The overall goal of this experimental protocol is to determine whether the VTA, and it's major mesotelencephatic DA projection zones would display coordinate and simultaneous c-Fos activation after novel intake of corn oil, glucose, fructose, or saccharine solutions. This method can help answer key questions in the neurobiology field, such as motivation, and identify whether multiple brain sites are activated in a coordinated fashion in response to palatable stimuli. The main advantage of this technique is that it combines the established behavioral assays, cellular activation techniques, and the statistical analyses.
To set up the apparatus, use a calibrated centrifuge tube with a robber stopper, and a 45-degree angle metal sipper tube to provide an accurate measurement of the solution presented to the rat. Secure it to the home cage by a taut metal spring, and to allow visibility of the calibration. Three to five days before the training, restrict food rations to the rat to reduce its body weight to 85%of its original weight, in order to increase motivation to consume the solution.
In the meantime, provide 10 milliliters of pre-training solution, with a 0.2%saccharine, to the animal for four days, over a one-hour session, to maximize the probability that the rat will sample the subsequent test solution with short latency. Weigh the tube before and after each session to obtain the intake measurement. Then, perform an intake test on the fifth day on subgroups receiving one of the six solutions.
Ensure that the rats sample solution with short latency, and exclude the subjects from the study if this requirement is not met. To prepare the tissue, after sacrificing the animal, remove the brain from the skull quickly. Use the rongeurs to carefully crack and remove the bone from the brain, starting from rear to front.
Work in the area below the cerebellum, and ensure the rongeurs are between the bone and the meningeal pia mater. Once the top and the sides of the skull are removed, use a small spatula to lift the brain from its base, and snip the cranial nerves with small scissors. Then, fix the brain in 4%paraformaldehyde solution overnight at four degrees Celsius.
The next day, place the brain in the sucrose solution at room temperature, until it settles at the bottom of the container. After that, remove the olfactory bulb. Subsequently, remove the cerebellum and the pons.
Then, mount the brain coronally, with the caudal part fixed to the stage of the microtome, and cut coronal sections of 40 mirometers. In this procedure, treat the sections with five milliliters of 5%normal goat serum, and 0.2%Triton x-100 in PBS, for one hour. Next, incubate the treated sections with primary antibody at four degrees Celsius for 36 hours, in the wells containing one milliliter of PBS.
After 36 hours, rinse the sections three times with five milliliters of PBS for 10 minutes each time. Then, incubate the sections with secondary antibody at room temperature for two hours, in the wells containing one milliliter of PBS. Afterward, rinse the sections three times in five milliliters of PBS for 10 minutes each time.
Next, incubate the rinsed sections for two hours in a commercially-available Avidin horseradish peroxidase mixture. After two hours, rinse the sections three times in five milliliters of PBS, for ten minutes each time. Subsequently, react the sections with DAB in the presence of hydrogen peroxide, for five to ten minutes.
After that, double label the VTA sections by incubating them with a tyrosine hydroxylase antibody, in five millilters of PBS, overnight at four degrees Celsius. The next day, rinse the sections three times in five milliliters of PBS, for ten minutes each time. Following that, incubate the sections with secondary antibody in five milliliters of PBS, at room temperature, for two hours.
After two hours, rinse the sections three times in five milliliters of PBS, for ten minutes each time. To visualize the antibody using a secondary antibody peroxidase complex, react it with a combination of DAB, and a 0.3%nickel sulfate solution, for five to ten minutes. Count the cells by double-clicking the software icon.
Go to the menu bar, click on Acquisition, and then Live Image. After that, bring the region of interest into focus, and click the screen to establish a reference point. Next, go to the grid toolbar, and click Display Grid, and Use Grid Labels.
Outline the region of interest with a predetermined trace. Then, count all the cells in each region of interest, and select Plus in the left-hand sidebar to register counts of the c-Fos cells. Consider a cell positive for c-Fos when a defined dark-red circle is observed.
Repeat this process for each site, and ensure the inter-rater reliability of the two uninformed raters for each section in each region of interest, always exceeds 0.8. Afterward, count the c-Fos positive neurons in the regions of interest. Delineate whether c-Fos immunoreactivity is present in the TH-positive and TH-negative cells in the VTA.
This image shows that the fat or sugar intake differentially increases c-Fos activation in the entire amygdala, as well as in the basolateral, and central-cortico-medial amygdala subareas. In this image, actual amygdala c-Fos activation was observed in animals exposed to intakes of corn oil, glucose, as well as fructose. C-Fos activation in the VTA was also observed in animals exposed to corn oil and water.
Black arrows indicate the representative, double-labeled, TH c-Fos positive cells, while gray arrows indicate the representative c-Fos only cells. Once mastered, this technique can be done in three to four weeks, if performed properly. While attempting this procedure, it's important to remember to follow precise methodology, storage and counting.
Thanks for watching, and good luck with your experiments.
