In vitro uptake assays in synaptosomes

Rats (n = 16) were euthanized by CO₂ narcosis and their brains were processed to produce synaptosomes as previously described [3, 30, 31]. Neurotransmitter uptake by DAT, NET, or SERT was assessed using 5 nM [³H]dopamine, 10 nM [³H]norepinephrine or 5 nM [³H]serotonin, respectively. For DAT, the selectivity of assays was established by surgical isolation of the caudate putamen, a region that is so enriched in DAT that measurable uptake of [³H]dopamine by NET or SERT does not occur. For SERT, the selectivity of the assay was established by adding GBR 12935 (50 nM) and nomifensine (100 nM) to block uptake of [³H]serotonin by DAT and NET, respectively. For NET, the selectivity of the assay was established by adding GBR 12935 (50 nM) to block uptake of [³H]norepinepherine by DAT; [³H]norepinepherine has low affinity for SERT, and therefore selective blockade of SERT is not required. The potent non-selective uptake inhibitor indatraline (1 µM) was used to define non-specific binding at DAT, NET, and SERT. Tissue suspension (100 μl) was added to tubes containing the test drug, [³H]neurotransmitter, and 900 μl Krebs-phosphate assay buffer (126 mM NaCl, 2.4 mM KCl, 0.83 nM CaCl₂, 0.8 nM MgCl₂, 0.5 nM KH₂PO₄, 0.5 mM Na₂SO₄, 11.1 mM glucose, 0.05 nM pargyline, 1 mg/ml bovine serum albumin, and 1 mg/ml ascorbic acid, pH 7.4) to begin the uptake inhibition assay. Rapid vacuum filtration through Whatman GF/B filters terminated the assay, and liquid scintillation counting was used to quantify the retained radioactivity.