The overall goal of this procedure is to maximize the information of the molecular readouts associated with the regulation of dopamine from a single tissue dissection from the somato dendritic regions of the midbrain rain. This is accomplished by first making a precise dissection to segregate the substantial nigra from the ventral segmental area. The second step is to process the tissues for the analysis of dopamine by HPLC and further processing of the precipitated endogenous protein for analysis.
Next, the processing of the precipitated protein for total protein content allows the determination of which dopamine regulating proteins or protein phosphorylation can be measured in conjunction with the readout of dopamine tissue content in the sample. Ultimately, the analyses of dopamine and its regulating proteins in the discrete midbrain. Somato dendritic regions enable conclusions to be drawn regarding the molecular mechanisms associated with dopamine regulation.
The monoamines affect many behavioral outcomes, and indeed researchers in the field of addiction, biology and locomotor dysfunction such as notably in Parkinson's disease are very interested in how mono means like dopamine, which we study in my lab, impact behavioral outcomes. But the importance of studying the mono means can be further embellished if we understand how they are regulated on a molecular level. The benefit of our technique is that we can take from a single sample obtained from CNS tissue, multiple readouts from that one sample so we can get a full readout of how molecular events affect the dysregulation of mono means.
We can take that data and then put it into context of how behavior is gonna be affected. Generally, individuals new to this method will struggle because determination of protein and phosphorylation require knowledge of how much total protein is gonna be needed for an accurate readout depending on the antibodies chosen by the investigator. Visual demonstration of this method is critical.
The dissection techniques and the determination of mono means with the regulating proteins is a relatively new approach to analyzing regulation in vivo. To begin this procedure, chill er rodent brain matrix, a Petri dish containing five razors and a number 11 scalpel on a bed of wet ice. Also pre-cool as many tubes as needed for the dissection on dry ice.
Then after sacrificing a rat, remove its brain and place the brain upside down with the lateral cerebellum fitting into notches in the ice cold matrix. After a minute, insert the cold razor blades into the cool brain, beginning around two millimeters, rostral to the optic chiasm for the analysis of the striatum and nucleus accumbens. Then continue to insert the cold razor blades at every one millimeter section until the midway of the ponds next, stagger the placement of each razor blade to facilitate easier removal from the matrix.
When pulling out the razors, look for the beginning of the hippocampus as it is the best guide for ensuring the presence of the SN and the VTA at the location where the hippocampus completely wraps around the midbrain. Take sections with the number 11 scalpel blade to dissect the SN from the VTA first. Remove the overlying cortex and hippocampus.
Then make a vertical cut separating the pigmented area of the SN from the VTA, making sure that the razor has constant contact with the wet ice during the dissection. Next, make a horizontal cut at or near the midline, just above the dorsal and lateral most part of the sn. Finally, tease the SN away from the rest of the brainstem.
Once the SN has been removed, tease the VTA away from the rest of the midbrain. Place the dissected tissues into the pre-cool micro fuge tubes immediately. Then store the tissues at minus 80 degrees Celsius until they're ready for processing.
In this procedure, gently homogenize the tissue immediately after removal from dry ice using ice cold 0.1 molar per chloric acid EDTA solution. After that, keep the sample on dry ice again. Next, centrifuge the samples at 12, 000 RPM to sediment.
The protein pellet to be used for western lot determinations subsequently aspirate the supernatants into the HPLC vial. Place the still frozen precipitated protein pellets derived from the HPLC buffer treatment on the wet ice to thaw and ensure that all of the buffer has been removed and archived. For bilateral processing of the SN add 300 microliters of 1%SDS containing five millimolar triss, and one millimolar EDTA to the pellet and sonicate, while for bilateral processing of the VTA 200 microliters of 1%SDS would be enough.
Next, place the samples into the boiling water for about five minutes to complete the protein denaturation. Then allow them to cool to room temperature, ensure that the caps are secured at this step. Then use the BCA assay with a protein standard curve range of two to 40 micrograms of protein.
To determine the protein concentration in the five microliter samples, use the median value obtained from a triplicate analysis to determine the protein quantity against the standard curve. Divide the result by the sample volume for protein concentration. After that, prepare the samples with the sample buffer containing DIO three etol for reducing SDS electrophoresis on 10%acrylamide gel if necessary, as indicated by a sample buffer color.
Change to yellow, add one molar tris base pH 8.2 in 10 microliter increments to the sample until the blue color is apparent. To ensure proper pH run the appropriate quantity of total protein using SDS page on 10%acrylamide gel, then transfer the proteins onto 0.45 micron pore size. Nitrocellulose, set the hoffer tank to at least 27 volts and allow the transfer to take place overnight.
In the next day, dry the blot for at least 30 minutes. Then stain it with poncho S solution for about five minutes. After that, detain it vigorously with 0.2%hydrochloric acid solution until the individual protein bands are clearly resolved and the background staining is removed.
Then obtain an image of the poncho S staining using image J in order to further quantify the relative protein loads in each lane and normalize the results of tyrosine hydroxylase, dopamine transporter, or vesicular monoamine transporter two shown here is the western blot of Syrian 31 phosphorylated tyrosine hydroxylase in the samples of rat substantial nigra. The calibrated Syrian 31 phosphorylated tyrosine hydroxylase standard curve ranges from 0.3 nanograms to 2.0 nanograms and is within the linear range of detection. Here is the western blot of Syrian 40 phosphorylated tyrosine hydroxylase in the rat ventral tegmental samples.
The calibrated SN 40 phosphorylated tyrosine hydroxylase standard curve ranges from 0.2 nanograms to 1.5 nanograms and is within the linear range of detection. Maintaining proper temperature is important as well as labeling the tubes. It's also important to normalize protein to decrease variance of your results.
This protocol, when thoroughly followed, can give investigators in the field of locomotive function and addiction behavior, extensive molecular readouts that affect mono mean regulation and brain, especially dopamine, which is what we're interested in. And as a result, these readouts can give a very detailed, thorough analysis of what it means for behavioral outcomes. After watching this video, you should have a good understanding of how to maximize readouts of monoamine regulation in the substantial nigra and ventral tegmental area from a single tissue dissection.
