We are introducing two techniques in this protocol. The goal of the first, is to assess dopamine transporter function by measuring dopamine uptake from synaptosomal preparations. The second, is for assessing levels of dopamine in tissue by HPLC analysis.
These methods provide important parameters of dopamine homeostasis by demonstrating how to measure dopamine tissue content and assess functionality of the dopamine transporter in mice. The main advantage of these techniques is that they can be performed post in-vivo manipulations such as, chemogenetic manipulations, in-vivo drug treatments or behavioral training of the animals. Demonstrating the HPLC procedure will be Pia Weikop, Associate Professor at the Laboratory of Neuropsychiatry.
To begin this procedure, reveal the skull by cutting the skin and removing any excess tissue. Next, cut the skull along the sutura sagittalis, all the way anteriorly. Place the scissor tips in the eyes and remove the remaining skull by cutting through it.
Rapidly remove the brain and place it in ice cold PBS. After that, using a brain matrix, dissect a three millimeter coronal slice of the striatum. Followed by finer dissection with a puncture.
Subsequently, transfer the tissues to a 1.5 millimeter micro centrifuge tube for weighing. Then, repeat the procedures for the second brain. Once wait has been obtained for both brains, transfer the tissues to a homogenization glass containing one milliliter of ice cold homogenization buffer.
After that, homogenize the tissue using a motor driven pestle. Next, transfer the homogeny to a 1.5 milliliter micro centrifuge tube and collect the remaining homogeny from the homogenization glass by rinsing it with 0.5 milliliters of additional homogenization buffer. Following that, pellet the nuclei and cell debris by centrifugation.
Then, transfer the supernatant containing the cell membranes and cytoplasm to the new 1.5 milliliter micro centrifuge tubes and centrifuge for 20 minutes at four degree celsius. Afterward, discard the supernatant containing the cytoplasm and re-suspend the pellet containing crude synaptosomes in 40 microliters per milligram tissue in homogenization buffer. In this step, add 440 microliters of uptake buffer, ligand and cocaine to 12 designated 1.5 milliliter micro centrifuge tubes and 440 microliters of uptake buffer and ligand to the 36 remaining 1.5 milliliter micro centrifuge tubes.
Label six two milliliter micro centrifuge tubes as 10, 5, 2.5, 1.25, 0.62 and 0.31. Then, add 750 microliters of uptake buffer and ligand to the five micro centrifuge tubes with the lowest number. Next, add 1, 455.4 microliters of uptake buffer and ligand, 14.6 microliters of unlabeled dopamine and 30 microliters of tritiated dopamine to the tube labeled, 10.
Next, transfer 750 microliters of the mixture from the tube labeled 10, to the tube labeled 5. Mix well and transfer 750 microliters of the mixture from this tube to the tube labeled 2.5. Repeat this dilution with the remaining tubes.
After that, pre-rinse the glass microfiber filters with four milliliters of uptake buffer after placing on a 12 well filter bucket. Following that, add 10 microliters of the synaptosomal membrane suspension to the first 24 1.5 milliliter micro centrifuge tubes containing 440 microliters buffer. Vortex carefully and spin down shortly to ensure that the synaptosomes are submerged in the buffer.
Then, leave them at 37 degree celsius for 10 minutes. After 10 minutes, add 50 microliters of dopamine of 10 micromolar and five micromolar to the first two columns and leave them at 37 degrees celsius for five minutes with shaking. After five minutes, stop the reaction by adding one milliliter of ice cold uptake buffer, repeat it with the 2.5 micromolar and 1.25 micromolar and then, with the 0.62 micromolar and 0.31 micromolar.
Add the samples to the pre-rinsed micro fiber filters and wash them with five, four milliliters of ice cold uptake buffer. After the first 12 samples have been added to the filters, move the filters to scintillation tubes. Repeat this with the next 12 samples.
Prepare three max counting tubes by placing a microfiber filter in the bottom of scintillation tubes, 49 to 51 and adding 25 microliters of the maximum dopamine of 10 micromolar on top. Following this, leave all 51 scintillation tubes in a fume hood for one hour. Then, add three milliliters of scintillation fluid to each scintillation tube and shake vigorous on a shaker for one hour.
To count tritiated dopamine in a beta counter for one minute, open the program and choose the suitable settings. For protein determination, use a standard BCA protein assay kit, to determine protein concentrations of the synaptosomes for adjustments and for proper comparison of uptake between samples. For tissue preparation, add 500 microliters of homogenization solution to each sample and homogenize all the samples using an ultra homogenizer on full speed for approximately 30 seconds.
Keep the samples in iced water during homogenization. Next, centrifuge the samples for 30 minutes at four degree celsius, 14, 000 x G.After that, transfer approximately 200 microliters of each sample to a glass 0.22 micron filter before analyzing the samples by EC-HPLC methodology. To set up the detector, set the output to 700 millivolts and temperature at 32 degree celsius on the detector oven.
Place the vials in the autosampler. Make a range program using the online manual and add the time program according to table two. Set up the system by opening the program.
Next, type user ID and password and click okay. Then, wait for the program to connect to the instruments. Then, go to batch table and fill in data information.
Save file by going to file, then click, save batch file as, followed by save. Subsequently, inject the first sample by pressing start batch. Afterward, go to data acquisition to follow the chromatogram when the samples are finished.
Then, go to LC post run analysis and choose the file name. Then, click on view. On the manual integration bar, add all the peaks to be integrated, then, go to data report and print the readout.
After the program has been added to the detector, make a three point calibration curve by injecting the prepared standard in five, 10 and 15 microliters. In this figure, the representative data depicts the saturation curve of dopamine uptake through the dopamine transporter from synaptosomal preparations in wild type mice. Black dots are the values of the four mice shown separately.
The green curve shows how data would normally be depicted by combining data from four to six mice per group. Here is the saturation graph of the raw data before adjusting for actual protein concentration of the samples. This graph shows the counts from the cocaine containing samples which are used to subtract background from the uptake data.
This control is essential to determine the reliability of the uptake data. This histogram shows the uptake capacity of the dopamine transporter and this histogram shows that Km for the dopamine transporter to be 0.1 plus or minus 0.03 micromolar which corresponds well to the rotating disk voltammetry method that depicts Km values of the dopamine transporter to be 0.6 to 1.2 micromolar. While attempting the synaptosomal optic procedure, it's important to keep tissue on ice at all times by preparing the synaptosomes.
Following the synaptosome of dopamine uptake assay, additional procedures such as the surface biotinylation assay can be performed in order to determine whether a perturbed dopamine uptake can be explained by an altered surface expression of the dopamine transporter. After watching this video, you should have a good understanding of how to investigate dopamine homeostasis in mice by accessing the functionality of the dopamine transporter and dopamine levels in striatal tissue preparations.
