DAT oligomers are concomitant with cocaine tolerance.

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DAT oligomers are concomitant with cocaine tolerance. DAT oligomers are concomitant with cocaine tolerance. A, N2A cells were treated for 3 d with cocaine (10 μm) followed by AMPH (10 μm) on the fourth day before assessing cocaine-induced inhibition of [3H]dopamine uptake. We found that cocaine exposure produced marked tolerance to cocaine effects, that this effect was ameliorated by AMPH. Inset, Baseline uptake rate for 10 nm [3H]dopamine was unchanged in any group. B, IC50 values for cocaine effects on dopamine uptake across groups. C, Schematic describing the use of FRET imaging to measure DAT-DAT interactions. Cells were transfected with YFP-DAT and CFP-DAT or appropriate control constructs as described in Materials and Methods. D, Control conditions for FRET measurements. E, Group data showing that cocaine exposure increases FRET efficiency, and this was decreased by AMPH treatment. F, Representative computed FRET images generated by Nikon NIS Elements software using the FRET efficiency equation described above, showing effects of cocaine exposure and AMPH treatment on FRET efficiency between YFPDAT/CFPDAT. BL, baseline. Error bars indicate ± SEM. *p < 0.05 versus Vehicle+Vehicle. **p < 0.01 versus Vehicle+Vehicle. ***p < 0.001 versus Vehicle+Vehicle. ▵p < 0.05 versus Cocaine+AMPH. ▵▵p < 0.01 versus Cocaine+AMPH. ▵▵▵p < 0.001 versus Cocaine+AMPH. A, B, n = 3 per group. C–F, YFPDAT-CFP, n = 15; CFPDAT-YFP, n = 15; FRET8, n = 29; Vehicle+Vehicle, n = 26; Cocaine+Vehicle, n = 28; Cocaine+AMPH, n = 25. Cody A. Siciliano et al. J. Neurosci. 2018;38:484-497 ©2018 by Society for Neuroscience