ms, Yes only in CS-only and Paired trials).</li>
		<li>To create a different protocol, choose Set up | Experiment protocol | New | From scratch or Copy from the EMC protocol and simply modify it, editing the table lines related to number of sessions (days of experiment) and number and type of trials per day. 
		NOTE: Resting time, cues type and activation, duration, intensity, and interval can also be adapted according to the experimental questions.</li>
		<li>To open the session list and name the subjects, choose Setup | Session List.</li>
		<li>Click on Add Subjects and Variables.</li>
		<li>Enter each specific Mouse Identifier, Birth Date, Sex, Genotype, and relevant categories, following the ordered list of mice.</li>
	</ol>
	</li>
	<li>Start the session (Supplementary Figure S2).
	<ol>
		<li>Before starting, check that the software is open, then turn on the Ladder.</li>
		<li>Check that the air compressor is connected and switched on.</li>
		<li>To open the Acquisition window, open the Experiment created.</li>
		<li>Choose Acquisition | Open Acquisition.</li>
		<li>Place the mouse with the identifier indicated by the software in the starting goal box (right side of the ladder).</li>
		<li>Select the mouse identifier to acquire in the first session.</li>
		<li>Click on Start Acquisition.</li>
		<li>Press the red ladder menu knob 3x. Check that the session starts and automatically controls and records mouse movements until the end of the last trial of the session.</li>
	</ol>
	</li>
	<li>End the session.
	<ol>
		<li>Check that at the end of the 42nd trial, the display shows the messages Sending Data and Acquired.</li>
		<li>Return the mouse to the home cage.</li>
		<li>Clean the ladder and the goal boxes.</li>
		<li>Place the next mouse and repeat from step 2.2.6 onwards.</li>
	</ol>
	</li>
	<li>Perform the selected type of session every day until the end of the protocol. Repeat steps 2.2 and 2.3 every day according to the selected protocol.</li>
	<li>Export the data (Supplementary Figure S2).
	<ol>
		<li>To visualize the recorded data, choose from the Analysis menu, Trial Statistics, Session statistics, and Group Statistic &#38; Charts. 
		NOTE: Data can be downloaded as a spreadsheet with data for individual trials and the means of the same trial types within a session. Sessions can also be filtered by variables chosen for specific analysis.</li>
		<li>Click on the Export button at the top-right corner choosing the file format (spreadsheet) and folder location.</li>
		<li>Right-click on the automatically generated charts and select Save to File as *.jpg.</li>
	</ol>
	</li>
</ol>
3. Data analysis
NOTE: A list of parameters is automatically measured by the Erasmus Ladder based on the instantaneous recording of the activities of the touch-sensitive sensors. For analysis, output parameters selected by the user are organized and processed in the spreadsheets. Along with the software-generated graphs, users can generate graphs using the graphing software of choice to visualize specific changes in different parameters over sessions.
<ol>
	<li>Choose specific parameters to analyze basal motivation or anxiety states, sensory responses, motor performance, and fine motor learning over the first 4 days.
	<ol>
		<li>Select and plot control parameters, including resting time in the goal box and time to exit the goal box after the resting period in response to light and air cues (Figure 2A). 
		NOTE: Resting times or response to cues are relatively constant in WT mice. Other parameters such as frequency of exits are basically negligible in WT mice-the animals rarely leave the rest box without the cues or come back once in the ladder, resulting in frequencies of exit equal to 1 per trial. If an animal goes out before cues are applied, an airflow gets activated forcing the mouse to return to the goal box; this is not counted as a trial by the software.</li>
		<li>Select and plot the time on ladder after cues, measured as the time spent crossing the ladder once the mouse leaves the goal box (Figure 2B). 
		NOTE: A power non-linear regression is a robust method for evaluating learning. The Pearson or Spearman coefficients (R) will provide a measure of whether the data fitting is good (R values close to one when the animals learn/improve over sessions; R values close to 0 imply that the data are constant and mice do not learn).</li>
		<li>Select and plot stepping pattern parameters such as the percentage of trials with missteps as a sensitive learning parameter (Figure 2C).
		<ol>
			<li>Define a correct step as a step from a high rung to another high rung (H-H), irrespective of the length of the step. Consider step types that involve a lower rung as&#160;missteps.</li>
			<li>Divide correct steps and missteps into short and long steps, backsteps, and jumps depending on the length and directionality of the step between the rungs pressed (see Figure 1A).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Select and plot specific parameters to evaluate challenged motor learning (US-only trials) and associative learning (paired trials) over the last 4 days.
	<ol>
		<li>Select and plot the time on ladder after cues (Figure 3).</li>
		<li>Select and plot the percentage of trials with missteps (Figure 4A).</li>
		<li>Select and plot the pre and post perturbation step times, defined as a ms precision difference between rung activation just before (control step) and after the obstacle (adapted step) on the same side of the ladder (Figure 4B). 
		NOTE: Pre vs post-perturbation step times analysis should be performed to compare data inside each type of session. The parameter measures the ability of the mice to predict and overcome the obstacles during associative learning.</li>
	</ol>
	</li>
	<li>Analyze the data with dedicated statistical software (e.g., SigmaPlot). Perform a power non-linear regression analysis of data collected from the same trial type across sessions to describe the learning process more efficiently and use Two-Way Repeated Measures (RM) ANOVA to compare between trial types.</li>
</ol>

Automated Assessment of Fine Motor Performance in Mice Using the Erasmus Ladder

<ol>
	<li>Brooks, S. P., 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=Tests+to+assess+motor+phenotype+in+mice:+a+user's+guide.">Tests to assess motor phenotype in mice: a user's guide.</a> Nat. Rev. Neurosci. 10 (7), 519-529 (2009).</li><li>. Available from: <a target="_blank" href="https://www.noldus.com/erasmusladder">https://www.noldus.com/erasmusladder</a> (2023)</li><li>Cupido, 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=.">.</a> Detecting cerebellar phenotypes with the Erasmus ladder[dissertation]. , (2009).</li><li>Van Der Giessen, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+olivary+electrical+coupling+in+cerebellar+motor+learning.">Role of olivary electrical coupling in cerebellar motor learning.</a> Neuron. 58 (4), 599-612 (2008).</li><li>Vinueza Veloz, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+an+mGluR5+inhibitor+on+procedural+memory+and+avoidance+discrimination+impairments+in+Fmr1+KO+mice.">The effect of an mGluR5 inhibitor on procedural memory and avoidance discrimination impairments in Fmr1 KO mice.</a> Genes Brain Behav. 11 (3), 325-331 (2012).</li><li>Vinueza Veloz, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+control+of+gait+and+interlimb+coordination.">Cerebellar control of gait and interlimb coordination.</a> Brain Struct. Funct. 220 (6), 3513-3536 (2015).</li><li>Sathyanesan, A., Kundu, S., Abbah, J., Gallo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+brain+injury+causes+cerebellar+learning+deficits+and+Purkinje+cell+dysfunction.">Neonatal brain injury causes cerebellar learning deficits and Purkinje cell dysfunction.</a> Nat. Commun. 9 (1), 3235 (2018).</li><li>Sathyanesan, A., Gallo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+contribution+to+locomotor+behavior:+A+neurodevelopmental+perspective.">Cerebellar contribution to locomotor behavior: A neurodevelopmental perspective.</a> Neurobiol. Learn Mem. 165, 106861 (2019).</li><li>McKenzie, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+skill+learning+requires+active+central+myelination.">Motor skill learning requires active central myelination.</a> Science. 346 (6207), 318-322 (2014).</li><li>Xiao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+production+of+new+oligodendrocytes+is+required+in+the+earliest+stages+of+motor-skill+learning.">Rapid production of new oligodendrocytes is required in the earliest stages of motor-skill learning.</a> Nat. Neurosci. 19 (9), 1210-1217 (2016).</li></ol>

The authors have no conflicts of interest to disclose.

The Erasmus Ladder presents major advantages for motor phenotype assessment beyond current approaches. Testing is easy to conduct, automated, reproducible, and allows researchers to assess various aspects of motor behavior separately using a single mouse cohort. In the current study, reproducibility allowed the generation of robust data with a small number of WT mice taking advantage of the features of the device, experimental design, and analysis methods. For instance, when compared to traditional beam-walk assays, the ...

A variety of tests have been developed to assess motor phenotypes in mice. Each test gives information on a specific aspect of motor behavior1. For example, the open field test informs on general locomotion and anxiety state; the rotarod and walking beam tests on coordination and balance; footprint analysis is about gait; the treadmill or running wheel on forced or voluntary physical exercise; and the complex wheel is about motor skill learning. To analyze mouse motor phenotypes, investigators must perform these tests sequentially, which involves a lot of time and effort and often several animal cohorts. If there is information at the cellular or circuitry level, the investigator normally opts for a test that monitors a related aspect and follows from there. However, paradigms that discriminate different aspects of motor behavior in an automated way are lacking.
This article describes a protocol to use the Erasmus Ladder2,3, a system that allows comprehensive assessment of a variety of motor learning features in mice. The main advantages are the reproducibility and sensitivity of the method, along with the ability to titrate motor difficulty and to separate deficits in motor performance from impaired associative motor learning. The main component consists of a horizontal ladder with alternate high (H) and low (L) rungs equipped with touch-sensitive sensors that detect the position of the mouse on the ladder. The ladder is made of 2 x 37 rungs (L, 6 mm; H, 12 mm) spaced 15 mm apart from each other and positioned in a left-right alternating pattern with 30 mm gaps (Figure 1A). Rungs can be moved individually to generate various levels of difficulty, that is, creating an obstacle (raising the high rungs by 18 mm). Coupled with an automated recording system and associating modifications of the rung pattern with sensory stimuli, the Erasmus ladder tests for fine motor learning and adaptation of motor performance in response to environmental challenges (appearance of a higher rung to simulate an obstacle, an unconditioned stimulus [US]) or association with sensory stimuli (a tone, a conditioned stimulus [CS]). Testing involves two distinct phases, each assessing improvement in motor performance over 4 days, during which mice undergo a session of 42 consecutive trials per day. In the initial phase, mice are trained to navigate the ladder to assess &#34;fine&#34; or &#34;skilled&#34; motor learning. The second phase consists of interleaved trials where an obstacle in the form of a higher rung is presented in the path of the moving animal. The perturbation can be unexpected to assess &#34;challenged&#34; motor learning (US-only trials) or announced by an auditory tone to assess &#34;associative&#34; motor learning (Paired trials).
The Erasmus ladder has been developed relatively recently2,3. It has not been extensively used because setting up and optimizing the protocol required focused effort and was specifically designed to assess cerebellar-dependent associative learning without exploring in detail its potential to reveal other motor deficits. To date, it has been validated for its ability to unveil subtle motor impairments linked to cerebellar dysfunction in mice3,4,5,6,7,8. For instance, connexin36 (Cx36) knockout mice, where gap junctions are impaired in olivary neurons, display firing deficits due to lack of electrotonic coupling but the motor phenotype had been hard to pinpoint. Testing using the Erasmus ladder suggested that the role of inferior olivary neurons in a cerebellar motor learning task is to encode precise temporal coding of stimuli and facilitate learning-dependent responses to unexpected events3,4. Fragile X Messenger Ribonucleoprotein 1 (Fmr1) knockout mouse, a model for Fragile-X-Syndrome (FXS), exhibits a well-known cognitive impairment along with milder defects in procedural memory formation. Fmr1 knockouts showed no significant differences in step times, missteps per trial, or motor performance improvement over sessions in the Erasmus Ladder but failed to adjust their walking pattern to the suddenly appearing obstacle compared to their wild-type (WT) littermates, confirming specific procedural and associative memory deficits3,5. Furthermore, cell-specific mouse mutant lines with defects in cerebellar function, including impaired Purkinje cell output, potentiation, and molecular layer interneuron or granule cell outputs, exhibited problems in motor coordination with altered acquisition of efficient step patterns and in the number of steps taken to cross the ladder6. Neonatal brain injury causes cerebellar learning deficits and Purkinje cell dysfunction that could also be detected with the Erasmus Ladder7,8.
In this video, we present a comprehensive step-by-step guide, which details the setup of the behavioral room, the behavioral test protocol, and subsequent data analysis. This report is crafted to be accessible and user-friendly and is designed specifically to assist newcomers. This protocol provides insight into different phases of motor training and expected motor patterns that mice adopt. Finally, the article proposes a systematic workflow for data analysis using a powerful non-linear regression approach, complete with valuable recommendations and suggestions for adapting and applying the protocol in other research contexts.

<table><tbody><tr><td>C57BL/6J mice (&lt;em&gt;Mus musculus&lt;/em&gt;)</td><td>Charles Rivers</td><td></td><td></td></tr><tr><td>Erasmus Ladder device</td><td>Noldus, Wageningen, Netherlands</td><td></td><td></td></tr><tr><td>Erasmus Ladder 2.0 software</td><td>Noldus, Wageningen, Netherlands</td><td></td><td></td></tr><tr><td>Excel software</td><td>Microsoft&amp;nbsp;</td><td></td><td></td></tr><tr><td>Sigmaplot software</td><td>Systat Software, Inc.</td><td></td><td></td></tr></tbody></table>

monitoring fine and associative motor learning in mice using the erasmus ladder

Behavior is shaped by actions, and actions necessitate motor skills such as strength, coordination, and learning. None of the behaviors essential for sustaining life would be possible without the ability to transition from one position to another. Unfortunately, motor skills can be compromised in a wide array of diseases. Therefore, investigating the mechanisms of motor functions at the cellular, molecular, and circuit levels, as well as understanding the symptoms, causes, and progression of motor disorders, is crucial for developing effective treatments. Mouse models are frequently employed for this purpose.
This article describes a protocol that allows the monitoring of various aspects of motor performance and learning in mice using an automated tool called the Erasmus Ladder. The assay involves two phases: an initial phase where mice are trained to navigate a horizontal ladder built of irregular rungs (&#34;fine motor learning&#34;), and a second phase where an obstacle is presented in the path of the moving animal. The perturbation can be unexpected (&#34;challenged motor learning&#34;) or preceded by an auditory tone (&#34;associative motor learning&#34;). The task is easy to conduct and is fully supported by automated software.
This report shows how different readouts from the test, when analyzed with sensitive statistical methods, allow fine monitoring of mouse motor skills using a small cohort of mice. We propose that the method will be highly sensitive to evaluate motor adaptations driven by environmental modifications as well as early-stage subtle motor deficits in mutant mice with compromised motor functions.

The Erasmus Ladder device, setup, and protocol applied are presented in Figure 1. The protocol consists of four undisturbed and four challenge sessions (42 trials each). Each trial is one run on the ladder between the starting and ending goal boxes. At the beginning of the session, a mouse is placed in one of the starting boxes. After a set time of 15 &#177; 5 s (&#34;resting&#34; state), the light is turned on (cue 1, for a maximum of 3 s). A light air cue (cue 2, 45 s maximum) is then appl...

Watch this Scientific Journal Video about Unveiling Neural Mechanisms Through Automated Evaluation of Motor Learning and Myelin Plasticity Studies Using the Erasmus Ladder at JoVE.com

Author Spotlight: Unveiling Neural Mechanisms Through Automated Evaluation of Motor Learning and Myelin Plasticity Studies Using the Erasmus Ladder 

This article presents a protocol that allows a non-invasive and automated assessment of fine motor performance, as well as adaptive and associative motor learning upon challenges, using a device called the Erasmus Ladder. Task difficulty can be titrated to detect motor impairment ranging from major to subtle degrees.

Monitoring Fine and Associative Motor Learning in Mice Using the Erasmus Ladder

Behavior is shaped by actions, and actions necessitate motor skills such as strength, coordination, and learning. None of the behaviors essential for ...

monitoring-fine-associative-motor-learning-mice-using-erasmus

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

1.1K Views.  Spain - Consejo Superior de Investigaciones Científicas and Universidad Miguel Hernández. This article presents a protocol that allows a non-invasive and automated assessment of fine motor performance, as well as adaptive and associative motor learning upon challenges, using a device called the Erasmus Ladder. Task difficulty can be titrated to detect motor impairment ranging from major to subtle degrees. 

Video: Author Spotlight: Unveiling Neural Mechanisms Through Automated Evaluation of Motor Learning and Myelin Plasticity Studies Using the Erasmus Ladder 

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. ...

Behavior

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 ...

The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their behavioral flexibility and required memory duration, the number of goals and pathways, and also the overall task complexity. A confounding feature in many of these tasks is the lack of control over the strategy employed by the rodents to reach the goal, e.g., allocentric (declarative-like) or egocentric (procedural) based strategies. The double-H maze is a novel water-escape memory task that addresses this issue, by allowing the experimenter to direct the type of strategy learned during the training period. The double-H maze is a transparent device, which consists of a central alleyway with three arms protruding on both sides, along with an escape platform submerged at the extremity of one of these arms.
Rats can be trained using an allocentric strategy by alternating the start position in the maze in an unpredictable manner (see protocol 1; &#167;4.7), thus requiring them to learn the location of the platform based on the available allothetic cues. Alternatively, an egocentric learning strategy (protocol 2; &#167;4.8) can be employed by releasing the rats from the same position during each trial, until they learn the procedural pattern required to reach the goal. This task has been proven to allow for the formation of stable memory traces.
Memory can be probed following the training period in a misleading probe trial, in which the starting position for the rats alternates. Following an egocentric learning paradigm, rats typically resort to an allocentric-based strategy, but only when their initial view on the extra-maze cues differs markedly from their original position. This task is ideally suited to explore the effects of drugs/perturbations on allocentric/egocentric memory performance, as well as the interactions between these two memory systems.

The goal of this protocol is to investigate spatial cognition in rodents. The double-H water maze is a novel test, which is particularly useful to elucidate the different components of learning, consolidation and memory, as well as the interplay of memory systems.

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their ...

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 ...

Measuring Motor Coordination in Mice

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such as emotionality or cognition can be assessed, however, it is vital to determine whether the motor capabilities of e.g. a mutant or lesioned mouse allow such an assessment. Performance on a maze task requiring strength and coordination, such as the Morris water maze, might well be impaired in a mouse by motor, rather than cognitive, impairments, so it is essential to selectively dissect the latter from the former. For example, sensorimotor impairments caused by NMDA antagonists have been shown to impair water maze performance2. 
Motor coordination has traditionally been assessed in mice and rats by the rotarod test, in which the animal is placed on a horizontal rod that rotates about its long axis; the animal must walk forwards to remain upright and not fall off. Both set speed and accelerating versions of the rotarod are available.
The other three tests described in this article (horizontal bar, static rods and parallel bars) all measure coordination on static apparatus. The horizontal bar also requires strength for adequate performance, particularly of the forelimbs as the mouse initially grips the bar just with the front paws. 
Adult rats do not perform well on tests such as the static rods and parallel bars (personal observations); they appear less well coordinated than mice. I have only tested male rats, however, and male mice seem generally less well coordinated than females. Mice appear to have a higher strength:weight ratio than rats; the Latin name, Mus musculus, seems entirely appropriate. The rotarod, the variations of the foot fault test12 or the Catwalk (Noldus)15 apparatus are generally used to assess motor coordination in rats.

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

A Novel Single Animal Motor Function Tracking System Using Simple, Readily Available Software

We have recently demonstrated that implanting intracortical microelectrodes in the motor corteces of rats&#160;results in immediate and lasting motor deficits. Motor impairments were manually quantified through an open field grid test to measure the gross motor function and through a ladder test to measure the fine motor function. Here, we discuss a technique for the automated quantification of the video-recorded tests using our custom Capadona Behavioral Video Analysis System: Grid and Ladder Test, or BVAS. Leveraging simple and readily available coding software (see the Table of Materials), this program allows for the tracking of a single animal on both the open field grid and the ladder tests. In open field grid tracking, the code thresholds the video for intensity, tracks the position of the rat over the 3 min duration of the grid test, and analyzes the path. It then computes and returns measurements for the total distance traveled, the maximum velocity achieved, the number of left- and right-handed turns, and the total number of grid lines crossed by the rat. In ladder tracking, the code again thresholds the video for intensity, tracks the movement of the rat across the ladder, and returns calculated measurements including the time it took the rat to cross the ladder, the number of paw slips occurring below the plane of the ladder rungs, and the incidence of failures due to stagnation or reversals. We envision that the BVAS developed here can be employed for the analysis of motor function in a variety of applications, including many injury or disease models.

The current study aimed to automate the quantification of motor deficits in rats. The initial evaluation model assesses motor loss resulting from an intracortical microelectrode implantation in the motor cortex. We report on the development and use of a tracking algorithm using easily adaptable, simple, and readily available coding software.

We have recently demonstrated that implanting intracortical microelectrodes in the motor corteces of rats&#160;results in immediate and lasting motor ...

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

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

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

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

Biology

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

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

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

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

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

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

A Flexible Platform for Monitoring Cerebellum-Dependent Sensory Associative Learning

Climbing fiber inputs to Purkinje cells provide instructive signals critical for cerebellum-dependent associative learning. Studying these signals in head-fixed mice facilitates the use of imaging, electrophysiological, and optogenetic methods. Here, a low-cost behavioral platform (~$1000) was developed that allows tracking of associative learning in head-fixed mice that locomote freely on a running wheel. The platform incorporates two common associative learning paradigms: eyeblink conditioning and delayed tactile startle conditioning. Behavior is tracked using a camera and the wheel movement by a detector. We describe the components and setup and provide a detailed protocol for training and data analysis. This platform allows the incorporation of optogenetic stimulation and fluorescence imaging. The design allows a single host computer to control multiple platforms for training multiple animals simultaneously.

We have developed a single platform to track animal behavior during two climbing fiber-dependent associative learning tasks. The low-cost design allows integration with optogenetic or imaging experiments directed towards climbing fiber-associated cerebellar activity.

Climbing fiber inputs to Purkinje cells provide instructive signals critical for cerebellum-dependent associative learning. Studying these signals in ...

An Open-Source Virtual Reality System for the Measurement of Spatial Learning in Head-Restrained Mice

Head-restrained behavioral experiments in mice allow neuroscientists to observe neural circuit activity with high-resolution electrophysiological and optical imaging tools while delivering precise sensory stimuli to a behaving animal. Recently, human and rodent studies using virtual reality (VR) environments have shown VR to be an important tool for uncovering the neural mechanisms underlying spatial learning in the hippocampus and cortex, due to the extremely precise control over parameters such as spatial and contextual cues. Setting up virtual environments for rodent spatial behaviors can, however, be costly and require an extensive background in engineering and computer programming. Here, we present a simple yet powerful system based upon inexpensive, modular, open-source hardware and software that enables researchers to study spatial learning in head-restrained mice using a VR environment. This system uses coupled microcontrollers to measure locomotion and deliver behavioral stimuli while head-restrained mice run on a wheel in concert with a virtual linear track environment rendered by a graphical software package running on a single-board computer. The emphasis on distributed processing allows researchers to design flexible, modular systems to elicit and measure complex spatial behaviors in mice in order to determine the connection between neural circuit activity and spatial learning in the mammalian brain.

Here, we present a simplified open-source hardware and software setup for investigating mouse spatial learning using virtual reality (VR). This system displays a virtual linear track to a head-restrained mouse running on a wheel by utilizing a network of microcontrollers and a single-board computer running an easy-to-use Python graphical software package.

Head-restrained behavioral experiments in mice allow neuroscientists to observe neural circuit activity with high-resolution electrophysiological and ...