Fig. 1
From: An unsuspected role for organic cation transporter 3 in the actions of amphetamine
Identification of OCT3 in dopaminergic neurons and VMAT2-positive cells. a Fluorescence activated cell sorting (FACS) of a single cell solution of transgenic Dat1-eGFP midbrain cells, with the relative intensity of fluorescence at 513/26 nm against 576/21 nm after excitation at 488 nm. Inset shows similar FACS of a single-cell solution from a wild-type littermate. b Relative expression of DA and non-DA markers in the eGFP positive (eGFP+) sorted population compared with the eGFP negative (eGFP−) sorted population after normalization to housekeeping genes (ΔΔCq), dopamine transporter (Dat), tyrosine hydroxylase (Th), vesicular monoamine transporter 2 (Vmat2), glutamate decarboxylase 1 (Gad1), and nuclear receptor related 1 protein (Nurr1). Data are represented as mean obtained from three experiments. Data reveal enrichment of DA markers (Dat, Th, Vmat2, Nurr1) and a decrease in expression of the non-DA marker (Gad1) in the eGFP+ population. Organic cation transporter 3 (Oct3) was expressed in equivalent amounts in eGFP+ and eGFP− populations, as highlighted further in c, showing the expression of Oct3 relative to a fixed detection limit with Cq = 35 set to 1. Oct3 is detected in both populations (n = 3, dashed line = detection limit) with no difference in expression between the two populations (two-tailed Mann–Whitney test, p = 0.9, n.s. not significant). Importantly, the Oct3 primer pair was designed to be intron-spanning, eliminating the possibility of amplification of genomic DNA, which was also evident in data that showed no signal in the no-RT samples. Melt curve analysis of the product showed amplification of a specific target (data not shown), and primer blast analysis of the Oct3 primers showed only one product for the primer pair, that product being Oct3. d–f Expression of Oct3 in Vmat2-positive neurons in VTA. Multiplex fluorescent in situ hybridization (FISH) using RNAscope probes directed against Vmat2 (green) and Oct3 (red) performed on adult fresh frozen sections. In VTA, approximately 50% of Vmat2-positive cells were also positive for Oct3. Arrow points to a cell expressing both Oct3 and Vmat2. Arrowhead points to a cell expressing Vmat2 only. Blue: DAPI
