Measuring Dopamine Uptake in Mouse Brain Synaptosomes Using Radiolabeled Dopamine

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.