Transporter Release and Uptake Assays in Rat Brain Synaptosomes

Twenty-four adult male Sprague-Dawley rats (Envigo, Frederick, MD) were used for the synaptosome assays (rats weighing 250–350 g). Rats were group housed in a temperature (22.2 ± 1.1°C)- and humidity (45% ± 10%)-controlled room on a 12-hour light/dark cycle (lights on at 0700) with free access to water. Rats were killed by CO₂ narcosis, and synaptosomes were prepared from brains using standard procedures (Rothman et al., 2003). Transporter uptake inhibition assays were performed as described previously (Solis et al., 2017; Schindler et al., 2021). In brief, synaptosomes were prepared from caudate tissue for DAT assays or from whole brain minus caudate and cerebellum for NET and SERT assays. For release assays, 9 nM [³H]1-methyl-4-phenylpyridinium (MPP+; PerkinElmer, Waltham MA) was used as the radiolabeled substrate for DAT and NET, whereas 5 nM [³H]serotonin ([³H]5-HT; PerkinElmer) was used for SERT. All buffers used in the release assay contained 1 μM reserpine to block vesicular uptake of substrates. The selectivity of release assays was optimized for a single transporter 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 hour. Release assays were initiated by incubating preloaded synaptosomes with the test drug. Release was terminated by vacuum filtration, and retained radioactivity was quantified by liquid scintillation counting. Effects of test drugs on release were expressed as percent maximum release, with maximal release (i.e., 100% E_max) defined as the release produced by tyramine at doses that evoke the efflux of all ‘releasable’ tritium by synaptosomes (10 μM tyramine for DAT and NET assay conditions and 100 μM tyramine for SERT assay conditions). For uptake inhibition assays, 5 nM [³H]dopamine, 10 nM [³H]norepinephrine, and 5 nM [³H]5-HT (all radiolabeled neurotransmitters from PerkinElmer) were used for DAT, NET, and SERT assays, respectively. To optimize uptake for a single transporter, unlabeled blockers were included that prevented uptake of [³H]transmitter by competing transporters. Uptake inhibition was initiated by incubating synaptosomes with test compound and [³H]transmitter in Krebs-phosphate buffer. Uptake assays were terminated by rapid vacuum filtration, and retained radioactivity was quantified with liquid scintillation counting (Baumann et al., 2013). Effects of test drugs on release and uptake inhibition were analyzed by nonlinear regression using GraphPad Prism v9.5.0 (GraphPad Scientific, San Diego, CA). Dose–response values for the uptake inhibition and release were fit to the equation Y(x) = Y_min + (Y_max – Y_min)/(1 + 10 exp[(logP₅₀ – logx)] × n), where x is the concentration of the compound tested, Y(x) is the response measured, Y_max is the maximal response, P₅₀ is either IC₅₀ (the concentration that yields half-maximal uptake inhibition response) or EC₅₀ (the concentration that yields half-maximal release), and n is the Hill slope parameter.