The overall goal of this proteomic approach is the characterization of molecular reorganization at synapses after learning and memory formation to understand basic mechanisms and signaling pathways. The main advantage of this technique is a hypothesis-free approach, permitting large-scale quantification and characterization of post-translation and modifications within synaptic proteomes. Our established workflow allows a correlation between a certain molecular change and a particular behavior.
Learning and memory formation are based on changes of synaptic efficacy and therefore proteomic studies should focus more on analysis of synaptic structures like synaptosomes than on homogenates from brain tissue. Proteomic results represent highly complex datasets therefore require bioinformetic processing. Begin training by placing the test mouse in a dimly lit shuttle box within a soundproof chamber.
Use a fully computer-controlled learning schedule for auditory discrimination learning. Begin with a habituation period of three minutes of silence before starting the training session. During go trials, the animal has to cross the hurdle within six seconds of tone presentation to score a hit.
During no-go trials, the animal has to remain in the current compartment of the shuttle box during the six seconds of tone presentation. Perform 30 go trials and 30 no-go trials in a pseudo-randomized order, with an inter-trial interval of 20 seconds so that one session consists of 60 trials and lasts about 25 minutes. Once all of the trials have been performed, return the trained mouse to the home cage until sacrifice.
After sacrificing the mouse and removing the brain, localize the auditory cortex using visual landmarks including blood vessels and the shape of the surface. Then use a scalpel and needle to bilaterally dissect the auditory cortex as a rectangular tissue block. Using the chiasma opticum as a landmark, dissect the frontal cortex as a brain slice between Bregma 3.56 and 1.54.
Dissect the striatum as a brain slice between Bregma 1.54 and 0.5. And carefully remove the cortical tissue. Dissect the hippocampus by stabilizing the brain with the needle through the cerebellum and uncoiling the cortex starting at the occipital lobe.
Shock-freeze dissected brain samples in liquid nitrogen. Begin synaptosome purification by transferring the dissected brain tissue into homogenization vessels containing one milliliter of ice-cold Buffer A.Then homogenize the tissue at 900 RPM using around 12 strokes. Centrifuge the samples at 1, 000 times G for 10 minutes.
After the centrifugation, retain the supernatants. Re-homogenize the pellet as before, and combine the supernatants. Spin the combined supernatants for 20 minutes at 12, 000 times G.Re-suspend the pellets in one milliliter of homogenization buffer and homogenize with six strokes at 900 RPM followed by centrifugation at 1, 200 times G for 20 minutes.
During the centrifugation to produce the P2 pellets prepare sucrose step gradients in ultracentrifuge tubes. Start with 2.5 milliliters of 1.0 molar sucrose buffer and then use a glass Pasteur pipette to sublayer 1.5 milliliters of 1.2 molar sucrose buffer. Following the centrifugation, add 0.5 milliliters of 0.32 molar sucrose buffer to the P2 pellets.
Then re-homogenize using six strokes. Load the homogenized fractions on top of the gradients. Place the loaded gradients in a swinging bucket rotor and then spin at 85, 000 times G for two hours in an ultracentrifuge.
Once centrifugation is complete, discard the top 0.32 molar sucrose layer, including the material at the interface to the 1.0 molar sucrose buffer. Collect synaptosomes at the one to 1.2 molar sucrose buffer interface. Add 0.32 molar sucrose buffer to the synaptosomal fraction at a 1.1 ratio.
Mix carefully and spin at 150, 000 times G for one hour. Synaptosomes are in the pellet, and can be re-suspended in a buffer for further processing. Dissolve the synaptosomes of each brain area of an animal in 20 to 50 microliters of 8 molar urea by incubating on ice for one hour in an ultrasonic bath.
Dilute with one percent of a removable detergent to ensure a final concentration of two molar urea. After determining relative protein amounts using SDS-PAGE pipette one third of the remainder of each sample into a fresh tube. Perform the in-solution digest by adding two millimolar DTT and 25 millimolar ammonium bicarbonate and gently vortex the sample.
Reduce the samples for 45 minutes at 20 degrees Celsius. Add 10 millimolar iota acedomide to carbamidomethylate cysteine residues and mix. Incubate for 30 minutes in the dark at 20 degrees Celsius.
Finally, add one microliter of a trypsin stock solution and incubate at 20 degrees Celsius for 12 hours. To remove the acid-cleavable detergent, add trifluoroacetic acid to a final concentration of one percent and incubate for a further hour at 20 degrees Celsius. After centrifuging the samples at 16, 000 times G for 10 minutes, carefully collect the supernatants.
Place the solid-phase extraction column in a rack and equilibrate the matrix with two milliliters of methanol. After washing, add an additional two milliliters of Buffer B and load the sample. After washing three times with Buffer B, elute the peptides by adding 200 microliters of 70 percent acetonitrile and 0.1 percent triflouroacetic acid.
Then dry the purified samples in a vacuum centrifuge. In this example, animals trained on the FM tone discrimination task show an increasing rate of hits and a decreasing rate of false alarms over the course of training sessions. Significant discrimination occurs from the fourth session.
The relative synaptic abundances of selected proteins are compared between mice trained on the FM tone discrimination task and naive control mice 24 hours after the first training session. After training, levels of the CYFP2 protein are altered in all regions studied. Don't forget that the animals often exhibit often intra-individual variabilities.
Therefore it is highly recommended to include at least five or more biological replicates of well-matched animal groups in the study. Once established, this technique can easily be adapted over to other species. For example, it has been used to monitor learning-dependent protein expression changes in single fruit fly brains.
