The monoamine stabilizer (-)-OSU6162 counteracts downregulated dopamine output in the nucleus accumbens of long-term drinking Wistar rats

Abstract

We recently established that the monoamine stabilizer (-)-OSU6162 (OSU6162) decreased voluntary alcohol-mediated behaviors, including alcohol intake and cue/priming-induced reinstatement, in long-term drinking rats, while blunting alcohol-induced dopamine output in the nucleus accumbens (NAc) of alcohol-naïve rats. Therefore, we hypothesized that OSU6162 attenuates alcohol-mediated behaviors by blunting alcohol's rewarding effects. Here, we evaluated the effects of long-term drinking and OSU6162 treatment (30 mg/kg, sc) on basal and alcohol-induced (2.5 g/kg, ip) NAc dopamine outputs in Wistar rats after 10 months of intermittent access to 20% alcohol. The results showed that basal and alcohol-induced NAc dopamine outputs were significantly lower in long-term drinking rats, compared with alcohol-naïve rats. In the long-term drinking rats, OSU6162 slowly increased and maintained the dopamine output significantly elevated compared with baseline for at least 4 hours. Furthermore, OSU6162 pre-treatment did not blunt the alcohol-induced output in the long-term drinking rats, a finding that contrasted with our previous results in alcohol-naïve rats. Finally, OSU6162 did not induce conditioned place preference (CPP) in either long-term drinking or alcohol-naïve rats, indicating that OSU6162 has no reinforcing properties. To verify that the CPP results were not due to memory acquisition impairment, we demonstrated that OSU6162 did not affect novel object recognition. In conclusion, these results indicate that OSU6162 attenuates alcohol-mediated behaviors by counteracting NAc dopamine deficits in long-term drinking rats and that OSU6162 is not rewarding on its own. Together with OSU6162's beneficial side-effect profile, the present study merits evaluation of OSU6162's clinical efficacy to attenuate alcohol use in alcohol-dependent patients.

Keywords: Alcohol dependence; condition place preference; ethanol; medication development; microdialysis.

Figure 1

The dopamine output in the nucleus accumbens following a systemic alcohol challenge was blunted in long‐term drinking rats. To evaluate the effects of an acute alcohol challenge (2.5 g/kg, ip) on the dopamine output in the nucleus accumbens following long‐term voluntary alcohol consumption, microdialysis was applied in awake Wistar rats after 10 months of intermittent access to 20% ethanol in a two‐bottle‐choice paradigm (n = 7) and in age‐matched alcohol‐naïve rats (n = 5). (a) The alcohol challenge affected dopamine output (mean ± SEM, fmol/min) differently in long‐term drinking and alcohol‐naïve rats as shown by a significant overall condition and a condition * time interaction effect (two‐way repeated‐measures ANOVA with condition as the between‐subject factor and time as the within‐subject factor). (b) The alcohol‐induced dopamine peak was significantly blunted in the alcohol‐drinking compared with alcohol‐naïve rats as revealed by comparison of the area under the curve (ΔAUC) between timepoints 0 and 45 minutes in relation to timepoint 0 within each group (Student's t‐test, *P < 0.05 compared with alcohol‐naïve rats)

Figure 2

(−)‐OSU6162 (OSU6162) counteracts the hypo‐dopaminergic state in the nucleus accumbens induced by long‐term voluntary alcohol drinking. Microdialysis was used to measure nucleus accumbens output of dopamine and the dopamine metabolites 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) following treatment with vehicle–alcohol (2.5 g/kg, ip) (a), OSU6162 (30 mg/kg, sc)–vehicle (b) and OSU6162–alcohol (c) (n = 7–8 per treatment) in awake Wistar rats, which had lower basal dopamine output after 10 months of drinking in the intermittent access to 20% ethanol two‐bottle‐choice paradigm (during 24‐hour withdrawal) than alcohol‐naïve age‐matched controls. (a) The acute alcohol challenge significantly increased and decreased dopamine output compared with baseline between 15 and 30 minutes and between 90 and 165 minutes after injection, respectively. DOPAC and HVA outputs were significantly increased compared with baseline at 30 and 30–135 minutes following the alcohol challenge, respectively. (b) OSU6162 administration significantly increased and maintained dopamine (from timepoint −45 minutes), DOPAC and HVA (from timepoint −30 minutes) outputs elevated compared with baseline throughout the experiment (i.e. at least 4 hours after the OSU6162 injection). (c) When OSU6162 was administered 60 minutes before the alcohol challenge, dopamine was significantly elevated compared with baseline from −45 to 60 minutes, and DOPAC and HVA outputs were significantly increased compared with baseline at all measured timepoints. Values are presented as percent of respective individual baseline (mean ± SEM), because basal values (fmol/min) did not differ between treatment groups (one‐way ANOVA). Data were analyzed using repeated‐measures one‐way ANOVA within each separate treatment group with time as the within‐subject factor followed by Fisher's least significant difference post hoc test. Arrows indicate time of injections. Asterisks indicate timepoints when dopamine output was significantly different from baseline (*P < 0.05, **P < 0.01, ***P < 0.001). To avoid clutter, asterisks were omitted for DOPAC and HVA. OSU, OSU6162; Alc, alcohol; Veh, vehicle

Figure 3

Pre‐treatment with (−)‐OSU6162 (OSU6162) had no significant effect on alcohol‐induced dopamine output in the nucleus accumbens of long‐term drinking rats. To evaluate the effects of OSU6162 specifically on the alcohol‐induced dopamine output in the nucleus accumbens in the long‐term drinking rats, the percent change in dopamine output in relation to the output at the timepoint immediately prior to the alcohol injection (timepoint 0 minutes) was compared between the rats treated with vehicle–alcohol (2.5 g/kg, ip) or OSU6162 (30 mg/kg, sc)–alcohol. There was no significant difference in the OSU6162‐pretreated compared with vehicle‐pretreated groups (repeated‐measures two‐way ANOVA with treatment as the between‐subject factor and time as the within‐subject factor). Values are presented as mean ± SEM (n = 7 per treatment)

Figure 4

(−)‐OSU6162 (OSU6162) did not induce conditioned place preference (CPP) in either long‐term drinking or alcohol‐naïve rats. To evaluate the reinforcing effects of OSU6162, alcohol‐naïve rats and rats that had been drinking alcohol (intermittent access to 20% ethanol) for 3 months prior to the experiment were subjected to the CPP paradigm. (a) First, the CPP boxes were validated by showing that morphine conditioning (10 mg/kg, sc) induced CPP as shown by a significant increased time spent on the morphine‐paired side during post‐conditioning compared with pre‐conditioning. In contrast, there was no expression of CPP in the vehicle‐treated rats. OSU6162 (30 mg/kg, sc) and vehicle treatment did not induce CPP in either (b) alcohol‐naïve or (c) alcohol‐drinking rats. All values are presented as mean ± SEM (n = 8–9 per treatment). Data were analyzed using paired Student's t‐test within each treatment group (comparing time spent on the drug‐paired side during post‐conditioning with the same side during pre‐conditioning); ***P < 0.001 compared with corresponding pre‐conditioning

Figure 5

(−)‐OSU6162 (OSU6162) did not impair object memory 2 hours after object presentation. Rats were allowed to explore two identical objects for 2 minutes during a training session, and following a 2‐ or 24‐hour intersession interval, rats were presented with one familiar object from the training session and one novel object during the 5‐minute testing session. A discrimination ratio (mean ± SEM, exploration of novel object divided by exploration of both objects) above 0.50 during the testing phase indicates remembrance of the familiar object. (a) Rats remembered the familiar object after a 2‐hour, but not after a 24‐hour, intersession interval (n = 16 per condition). (b) Total time spent to explore both objects during the training session was not significantly different between OSU6162‐treated (30 mg/kg, sc; n = 8, 60 minutes before S1) and vehicle‐treated rats (n = 8). (c) Using a 2‐hour intersession interval, there was no significant difference on discrimination ratio between OSU6162‐treated and vehicle‐treated rats during the testing session. All values are expressed as mean ± SEM. Data were analyzed using unpaired Student's t‐test within each session;***P < 0.001 compared with the 24‐hour intersession interval