Biased Signaling Agonists of Dopamine D3 Receptor Differentially Regulate the Effects of Cocaine On Dopamine Transporter Function

Abstract

Cocaine use disorder is a major healthcare issue with no effective FDA-approved treatments. Cocaine exerts its effects - in part - by blocking dopamine transporters (DAT) and subsequently dysregulating DAT function. Several molecular targets have been identified as key regulators of DAT function and expression including dopamine D3 receptors (D3R) that are highly expressed in the mesolimbic dopamine pathway. Although D3R partial agonists and antagonists have been shown to influence cocaine seeking in rodents, effects have been inconsistent with studies reporting varying outcomes on cocaine-associated behavior. In this study, we tested the effects of SK609, a novel G-protein biased D3R agonist, and pramipexole, an unbiased agonist of D3R, on DAT expression and function and cocaine-seeking behavior. Results indicated that SK609 reduced phosphorylation of DATs following cocaine and the uptake inhibition effects of cocaine on dopamine transmission in in vitro and ex vivo studies, respectively. By comparison, pramipexole augmented the effects of cocaine on DAT phosphorylation, enhanced dopamine levels, and increased cocaine seeking in rats. These results suggest that unbiased D3R activation promotes the effects of cocaine and that limiting D3R agonists to G-protein signaling pathways may have the potential to reduce these effects.

Keywords: G-protein biased agonist; SK609; beta-arrestin2; cocaine seeking; cocaine self-administration; dopamine D3 receptor; dopamine transporter; fast scan cyclic voltammetry; phosphorylation; pramipexole.

1

Temporal activation of DAT by D3R agonists in vitro. Time course profiles of phosphorylated DAT (pDAT) and total DAT (tDAT) expression from SH-SY5Y-D3R cells treated with D3R agonists with cocaine (CC) and without CC pretreatment. (A) Representative Western blots for tDAT, pDAT, and GAPDH (loading control). Quantification of (B) tDAT/GAPDH, (C) pDAT/GAPDH, and (D) pDAT/tDAT. (E) Representative Western blots for tDAT, pDAT, and GAPDH with cocaine pretreatment. Quantification of (F) tDAT/GAPDH, (G) pDAT/GAPDH, and (H) pDAT/tDAT. Data are represented as mean ± SEM (B–D) Šidák tests; **p < 0.01 PRX vs SK609. (F–H) Dunnett’s tests; *p < 0.05, ***p < 0.001 vs CC/SAL.

2

Effects of in vivo D3R agonists on DAT expression and phosphorylation without and following cocaine exposure. Male, Sprague–Dawley rats received ip pretreatment of either saline (SAL) or cocaine (CC) 15 min prior to treatment with saline (SAL/SAL n = 5; CC/SAL n = 6), PRX (0.25 mg/kg; SAL/PRX n = 5; CC/PRX n = 3), or SK609 (4 mg/kg; SAL/SK609 n = 3; CC/SK609 n = 6) 1x/day for 7 days. (A) Representative Western blots for total DAT (tDAT), phosphorylated DAT (pDAT), and GAPDH. Quantification of (B) tDAT/GAPDH, (C) pDAT/GAPDH, and (D) pDAT/tDAT. Data are represented as mean ± SEM normalized to SAL/SAL control. (B-D) Dunnett’s tests; *p < 0.05 vs SAL/SAL.

3

Acute ex vivo effects of D3R agonists on DAT dynamics in the NAc core. Male, Sprague–Dawley rats received an i.p. injection of saline (SAL, n = 8), (PRX, n = 8), or (SK609, n = 8) 30 min prior to euthanasia for FSCV. (A) Average baseline DA traces are depicted by solid lines with SEM represented as shading. (B) DA peak height (μM) and (C) DA uptake (μM/s, Vmax). (D) Average DA traces for 30 μM cocaine are depicted by solid lines with the SEM represented as shading. (E) DA peak height (μM) and (F) inhibition of DA uptake (μM) are quantified for each cocaine concentration. (A, D) Arrow depicts time of electrical stimulation. Data are represented as mean ± SEM (E, F) Dunnet’s tests; *p < 0.05, **p < 0.01 vs SAL.

4

Effects of D3R agonists on cue-induced cocaine seeking after repeated treatment during forced abstinence. Male, Long Evans rats were treated with saline (SAL; n = 7), pramipexole (PRX, 0.25 mg/kg; n = 6), or SK609 (4 mg/kg; n = 6) beginning on abstinence day (AD) 2. (A) Experimental timeline. (B) Average number of days to acquire self-administration behavior and (C) average active and inactive lever presses for the last two sessions of acquisition. (D) Average active and inactive lever presses as well as (E) average infusions during IntA self-administration across each session. (F) Cue-induced seeking was measured 24 h (AD1) and 7 days (AD7) into abstinence as the number of active lever presses within 1 h seeking tests. Data presented are mean ± SEM (D) Dunnett’s tests; ***p < 0.001 vs AD1. (WB, Western blot).

5

Effects of repeated D3R agonist treatment in vivo on DAT expression and phosphorylation during abstinence from cocaine. Male, Long Evans rats were treated with saline (SAL; n = 4), pramipexole (PRX, 0.25 mg/kg; n = 4), or SK609 (4 mg/kg, n = 4) on abstinence day (AD) 2 through AD8. (A) Representative Western blots for total DAT (tDAT), phosphorylated DAT (pDAT), and GAPDH (loading control) used NAc tissue collected on AD8. Quantification of (B) tDAT/GAPDH, (C) pDAT/GAPDH, and (D) pDAT/tDAT. Data are represented as mean ± SEM. Dunnett’s tests; **p < 0.05 vs SAL.

6

Effects of repeated D3R agonist treatment onex vivoDAT dynamics in the NAc core. Male, Long Evans rats were treated with saline (SAL; n = 7), pramipexole (PRX, 0.25 mg/kg; n = 6), or SK609 (4 mg/kg; n = 6) beginning on abstinence day (AD) 2 and through AD8. (A) Average DA traces for baseline recordings (i.e., no cocaine) are represented by the solid line with SEM presented as shading. (B) DA peak height (μM) and (C) DA uptake (μM/s, V _max). (D) Average DA traces for 30 μM cocaine are represented by the solid line with the SEM presented as shading. (E) DA peak height (μM) and (F) inhibition of DA uptake (μM). (A,D) Arrow depicts time of electrical stimulation. Data are represented as mean ± SEM. (D) Two-way repeated measures ANOVA, main effect of treatment; *p < 0.05.