This article describes a method for harvesting synaptosomes from mouse brain tissue to study dopamine uptake. The process involves the use of ligands and inhibitors to measure the activity of dopamine transporters.
Harvest synaptosomes, membrane-bound structures derived from the intact presynaptic terminals of neuronal synapses that contain dopamine transporters (DAT), from a mouse brain.
Fill test tubes with buffer containing ligands and reference tubes with buffer containing ligands and a DAT inhibitor.
Add the synaptosomes.
The ligands inhibit dopamine-degrading enzymes and other transporters, preventing dopamine degradation and nonspecific uptake.
Introduce unlabeled and radiolabeled dopamine.
In the test condition, synaptosomal DAT takes up the dopamine.
In the reference condition, the inhibitor prevents DAT-mediated dopamine uptake, allowing only background uptake.
Add the samples to microfiber filters that retain the synaptosomes.
Wash to remove unbound dopamine.
Transfer the filters to scintillation tubes.
Add scintillation fluid containing a radiosensitive compound.
Place the tubes in a scintillation counter.
Radioactive emissions from dopamine excite the compound to emit light, which is measured by the counter.
Subtract the reference from the test data to measure synaptosomal dopamine uptake.
In this step, add 440 microliters of uptake buffer, ligand, and cocaine to 12 designated 1.5-milliliter microcentrifuge tubes, and 440 microliters of uptake buffer and ligand to the 36 remaining 1.5-milliliter microcentrifuge tubes. Label 6 2-milliliter microcentrifuge tubes as 10, 5, 2.5, 1.25, 0.62 and 0.31. Then add 750 microliters of uptake buffer and ligand to the 5 microcentrifuge 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 4 milliliters of uptake buffer after placing them on a 12-well filter bucket. Following that, add 10 microliters of the synaptosomal membrane suspension to the first 24 1.5-milliliter microcentrifuge 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 degrees Celsius for 10 minutes. After 10 minutes, add 50 microliters of dopamine of 10 micromolar and 5 micromolar to the first 2 columns, and leave them at 37 degrees Celsius for 5 minutes with shaking. After 5 minutes, stop the reaction by adding 1 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 microfiber filters, and wash them with 5 x 4 milliliters of ice cold uptake buffer. After the first 12 samples have been added to the filters, move the filters to the scintillation tubes. Repeat this with the next 12 samples. Prepare 3 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 1 hour. Then, add 3 milliliters of scintillation fluid to each scintillation tube, and shake vigorously on a shaker for 1 hour. To count tritiated dopamine in a beta counter for 1 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.
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