This method builds upon established rat skilled reaching protocols to automate training and testing providing an efficient means to acquire large data sets relatively quickly. It minimizes experimenter effort while allowing three-dimensional reconstruction of forelimb kinematics. The kinematics can be used to evaluate the effects of precisely timed interventions or correlated with physiologic recordings.
With some adjustments, this technique can also be applied in mice. Begin by turning on the lights and place the rat in a skilled reaching chamber. Use forceps to hold a pellet through the reaching slot at the front of the box.
Allow the rat to eat three pellets from the forceps. Pull the pellet back the next time the rat tries to eat the pellet from the forceps and repeat until the rat reaches for the pellet with one paw the most out of 11 reaches indicating paw preference. Next, align the pellet delivery rod with the side of the reaching slot contralateral to the rat's preferred paw.
Place a pellet on the delivery rod. Then use forceps to bait the rat with the pellet but direct the rat towards the delivery rod so that its paw hits the pellet on the rod. After the rat has attempted 10 reaches to the delivery rod without being baited, advance the rat to the next phase.
Next, position the pellet delivery rod based on the rat's paw preference and set it to position two. Set the height position of the pellet delivery rod using the actuator remote. Once the delivery rod is at the correct height, hold down the desired number until the light blinks red to set.
Then place the rat in the chamber and bait the rat to the back with a pellet. When the rat moves far enough to the back of the chamber that it would break the infrared beam if the automated version was running, move the pellet delivery rod to position three. Finally, wait for the rat to reach for the pellet and then move the pellet delivery rod back to position two.
Place a new pellet on the delivery rod if knocked off. Repeat these steps gradually baiting the rat less and less until the rat begins to one, move to the back to request a pellet without being baited, and two, immediately move to the front after requesting a pellet in the back. After the rat has done this 10 times, begin training on the automated task.
To set up the automated system, turn on the lights in the chamber and refill the pellet reservoir. Position the pellet delivery rod according to the rat's paw preference. Check that the actuator positions are set correctly.
Next, turn on the computer and open the skilled reaching program. Enter the rat ID number under subject and select the paw preference from the hand dropdown menu. Specify the save path for the videos.
Then set the session time and the number of max videos. Set the pellet lift duration as well and enable the early reach penalty. Next, to take calibration images, place the helping hand inside the reaching chamber and poke the alligator clip through the reaching slot.
Hold the cube in front of the reaching slot with the alligator clip. Then position the cube so that the red side appears in the top mirror, the green side in the left mirror, and the blue side in the right mirror. In the behavioral program, ensure the ROI threshold is set to a very large value.
Click the run button and once the camera initialized button turns green, press start to begin video acquisition. Click Cal Mode then take an image by clicking Take Cal Image. Move the cube slightly and take another image.
Repeat again for a total of three images. Finally, stop the program by clicking stop and then click the stop sign button. Remove the helping hand and cube from the box.
Be careful not to bump anything in the behavioral chamber after calibration images have been taken that day. First, place the rat in the skilled reaching chamber. Click on the white arrow to run the program.
Then set the position of the ROI for paw detection by adjusting x offset, y offset, ROI width, and ROI height. Position the ROI in the side mirror that shows the dorsum of the paw directly in front of the reaching slot and click start to begin the program. While the rat is not reaching, adjust the low ROI threshold value until the live ROI trigger value is oscillating between zero and one.
Then set the ROI threshold to be significantly greater than the live ROI trigger value during nose pokes and lower than the live ROI trigger value when the rat reaches. Adjust until videos are consistently triggered when the rat reaches but not when it pokes its nose through the slot. Monitor the first few trials to ensure that everything is working correctly.
When a rat reaches before requesting a pellet, observe the early reaches number increase. When a rat reaches after requesting a pellet, observe the videos number increase while a copy of the video is saved as a bin file. Finally, after the session time or max number of videos is reached, press the stop sign button.
Results indicate that after 20 days, rats acquire the skilled reaching task. Here, the reach trajectories from a single session are shown. This represents only initial paw advancement for ease of presentation.
Further, if the paw detection trigger is not accurately set, there is significant variability in the frame at which the paw breaches the reaching slot which may lead to variability in when interventions are triggered during reaching movements. Other than careful handling of the animals, no precautions will be taken in this protocol. It is most important to set the ROI threshold correctly so that videos or other interventions are triggered at the appropriate moments.
This method can be paired with several other techniques such as optogenetics, calcium imaging, and genetic models which can address questions ranging from basic motor control to movement disorder pathophysiology. This technique permits correlation of detailed reach and grasp kinematics with physiologic recordings or interventions allowing researchers to answer not just if but how movements change with neural circuitry changes.
