The overall goal of this procedure is to use gold G Cox staining techniques to visualize the morphological changes in neurons that follow tactile stimulation early in life. This is accomplished by first administering the tactile stimulation three times daily from postnatal day three to postnatal day 21. The second step is to perfuse the animals once they reach the age of inquiry and store their brains in Goldy cos solution.
Next, the brains are sliced with Aome. The final step is to impregnate the brains with the GOGI co staining technique. Ultimately, the GOGI co procedure is used to visualize individual neurons, which permits the analysis of morphological changes.
The beauty of the Goy Cox technique is it allows us to quantify the structure of neurons and correlate that with behavioral outcomes. The implications of this technique extend toward developmental disorders as tactile stimulation has been shown to be effective in both behavioral and anatomical recovery from these disorders. Although this method can provide insight into the neuroanatomy of rats, it can also be applied to other model organisms and human brain tissue.
To help us further our understanding of experienced dependent plasticity, Animals are maintained on a 12 hour light dark cycle with free access to food and water after birth. Each female rat is housed individually with their offspring. To avoid additional stress related to handling, the pups receive tactile stimulation Starting on P three.
After weighing the pups prior to the morning tactile stimulation session, transport the pups in their home cages to a separate testing room. Place the home cage on a heating pad that is set to 24 degrees Celsius. When ready for tactile stimulation, remove dams from their home cage and place them in a temporary cage with food and water.
Keep the dam and temporary cage in the breeding room. Next, use a stiff board to divide the home cage into two halves. Then randomly assign half of the pups from each litter to undergo tactile stimulation with the other half serving as controls.
Mark their hind legs and tail with permanent marker to differentiate the groups and place the pups in the tactile stimulation group in one side of the cage and control pups in the other. Set a timer to 15 minutes using a soft feather like duster. Brush all pups in the tactile stimulation group at the same time for 15 consecutive minutes.
The young rat pups usually huddle together and appear to enter a deep sleep cycle, making it very easy to stimulate all pups at once. As the pups age, they become more active with some pups walking and investigating. During the session, move any wandering pups into the group to ensure that each pup receives equal stimulation.
After 15 minutes of tactile stimulation, return the mother to the cage and transport the cage back to the breeding room. This process is repeated three times a day. For 19 days After the final tactile stimulation session, wean the pups from their mothers Pups are housed in cages with five or six other weaned animals of the same sex leave the rats undisturbed under standard housing conditions until they reach the age of inquiry.
Samples are collected when the animals reach 100 days old. After euthanasia and intracardiac perfusion, the brain is extracted. Taking care to keep the cerebellum intact.
Place the brains in an opaque Nalgene bottle filled with 20 milliliters of Goldy Cox solution where they're kept for 14 days. After 14 days, replace the solution with a 30%sucrose solution. Keep the brains in sucrose solution for two to five days before sectioning for sectioning.
Blot the brain dry before fixing to a sectioning stage with Sano acrylic glue to prevent tearing or uneven sectioning, ensure that the entire brain is firmly secured to the stage. Next, fill the Vibram reservoir with 6%sucrose solution until it covers the sectioning blade. Set the speed and amplitude of the Vibram to five.
Slice the brain into 200 micron sections and place the sections on a 2%gelatinized microscope slide. Be sure to keep the sections wet during the course of sectioning when all of the sections of interest have been collected. Press the sections onto the slides by applying pressure with moistened ous paper.
Place the slides in a slide rack and store them in a humidity chamber for at least 12 hours, but not longer than four days. To begin the staining procedure, label and prepare 12 glass staining dishes. First, immerse the slides in distilled water for one minute.
Next, place the slides in ammonium hydroxide and keep them in the dark for 30 minutes. After an additional minute in distilled water, place the slides in Kodak Fix for 30 minutes with light protection. Place the slides in distilled water once more before dehydrating in a series of graded alcohol solutions.
After the last alcohol wash, place the slides in one third solution for 15 minutes. Lastly, place the slides in hemo D for 15 minutes. When ready, use paramount to place the cover slips on the slides and allow the slides to air dry before examining them with a microscope seen.
Here is a representative photograph of a Golgi Cox stained parametal neuron in a rat that received tactile stimulation during development. A magnified photograph shows dendritic spines on the terminal dendrites. The lighter image on the left is from a rat in the control group and the darker image is from a rat who received tactile stimulation.
This graph represents the average apical and basler dendritic complexity for neurons in the CG three area from adult rats that either received tactile stimulation during development or did not. By following this procedure, we have the advantage that if we want to ask additional questions, we've stained the entire brain and so we can go back and use the same brain tissue to answer those questions. By developing these techniques, we've paved the way for researchers to explore the usefulness of tactile stimulation as a therapy for treatment of head trauma or other forms of neurological injury After its development.
This technique paved the way for neuroscience researchers to explore its potential in remediating brain trauma and developmental disorders. After watching this video, you should have a good understanding of how to provide wrap pups with the enriching experience of tactile stimulation as well as a means by which to visualize individual neurons and synapses.
