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.
