setting up and implementing behavioral testing in mice using the erasmus ladder system

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

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.

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

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

In the lab, we study brain plasticity. One of our goals is to identify the neural circuits and mechanisms that are engaged and understand what's wrong in disease, so we can find suitable targets for interventions that are lacking. One of our critical needs is to have robust training protocols to evaluate, to induce plasticity, and evaluate the impact of genetic manipulations in healthy mice and in disease models.Current research needs sensitive, versatile, and automatic techniques to assess mice behavior. We are mainly interested in motor behavioral learning, and traditional tests requires sequential implementation, which consumes significant time and resources. Also, traditional tests don't always have enough accuracy.However, Erasmus Ladder allows motor learning future explanation and analysis in a single automated setup. While existing paradigms often focus on specific aspects of motor behavior, our approach aims to discriminate between fine motor learning, challenge motor learning, and associative motor learning in an automated and non invasive way, filling a gap in current methodologies. Testing is easy to conduct, automated, reproducible, and allow researchers to study different aspects of motor behavior separately using a single mouse cohort.The automatic software and adjustable parameters announce the precision of data collection and analysis, and also the versatility and customization of the protocol according to the scientific question. Our lab will focus on further refining the Erasmus Ladder protocols, combining cellular and molecular techniques to investigate motor adaptation and the underlying neuronal mechanisms. In particular, one of our projects focuses on myelin plasticity, a phenomenon triggered during complex motor skill learning that could help finding cures for patients with dysmyelinating disease.

Watch this Scientific Journal Video about Setting Up and Implementing Behavioral Testing in Mice Using the Erasmus Ladder System at JoVE.com

Setting Up and Implementing Behavioral Testing in Mice Using the Erasmus Ladder System  

To begin, place the experimental mice outside the testing room. In the testing room, ensure that all components of the Erasmus ladder system, including the network router, the computer with the necessary software, the air compressor, two goal boxes, and the ladder with all its rungs are ready to use. Turn on the Erasmus ladder software.To create an experiment protocol, choose File, then New Experiment. Click on File, and then New. Give the experiment a name and click Okay.Next, check that the default EMC protocol consists of four days of undisturbed sessions with 42 undisturbed trials per day, and four days of challenge sessions with 42 daily mixed trials, including undisturbed conditioned stimulus or CS-only tone, unconditioned stimulus or US-only obstacle, paired obstacle announced by tone. In the right side panel set the light cue, air cue, and tailwind, which are used to encourage the mouse to cross the ladder, and the tone. To create a different protocol, choose Setup, then Experiment Protocol and select New.Now from scratch or copied from the EMC protocol, edit the table lines related to the number of sessions and number and type of trials per day. To open the session list and name the subjects, under Setup, click Session List. Then click on Add Subjects and Variables.Enter each specific mouse identifier, birth date, sex, genotype, and relevant categories, following the ordered list of mice. Before starting the session, turn on the ladder. Ensure that the air compressor is connected and switched on.Open the previously created experiment protocol and from the acquisition window, choose Open Acquisition. Place the mouse with the identifier indicated by the software in the starting goal box. Select the mouse identifier to acquire in the first session and click on Start Acquisition.Press the red ladder menu knob three times to initiate the session. Monitor the start of the session and track mouse movements until the last trial of the session. After the completion of the 42nd trial, ensure the display shows messages Sending Data and Acquired.Then return the mouse to its home cage and clean the ladder and goal boxes. To visualize the recorded data, from the Analysis menu, select Trial Statistics, Session Statistics, and Group Statistic in Charts. Click on the Export button, then select the file format as spreadsheet in folder location.Right click on the automatically generated charts and select Save to file as star.png. Open the saved spreadsheet and choose parameters to assess basal motivation, anxiety states, sensory responses, motor performance, and fine motor learning during the initial four days. Select and plot control parameters, including resting time in the goal box, and time to leave the goal box after the resting period in response to light in their cues.Choose and plot the time on the ladder after cues, measured as the time spent crossing the ladder once the mouse leaves the goal box. Select and plot stepping pattern parameters, including the percentage of trials with missteps as an indicator of learning sensitivity. Next, choose parameters to evaluate challenged motor learning in US-only trials and associative learning in paired trials over the last four days.Select and plot the time on the ladder after cues. Then plot the percentage of trials with missteps. Finally, plot the pre-and post-perturbation step times, defining the millisecond precision difference between rung activation before and after the obstacle on the same ladder side.In wild-type mice, resting times and responses to the light cue remained consistent from days one to four. However, the response time to the air cue showed a slight decrease between days one and two. The time taken to cross the ladder exhibited a significant learning curve.Missteps during ladder crossing decreased over days one to four in undisturbed sessions. Mouse performance improved from days five to eight, yielding a significant learning curve across US-only sessions. In associative learning trials with an obstacle paired with a tone, mice completed sessions faster than in US-only trials.In control trials with tone-only presentations, a significant learning curve resembling that of undisturbed sessions was observed. The percentage of trials with missteps remained constant in US-only trials, while it significantly decreased in paired trials. A notable difference was seen between pre-and post-perturbation step times in US-only trials, but not in paired trials indicating, faster learning to overcome the obstacle in the paired trials.

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JoVE Journal

Neuroscience

132 Views.  Spain - Consejo Superior de Investigaciones Científicas and Universidad Miguel Hernández. To begin, place the experimental mice outside the testing room. In the testing room, ensure that all components of the Erasmus ladder system, including the network router, the computer with the necessary software, the air compressor, two goal boxes, and the ladder with all its rungs are ready to use. Turn on the Erasmus ladder software.To create an experiment protocol, choose File, then New Experiment. Click on File, and then New. Give the experiment a name and click Okay.Next, ...