Partial agonists of the α3β4* neuronal nicotinic acetylcholine receptor reduce ethanol consumption and seeking in rats

Abstract

Alcohol use disorders (AUDs) impact millions of individuals and there remain few effective treatment strategies. Despite evidence that neuronal nicotinic acetylcholine receptors (nAChRs) have a role in AUDs, it has not been established which subtypes of the nAChR are involved. Recent human genetic association studies have implicated the gene cluster CHRNA3-CHRNA5-CHRNB4 encoding the α3, α5, and β4 subunits of the nAChR in susceptibility to develop nicotine and alcohol dependence; however, their role in ethanol-mediated behaviors is unknown due to the lack of suitable and selective research tools. To determine the role of the α3, and β4 subunits of the nAChR in ethanol self-administration, we developed and characterized high-affinity partial agonists at α3β4 nAChRs, CP-601932, and PF-4575180. Both CP-601932 and PF-4575180 selectively decrease ethanol but not sucrose consumption and operant self-administration following long-term exposure. We show that the functional potencies of CP-601932 and PF-4575180 at α3β4 nAChRs correlate with their unbound rat brain concentrations, suggesting that the effects on ethanol self-administration are mediated via interaction with α3β4 nAChRs. Also varenicline, an approved smoking cessation aid previously shown to decrease ethanol consumption and seeking in rats and mice, reduces ethanol intake at unbound brain concentrations that allow functional interactions with α3β4 nAChRs. Furthermore, the selective α4β2(*) nAChR antagonist, DHβE, did not reduce ethanol intake. Together, these data provide further support for the human genetic association studies, implicating CHRNA3 and CHRNB4 genes in ethanol-mediated behaviors. CP-601932 has been shown to be safe in humans and may represent a potential novel treatment for AUDs.

Figure 1

Structures of (a) CP-601932, (b) PF-4575180, (c) varenicline, and (d) dihydro-β-erythroidine.

Figure 2

Concentration-dependent activation and inhibition curves of α3β4 nAChRs expressed in HEK293 cells measured by FLIPR methodology. Activation data (filled circles) are expressed as fraction of the response evoked by 100 μM ACh. Inhibition data (open squares) were generated by applying 30 μM ACh in the presence of varying concentrations of the test compound, and the data are normalized to the response evoked by 30 μM ACh in the absence of test compound. The activation and inhibition curves are the curves of best fit through the data points. (Inserts) Concentration-dependent fraction of activated α3β4 nAChRs calculated from the fitted activation and inhibition curves. Vertical gray bars correspond to the estimated unbound rat brain concentrations (in nM) measured at 30 min after 5 and 10 mg/kg of CP-601932 (a), 1 and 10 mg/kg of PF-4575180 (b), and 1 and 2 mg/kg of varenicline (c).

Figure 3

Time courses of unbound CP-601932 and PF-4575180 concentrations in rat brain (C_b,u) after s.c. administration of 5 mg/kg (▪-▪) and 10 mg/kg (▴-▴)CP-601932 (a) and 1 mg/kg (•-•) and 10 mg/kg (▴-▴) PF-4575180 (b). Data are expressed as nM (n=2).

Figure 4

Acute administration of CP-601932 decreases ethanol but not sucrose consumption and seeking. CP-601932 (10 mg/kg) decreased active lever presses for (a) 10% ethanol, but not (b) 5% sucrose in the operant self-administration paradigm. CP-601932 (5 and 10 mg/kg) significantly decreased high voluntary ethanol consumption (c) but not sucrose consumption (d) at 30 min after the onset of drinking in rats using the intermittent-access two-bottle-choice drinking paradigm. All values are expressed as mean number of active lever presses±SEM (a, b) and mean ethanol or sucrose intake (g/kg)±SEM (c, d) (repeated measures ANOVA followed by Newman–Keuls post hoc test. ^*P<0.05 compared with vehicle, n=10–13 for the operant paradigm and ^*P<0.05, ^***P<0.001 compared with vehicle, n=10–12 for the two-bottle-choice paradigm.

Figure 5

PF-4575180, an α3β4 partial agonist, decreased ethanol but not sucrose consumption and seeking. PF-4575180 (10 mg/kg s.c.) treatment decreased active lever presses for ethanol (a) but not for 5% sucrose self-administration (b) in the operant drinking paradigm. PF-4575180 (10 mg/kg s.c.) treatment decreased voluntary ethanol consumption (c) but not sucrose consumption (d) 30 min after the onset of drinking in the intermittent-access two-bottle-choice drinking paradigm. The values are expressed as mean number of active lever presses±SEM (a, b) or ethanol or sucrose intake (g/kg) ±SEM (c, d) (repeated measures ANOVA followed by Newman–Keuls post hoc test). ^*P<0.05, compared with vehicle, n=8–14 for the operant paradigm and ^***P<0.001 compared with vehicle, n=10 for the two-bottle-choice paradigm.

Figure 6

Multiple repeated administration of CP-601932 decreased ethanol consumption using the operant self-administration paradigm. Chronic administration of CP-601932 (10 mg/kg s.c.) but not vehicle decreased active lever presses for 10% ethanol. CP-601932 (10 mg/kg s.c.) or vehicle (saline s.c.) was administered on each of 5 consecutive days, 30 min before the start of the operant session. The values are expressed as ethanol intake (g/kg) ±SEM (two-way ANOVA followed by Newman–Keuls post hoc test). ^*P<0.05 compared with vehicle, n=6–7.