2.7. Monoamine Transporter Assays

Male Sprague-Dawley rats (Envigo, Frederick, MD, USA) weighing 250-350 g were housed three per cage with free access to food and water. Rats were maintained on a 12 h light/dark cycle with lights on from 7 AM to 7 PM. Animal facilities were fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, and procedures were carried out in accordance with the Animal Care and Use Committee and the National Institutes of Health guidelines on care and use of animal subjects. Rats were euthanized by CO₂ narcosis, and synaptosomes were prepared from brains as previously described.^3,11 Briefly, rat caudate tissue was used for dopamine transporter (DAT) assays whereas whole brain minus cerebellum and caudate was used for norepinephrine transporter (NET) and 5-HT transporter (SERT) assays. Tissue was homogenized in ice-cold 10% sucrose. After 12 strokes with a Potter-Elvehjem homogenizer, the homogenates were centrifuged at 1,000 g for 10 min at 4°C, and the supernatants (i.e., synaptosomes) were retained on ice.

For uptake inhibition assays, 5 nM [³H]dopamine, 10 nM [³H]norepinephrine or 5 nM [³H]5-HT was used as the radiolabeled neurotransmitter for DAT, NET or SERT, respectively. The uptake assays were optimized for the single transporter of interest by adding unlabeled blockers to prevent uptake of [³H]transmitter by competing transporters. Uptake inhibition assays were initiated by adding 100 μL of synaptosome suspension to 900 μL Krebs-phosphate buffer (126 mM NaCl, 2.4 mM KCl, 0.83 mM CaCl₂, 0.8 mM MgCl₂, 0.5 mM KH₂PO₄, 0.5 mM Na₂SO₄, 11.1 glucose, 0.05 pargyline, with 1 mg/mL bovine serum albumin and 1 mg/mL ascorbic acid, pH 7.4) containing the test compound and [³H]transmitter. Dose-response curves were generated by testing 8 different drug concentrations. Uptake inhibition was terminated by rapid vacuum filtration through Whatman GF/B filters and retained radioactivity in synaptosomes was quantified with liquid scintillation counting (Perkin Elmer).

For release assays, 9 nM [³H]MPP⁺ was used as the radiolabeled substrate for DAT and NET, whereas 5 nM [³H]5-HT was used as a substrate for SERT. All buffers used in the release assay methods contained 1 μM reserpine to block vesicular uptake of substrates. The selectivity of release assays was optimized for the single transporter of interest by including unlabeled blockers to prevent the uptake of [³H]MPP⁺ or [³H]5-HT by competing transporters. Synaptosomes were preloaded with radiolabeled substrate in Krebs-phosphate buffer for 1 h to reach steady state. Release assays were initiated by adding 850 μL of preloaded synaptosomes to 150 μL of test drug. Dose-response curves were generated by testing 8 different drug concentrations. Release was terminated by vacuum filtration, and retained radioactivity was quantified by liquid scintillation counting (Perkin Elmer). Effects of test drug concentrations were expressed as % maximum release, with maximum release (i.e., 100% Emax) defined as the release produced by 10 μM tyramine for DAT and NET assay conditions, and 100 μM tyramine for SERT assay conditions. These doses of tyramine evoke the efflux of all “releasable” tritium from synaptosomes.

Dose-response data from the uptake inhibition and release assays were subjected to non-linear curve fitting using the software program GraphPad Prism 6 (GraphPad Scientific, San Diego, CA, USA). The IC₅₀ value represents the drug concentration that yields half-maximal uptake inhibition whereas EC₅₀ value represents the drug concentration that yields half-maximal transmitter release.