The overall goal of this behavioral task is to train rats to turn a knob from palm down to palm up, called supination. This tool is used to test motor system function before and after an injury or intervention. We developed this task to test corticospinal function in rats.
We found the knob supination task is more sensitive to corticospinal injury than current tasks of dexterity, such as pellet reaching. This task is semi-automated, making it quantitative, objective, and high-throughput. By quantifying supination, the task measures a clinically important movement that assesses an important neural pathway.
Demonstrating the procedure will be Thelma Bethea, our lab manager and a research technician. Throughout the protocol, train the rats twice daily, once in the morning and once in the afternoon, with at least hours between sessions. Begin with habituating the rats to familiarize the rat with the testing box and with handling.
First, set up the box. Retract the knob device fully by pressing the down arrow key on the auto-positioner. Then place the rat in the behavioral box for 15 minutes.
After 15 minutes, handle each rat by hand for at least five minutes to familiarize the rat to the experimenter. Repeat this for five days before advancing the experiment. The next phase is to create a reward association.
The goal is to train the rat to associate turning the knob with a food reward. First, open the software and input the rat's identification under Name, then set the stage to K1:Knob Shaping No Pulley. This allows the rat to be fed as long as it turns the knob past three degrees during two-second reward windows.
Next, put the rat in the behavioral box and press Start to begin the session. Familiarize the rat to the reward trough by manually dispensing two or three pellets and tapping the side of the box where the reward trough is located. Once the rat knows the reward location, only manually dispense a pellet when the rat moves in front of the aperture.
This will associate the reward with the aperture. Once the rat reliably moves to the aperture in anticipation of a reward pellet, condition the rat to interact with the manipulandum. Prompt the rat to engage with it by placing a 45 milligram pellet nearby.
If this doesn't work, apply pellet dust directly onto the manipulandum. If the rat moves away from the aperture, tap on the box near the manipulandum to redirect its attention. Only reward the rat when it touches the knob with its hands, not when it bites or noses the manipulandum.
Once the rat has gotten 10 consecutive rewards, use the down arrow key on the auto-positioner to retract the manipulandum by 0.25 centimeters. Continue repositioning the manipulandum by 0.25 centimeter increments until it is 1.25 centimeters away from the aperture. This is the ultimate goal of reward association, and once a rat does this in two consecutive sessions advance to counterweight training.
After half an hour of training, end the session by pressing Stop in the software. To set up for counterweight training, start with placing a three gram counterweight on the manipulandum at the L-shaped attachment point. Then feed the counterweight string through the pulley until the counterweight hangs freely.
To run the session, first open the software, input the rat's name, and set the stage to K2:Knob Shaping Pulley. This puts the manipulandum 1.25 centimeters from the aperture. Next, put the rat into the behavioral box and press Start in the software to begin the session.
After 100 or more successful trials, end the session by pressing Stop in the software. Once the rat has completed two consecutive sessions with over 100 successful trials, increase the counterweight's mass by one gram in the next session. Following this program, gradually increase the weight to six grams over an average of six sessions.
During this training it is imperative that the correct supination movement gets rewarded and that an incorrect supination movement gets shaped out. Shaping the supination movement continues into the baseline training as well. When correcting supination behavior, manually restrict movement of the manipulandum and reward good supination behavior even when it is unsuccessful by pressing the Feed button on the software.
At this point, train the rats to supinate to the baseline criteria, which in this case is to turn the knob 75 degrees with a six gram counterweight at a success rate of 75%or greater. After setting the counterweight, use the KSB4:Knob Training Median 75 Max option in the software. This sets the initial threshold to five degrees.
It sets the hit threshold minimum to 15 degrees, and sets the hit threshold maximum to 75 degrees. The software will automatically increase the minimum threshold for turning the knob based on the identity of the rat and its past performance, so user input is minimized at this point. After 30 minutes, stop the session.
After about 10 days or 20 sessions, most rats complete the adaptive stage and are able to turn the knob to an average peak angle of 75 degrees or greater. Once baseline training is complete, advance the rat to the next phase of training, which is handled by the software stage K27:75 Degrees. To end sessions at this stage, press stop after 30 minutes or 100 trials, whichever comes first.
Keep each rat at this stage of training until it can complete four consecutive baselines with a success rate of 75%or higher. A trained rat can then be used for experimental manipulations using this task as a measure of forelimb function. From a pool of 56 rats, 75%acquired the initial task.
This stage involved the most hands-on training. Next, the rats progressed to using a six gram counterweight by day seven, and then spent 10 days learning to turn the knob with a generally upward trend in supination angle. By day 17, most rats recorded their first baseline, and if a rat did not achieve baseline by day 20 it was removed from the study.
An unsuccessful rat can show progression for a few days but will be unable to advance with the group because it isn't working with the correct grasp. In the first third of training, the rat might begin to supinate, but the wave form of their action can reveal that it is performing two turns where successful rats only need one. In the middle third of training, the normal increase in peak angle from 20 to 50 degrees which comes with a defined single-peak curve may only show a marginal increase in peak angle in rats failing to supinate properly.
By the final third of training there should be a very pronounced single wave form;however, an unsuccessful rat may show a peak angle well below the requisite success criterion in addition to having multiple peaks. The unsuccessful rat may eventually learn the task, but it may also do so with non-supination compensations, making the rat unadvisable for use in further studies. After watching this video you should have a good understanding of how to train rats to perform forelimb supination at baseline and troubleshoot incorrect supination movements.
Be sure to stay alert and observant while the rats learn how to supinate. Correcting supination behavior at an early stage is crucial to the success of this training procedure. Overall, the procedure is meant to be time efficient.
Once the rats reach baseline, the experimenter only needs to place the rat in the box, start the software, and then remove the rat when they've completed their session. In addition to this video there are several resources available on our laboratory website. Thank you, and good luck with your experiments.
