The overall goal of this behavioral test battery, is to assess paw use assymetry in Hemiparkinsonian rats. This method can help answer key questions in the experimental deep brain stimulation field such as the motor outcome and the different stimulation patterns. The main advantage of this technique is that the cylinder test and the staircase test are complimentary and they can be both used easily in animal stimulated via cable.
Demonstrating the procedure will be Dr.Milos Krstic, from my laboratory. Always perform behavioral tests at the same time of day to avoid circadian differences in activity. For the cylinder test, begin with cleaning a 40 centimeter tall, 19 centimeter diameter clear, plastic glass cylinder with a 0.1%acetic acid solution.
Once well-cleaned, position two mirrors at a 90 degree angle behind the cylinder. Place the camera in front of the cylinder such are the distance between camera and the cylinder allows for a good view of the animal's paws when in the cylinder. Then, place the rat in the cylinder.
If the animal is connected to the stimulus generator, make sure the cable is not twisted. Now, start the recording and flash a card to the camera with the actual date of the experiment and the rat's indentification number. After five minutes of recorded activity, return the rat to its home cage.
Then, clean the cylinder again before running the next rat. In the video, count how often the front left and front right paws contact the wall. Also, count how often the rat stands on its hind paws with or without supporting itself with its front paws.
These are called rearings. One day prior to the training, familiarize the rats with the staircase apparatus and pellet rewards. First, prepare a clear plastic glass staircase box by cleaning the box with 0.1%acetic acid.
The staircase box is a two-compartment box with a raised platform and two stairs in a narrow compartment. The left steps on the stairs can be reached only with the left paw. The right steps can only be reached with the right paw.
This is a standard commercially available box, but if the rats are to be stimulated via a cable, use a high box without a lid. Now, remove the staircase and fill the wells on each step with eight 45-milligram pellets. Then, replace the staircase and put eight additional pellets on the heightened platform.
Now, place the rat in the box and after five minutes, return the rat to its home cage. Score how many pellets were eaten from the platform. Before the next test, wash the staircase and reload it with pellets.
The acquisition phase should be performed once per day for three days. Over the next three days, run the free choice phase of the test. It is executed just like the acquisition phase, but now score how many pellets were eaten from the two staircases.
If the animals have problems with grasping the pellet, add some more on the platform where they can be easily reached. Then, for six days, run the forced choice phase of the test. During this phase, only load pellets into the same side of the staircase as the occurred impairment.
A lesion of the right hemisphere would require testing the left paw and thus loading the left staircase. Over the first three of these days, load one staircase side with eight pellets per step. But on the fourth through sixth days, only load that side with four pellets per step.
For a successful staircase training, it is crucial to motivate a animal during the acquisition phase. It is important that the rat is able to grab some pellets at first grasping approach because the immediate reward resides in good training effect in the later phases. Another critical step of course, when the rat is not interested in the task, it can be then necessary to apply a mild foot restriction until the proper motivation level is reached.
Tested animals were given dopaminergic lesions and STN-placed electrodes. Data was only included in the analysis when post-mortem analysis confirmed a correct electrode placement and lesion propagation. The cylinder test revealed that the lesioned paw use decreased to about 15%After 24 hours of deep-brain stimulation delivered to the STN for 60 microseconds, at 130 hertz frequency, the use of the lesioned paw increased to roughly 22%The result was encouraging, but not significant.
In the staircase test, normal animals retrieved about half the pellets after 12 days of testing. Lesioned animals could only retrieve about 10%of the pellets. After deep-brain stimulation however, a significant improvement was made and they retrieved about 25%of the pellets.
In neither test, did the stimulation cable effect the results. Animals reared as expected and ran the staircase as expected. After watching this video, you should have a good understanding how to train the rats in the staircase test and how to use the presented behavioral test battery to screen different stimulation patterns in rats.
While attempting this procedure, it is important to remember that to handle the animals before starting the behavioral test. The test should be always performed by the same experimenter to avoid stress in the animals. Don't forget that while working with animals with implanted deep-brain stimulation electrodes, you should be extremely careful and the animals should be daily controlled for signs of pain or distress during the whole procedure.
