Amphetamine Reverses Escalated Cocaine Intake via Restoration of Dopamine Transporter Conformation

Abstract

Cocaine abuse disrupts dopamine system function, and reduces cocaine inhibition of the dopamine transporter (DAT), which results in tolerance. Although tolerance is a hallmark of cocaine addiction and a DSM-V criterion for substance abuse disorders, the molecular adaptations producing tolerance are unknown, and testing the impact of DAT changes on drug taking behaviors has proven difficult. In regard to treatment, amphetamine has shown efficacy in reducing cocaine intake; however, the mechanisms underlying these effects have not been explored. The goals of this study were twofold; we sought to (1) identify the molecular mechanisms by which cocaine exposure produces tolerance and (2) determine whether amphetamine-induced reductions in cocaine intake are connected to these mechanisms. Using cocaine self-administration and fast-scan cyclic voltammetry in male rats, we show that low-dose, continuous amphetamine treatment, during self-administration or abstinence, completely reversed cocaine tolerance. Amphetamine treatment also reversed escalated cocaine intake and decreased motivation to obtain cocaine as measured in a behavioral economics task, thereby linking tolerance to multiple facets of cocaine use. Finally, using fluorescence resonance energy transfer imaging, we found that cocaine tolerance is associated with the formation of DAT-DAT complexes, and that amphetamine disperses these complexes. In addition to extending our basic understanding of DATs and their role in cocaine reinforcement, we serendipitously identified a novel therapeutic target: DAT oligomer complexes. We show that dispersion of oligomers is concomitant with reduced cocaine intake, and propose that pharmacotherapeutics aimed at these complexes may have potential for cocaine addiction treatment.SIGNIFICANCE STATEMENT Tolerance to cocaine's subjective effects is a cardinal symptom of cocaine addiction and a DSM-V criterion for substance abuse disorders. However, elucidating the molecular adaptions that produce tolerance and determining its behavioral impact have proven difficult. Using cocaine self-administration in rats, we link tolerance to cocaine effects at the dopamine transporter (DAT) with aberrant cocaine-taking behaviors. Further, tolerance was associated with multi-DAT complexes, which formed after cocaine exposure. Treatment with amphetamine deconstructed DAT complexes, reversed tolerance, and decreased cocaine seeking. These data describe the behavioral consequence of cocaine tolerance, provide a putative mechanism for its development, and suggest that compounds that disperse DAT complexes may be efficacious treatments for cocaine addiction.

Keywords: agonist therapy; behavioral economics; nucleus accumbens; self-administration; tolerance; voltammetry.

Figure 1.

AMPH treatment prevented escalation of cocaine self-administration. A, Timeline showing conditions across days (top) and within days (bottom). B, Total infusions per session were lowered by AMPH treatment during long-access, but not short-access, self-administration. C, Session 14 minus session 1 infusions reveal that LgA+Saline animals escalate intake over time, whereas all other groups do not. Error bars indicate ± SEM. *p < 0.05 versus LgA+Saline. **p < 0.01 versus LgA+Saline. ****p < 0.0001 versus LgA+Saline. ^#p < 0.05 versus 0. ShA+Saline, n = 6; ShA+AMPH, n = 4; LgA+Saline, n = 7; LgA+AMPH, n = 7.

Figure 2.

AMPH treatment prevented increase in motivation to administer cocaine. A, Experimental timeline showing when assessment of motivation to obtain cocaine (threshold procedure) was performed. Representative data from LgA+Saline (B) and LgA+AMPH (C) animals showing that the reinforcing efficacy of cocaine (P_max) is decreased by AMPH treatment. D, LgA-induced increases in reinforcing efficacy are prevented by AMPH treatment during LgA. Importantly, reinforcing efficacy stays depressed for up to 7 d following cessation of treatment. Similar results were seen with O_max (E), but not Q₀ (F). Error bars indicate ± SEM. *p < 0.05 versus LgA+Saline. LgA+Saline, n = 5; LgA+AMPH, n = 7.

Figure 3.

AMPH treatment prevented cocaine-evoked alterations to rapid dopamine signaling and cocaine potency. A, Timeline showing conditions across days (top) and within days (bottom). B, Dopamine release elicited by phasic-like stimulations (5 pulse, 100 Hz) is reduced in LgA animals, and this effect is prevented by treatment with AMPH during cocaine self-administration. C, Representative traces elicited by 100 Hz stimulation trains indicating that phasic dopamine release is attenuated in LgA+Saline animals, and that this effect is prevented by AMPH treatment. D, Phasic/tonic ratio (100 Hz/5 Hz) is reduced in LgA animals, and this effect is prevented by AMPH treatment. E, Maximal rate of dopamine uptake (V_max) is unaffected in all treatment groups when AMPH treatment is given during cocaine self-administration. F, Quinpirole concentration response curves show no change in autoreceptor sensitivity across all treatment groups. G, Representative traces following bath application of 30 μm cocaine. H, Pseudo-color plots for LgA+saline (top) and LgA+AMPH (bottom) animals following bath application of 30 μm cocaine. I, Group data demonstrating that LgA cocaine self-administration results in a reduction in the ability of cocaine to inhibit the DAT, and that this effect is selectively prevented by AMPH treatment. Error bars indicate ± SEM. *p < 0.05 versus ShA+Saline. **p < 0.01 versus ShA+Saline. ***p < 0.001 versus ShA+Saline. ShA+Saline, n = 6; ShA+AMPH, n = 4; LgA+Saline, n = 6; LgA+AMPH, n = 7.

Figure 4.

AMPH treatment reversed cocaine-induced changes to dopamine signaling. A, Experimental timeline depicting 14 d of cocaine self-administration (LgA or ShA) followed by a 7 d abstinence period. During the abstinence period, animals were treated with either saline or AMPH delivered via osmotic mini-pump. B, Dopamine release elicited by tonic-like frequency stimulations (5 pulse, 5 or 10 Hz) is unchanged in any treatment group. Dopamine release elicited by phasic-like frequency stimulations (5 pulse, 100 Hz) is increased in LgA animals after a 7 d abstinence period, and this effect is reversed by treatment with AMPH for 7 d following LgA. C, Representative traces indicating that phasic dopamine release is augmented in LgA animals after an abstinence period, and that this effect is reversed by AMPH treatment. D, Phasic/tonic ratio (100 Hz/5 Hz) is increased in LgA animals after a 7 d abstinence period, and this effect is reversed by AMPH treatment. E, Maximal rate of dopamine uptake (V_max) is unaffected in all treatment groups when AMPH treatment is given during abstinence. F, Quinpirole concentration response curves show no change in autoreceptor sensitivity across all treatment groups. G, Representative traces following bath application of 30 μm cocaine indicating that cocaine is less effective at inhibiting dopamine uptake following LgA cocaine self-administration and abstinence. H, Pseudo-color plots for LgA+Saline (top) and LgA+AMPH (bottom) animals following bath application of 30 μm cocaine. I, Group data demonstrating that LgA cocaine self-administration animals remain tolerant to cocaine's effects following a 7 d abstinence period, and that this effect is reversed by AMPH treatment during abstinence. Error bars indicate ± SEM. *p < 0.05 versus ShA+Saline. **p < 0.01 versus ShA+Saline. ****p < 0.0001 versus ShA+Saline. ShA+Saline, n = 6; ShA+AMPH, n = 5; LgA+Saline, n = 4; LgA+AMPH, n = 5.

Figure 5.

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.

Figure 6.

Cocaine reverses escalated cocaine intake. A, Experimental timeline of 28 d of cocaine self-administration split into three epochs: (1) 14 d of self-administration with no treatment (initial escalation period), (2) 7 d of AMPH treatment during cocaine self-administration (treatment period), and (3) 7 d of self-administration after cessation of treatment (post-treatment period). B, Average infusions per day over the 28 d of self-administration. Dotted line indicates average intake across groups on day 1. Shaded area represents the AMPH treatment period. C, Cocaine intake was decreased by AMPH treatment during the treatment and post-treatment periods. Error bars indicate ± SEM. *p < 0.05 versus LgA+Saline. **p < 0.01 versus LgA+Saline. LgA+Saline, n = 5; LgA+AMPH, n = 5.

Figure 7.

Summary of findings. Top, Model of the molecular underpinnings of the development of cocaine tolerance and restoration by AMPH. Bottom, Model of cocaine-induced changes to phasic dopamine signaling, cocaine potency, and reinforcing efficacy of cocaine over the course of LgA and abstinence. All three measures are normalized to control levels by AMPH.