The overall goal of this procedure is to investigate the function of the transcription factor NF kappa B in the adult brain. This is accomplished by first breeding transgenic mice that specifically inhibit NF kappa B activity in neurons. The second step of the procedure is to test the behavioral consequences of the NF kappa B inhibition using the spatial pattern, separation or barns, maze.
The third step is the careful profusion of the animals, removal of the brains, and the proper sectioning thereof. The final step is to analyze histological and structural effects, such as visualizing mossy fiber projections by immuno staining. Ultimately using the behavior test followed by histology can correlate changes in dentate gyrus dependent learning with structural changes in associated tissues.
The main advantage of this technique over existing methods like Mars Vase is that spatial pattern separation bonds MA offers the opportunity to specifically measure anti child dependent learning processes as well as neuro degeneration and regeneration in this brain area. This method can help to answer key questions in the field of adult neurogenesis, such as learning as special pattern separation. Demonstrating the maze will be done by Angela Kula, a technician from my lab.
The perfusion will be presented by Peter Hyman, a research associate. Then the sectioning of brains will be shown by Ulrich KA technician. And finally, the confocal scanning microscopy will be presented by Jeanine Muah, a PhD student of my lab Carry out the spatial pattern separation.
Barnes Maze Behavioral testing according to international and local guidelines. For this assay, it is important to use only male mice at least six months old. For each test series, the animals must be within four days of age and the same operator should conduct all the tests prior to the testing.
The mice should be fed a standard diet so that they are motivated by sweet food. To set up the test first, obtain a 120 centimeter diameter white plate made out of hard plastic. The extra maze cues can be made of different materials and may have different shapes.
Importantly, they must have high contrast to be clearly recognized by the animal running wheels. Ladders and tunnels are all good cues. Position the plate under several neon fluorescent lamps that are moderately intense, so the mice are motivated to get out of the light.
The room must be humidity controlled and at a constant 22 degrees Celsius. Now set up the video tracking system. Position the camera 115 centimeters above the center of the plate.
Next, attach multicolored extra maze cues to a white colored cloth that fits the plate. Position them near the center of the cloth so that they're easily visible to the animals. Now, carefully clean the plate with the rapid disinfectant that can remove any odor from the experimental setup up.
Position seven identical yellow food houses on the white plate. Their positions should be unequivocally marked one day before starting the tests, habituate each mouse to the disc. Place sweet food pellet rewards like a quartered fruit loop inside each of the seven food houses for the habitation trial.
Put each mouse on the start point of the plate and allow it 10 minutes to explore. Record this exploration between mice. Clean the circular plate and the food houses with rapid disinfectant.
Also replenish the food rewards. On the next seven consecutive days, run the test trials for the tests. All but one house is closed by transparent tape for the whole experiment.
It is always the same house. Each house is still loaded with a reward and the test is run Like the habituation trial. Run the tests at the same hour of the day for each animal to avoid circadian variations.
Later, analyze the latency, distance covered and errors using appropriate statistics. Software define errors as approaching the wrong food house or making contact with the proper box, but failing to enter and retrieve the reward. To compare the groups use a two-way ANOVA with a bon fer post hoc test.
After anesthetizing the animal with an interperitoneal injection of vertin, carefully perfuse it trans cordially with PBS containing heparin and propane. Make a hole in the left ventricle using a 26 gauge needle. Also make a cut in the right atrium to allow the blood and perfuse eight to flow out.
Insert the perfusion needle into the left ventricle at the hole with a straight angle and to a depth of one to two millimeters. Use 1.2 to 1.4 meters of hydrostatic pressure and perfuse the mouse over two to four minutes. Follow the heparin and propane with 4%paraldehyde in PBS for 10 to 15 minutes.
The perfusion should be a rate of 15 milliliters per minute. Now dissect out the brain. It should be pale white with no visible red blood vessels.
Fix all the collected brains in 4%para formaldehyde at four degrees Celsius for 24 hours. The next day. Cryo protect the brains in 30%sucrose in PBS store them at four degrees Celsius for at least 24 hours later.
The brains are frozen on a cryo tone stage and sliced into 40 micron thick horizontal sections. After staining the sections with an antibody for neuro filament M, the mossy fiber projections can be visualized using a confocal laser scanning microscope at the appropriate wavelength for the fluorescent antibody. Start the scanning at low magnification and orient to the hippocampus and mossy fiber projections.
Then at higher magnification, collect high resolution C-section scans of the mossy fibers for analysis to investigate the expression of the I kappa B alpha AA one transgene. In the double transgenic mouse brains were isolated, cryo sectioned and stained using an antibody against GFP confocal laser scanning. Microscopy revealed high expression of the transgene in the CA one region, the CA three region and in the DG to determine the earliest cell type expressing the CAMK two alpha driven I kappa B alpha AA one in the hippocampal neurogenic region.
Cryo sections of hippo Campi from I Kappa BTTA. Animals were stained for double courtin and the transgene confocal analysis revealed GFP signals in young DCX positive granule cells marked by the arrows indicating induction of the expression of the transgene by CAMK two alpha activity. Coronal sections of the hippocampus of i kappa BTTA and i Kappa B minus mice were stained for neurofilament M to visualize mossy fiber projections and reveal complex and vesicular organization in I kappa BTTA mice.
The mossy fiber projections were severely impaired. In addition, NF kappa B inhibition led to a significantly reduced speral blade thickness and a smaller DG volume to study the potential reasons for the dramatic structural defect. Fresh unfixed hippocampal slices from control and I kappa BTTA mice were stained with fluoro jade C, which allows the specific detection of neuronal cell death.
A dramatically increased number of degenerating neurites were observed in double transgenic animals compared with single transgenic controls. Moreover, a significant increase of cleaved capace three positive cells was detected in I kappa BTTA Mice. By contrast, immunohistochemical staining against DCX revealed significantly increased amounts of DCX positive cells in the brains of I kappa BT mice.
Furthermore, d CX positive cells in I kappa B TTA hippo Campi were not arranged exclusively in the sub granular zone, but were also found in the deeper regions of the dg. Whereas the D CX positive progenitors in the control animals were localized nearly exclusively within the sub granular zone. To test of the increased amount of DCX expressing cells was a result of maturation of earlier progenitors or directly due to proliferation of DCX positive cells.
BRDU was injected intraperitoneal of i kappa BTTA and control mice, which were then cryo sectioned and stained with DCX and BRDU for analysis of proliferation. BRDU was injected once followed by analysis after 24 hours. For analysis of differentiation, three injections were performed daily, followed by analysis after seven days after a single BRDU injection.
No significant differences in the total number of BRDU positive cells between I kappa BTTA negative and control animals were observed. By contrast, the three serial injections approach revealed a clearly increased amount of B-R-D-U-D-C-X positive cells in the DG of I capa, BTTA. Mice disturbed differentiation or integration of DCX expressing cells may have increased cell numbers and type three cells may have been involved to specifically test the behavior of the double transgenic mice.
In a DG dependent task, the S-P-S-B-M was employed. The I kappa B control animals explored the food houses serially on the first day of the experiment. After seven days of training, the animals were able to find the open food house directly.
In contrast, the IA BTTA animals continue to explore the food houses serially even after the training. The observation of significant increases in the distances covered and higher latencies and error rates in the I kappa BTTA animals compared with the i Kappa B control animals suggests that these animals had severe learning defects. Once established, this technique can be done in 30 minutes per animal if it's performed properly After its development.
This technique might pave its way to other neuroscientists interested in daid gyros dependent tasks such as adult neurogenesis degeneration or regeneration in mice.
