Adenosine transporter ENT1 regulates the acquisition of goal-directed behavior and ethanol drinking through A2A receptor in the dorsomedial striatum

Abstract

Adenosine signaling has been implicated in the pathophysiology of many psychiatric disorders including alcoholism. Striatal adenosine A2A receptors (A2AR) play an essential role in both ethanol drinking and the shift from goal-directed action to habitual behavior. However, direct evidence for a role of striatal A2AR signaling in ethanol drinking and habit development has not been established. In the present study, we found that decreased A2AR-mediated CREB activity in the dorsomedial striatum (DMS) enhanced initial behavioral acquisition of goal-directed behaviors and the vulnerability to progress to excessive ethanol drinking during operant conditioning in mice lacking ethanol-sensitive adenosine transporter ENT1 (ENT1(-/-)). Using mice expressing β-galactosidase (lacZ) under the control of seven repeated CRE sites in both genotypes (CRE-lacZ/ENT1(+/+) mice and CRE-lacZ/ENT1(-/-) mice) and the dominant-negative form of CREB, we found that reduced CREB activity in the DMS was causally associated with decreased A2AR signaling and increased goal-directed ethanol drinking. Finally, we have demonstrated that the A2AR antagonist ZM241385 dampened protein kinase A activity-mediated signaling in the DMS and promoted excessive ethanol drinking in ENT1(+/+) mice, but not in ENT1(-/-) mice. Our results indicate that A2AR-mediated CREB signaling in the DMS is a key determinant in enhancing the development of goal-directed ethanol drinking in mice.

Figure 1.

Decreased A_2AR-mediated CREB activity in the DMS of ENT1^−/− mice. A, Schematic showing brain regions in which tissue samples were taken from the DMS and DLS for Western blot analysis. B, Decreased pPKA (T197) in the DMS (t = 2.9, p < 0.05) of ENT1^−/− mice. No change in total PKA protein expression was observed. A_2AR antagonist (ZM241385, 20 mg/kg, i.p.) decreased pPKA levels in ENT1^+/+ mice to a level similar to that of ENT1^−/− mice (t = 2.7, p < 0.05), normalizing PKA activity between genotypes (n = 8). *p < 0.05 compared with ENT1^+/+ mice after normalization by GAPDH (unpaired, two-tailed t test). C, No significant pPKA or total PKA levels change were detected in the DLS. D, pCREB (Ser133; t = 3.4, p < 0.05) levels were reduced in the DMS of ENT1^−/− mice (n = 8). *p < 0.05 compared with ENT1^+/+ mice after normalization by GAPDH (unpaired, two-tailed t test). E, Decreased CRE-driven lacZ expression in the DMS of CRE-lacZ/ENT1^−/− mice compared with CRE-lacZ/ENT1^+/+ mice (n = 6; t = 4.3, p < 0.05). F, DMS-specific A_2AR inhibition (ZM241385, microinjection, 10 μm, 2 h) decreased the CREB activity in CRE-lacZ mice compared with that of vehicle control (n = 5; t = 3.2, p < 0.05). Representative DMS coronal brain sections of lacZ expression are shown by X-Gal staining. Scale bar, 100 μm. All data are presented as the mean ± SEM.

Figure 2.

Inhibition of A_2AR enhances the initial acquisition of operant responding for sucrose solution. A, B, ENT1^−/− mice showed an increased rate of initial acquisition in both the RR (A) and RI (B) schedules compared with that of ENT1^+/+ mice (n = 16). *p < 0.05 by two-way measures ANOVA followed by Tukey post hoc analysis; ^#p < 0.05 for the main effect of genotype. C, The rate of initial acquisition (RR10) was significantly increased by ZM241385 treatment (ZM241385, 20 mg/kg, i.p.) in ENT1^+/+ mice (t = 2.2, p < 0.05), whereas no significant change was observed in ENT1^−/− mice. D, Neither genotype showed any changes during an extended RR (RR20) schedule in response to A_2AR antagonist (n = 8). All data are presented as mean ± SEM.

Figure 3.

DMS-specific inhibition of A_2AR enhances the initial acquisition of operant responding for sucrose solution. A, Schematic representation of the microinjection cannula placements within the DMS and DLS in coronal sections (Franklin and Paxinos, 2007). The numbers on each illustration indicate millimeters from bregma. The location of the microinjector tips is represented by circles for DMS and rhombuses for DLS. B, DMS-specific A_2AR inhibition using ZM241385 (10 μm microinjection for 3 d) enhanced the nose poking for initial acquisition of goal-directed response (RR10) in C57BL/6J mice (Tukey post hoc analysis, two-way ANOVA), whereas no significant nose-poking change was observed after ZM241385 treatment in the DLS (n = 6). All data are presented as mean ± SEM.

Figure 4.

Attenuation of A_2AR-mediated CREB activity in the DMS promotes goal-directed behavior in operant responding for ethanol solution. A, Early acquisition (RR10) for 10% ethanol solution was increased by ZM241385 treatment (20 mg/kg, i.p.) in ENT1^+/+ mice (t = 2.2, p < 0.05), whereas no significant change was observed in ENT1^−/− mice (n = 7). B, Neither genotype showed any changes from ZM241385 during an extended RR (RR20) schedule in operant conditioning using ethanol solution (n = 10). All data are presented as mean ± SEM. C, Schematic illustrating the experimental procedures for operant conditioning for ethanol seeking in response to HSV-dnCREB. Mice were trained to respond on sucrose reward and then HSV-dnCREB was infused into DMS or DLS using stereotaxic surgery. After surgery, operant responding for sucrose solution measured basal nose poking (S) and then compared with response for 10% ethanol in sucrose solution (S+E), extinction (Ex) and reacquisition (Re). The operant conditioning was repeated after 1 month to compare the effect of dnCREB clearance in operant responding. D, Mice injected with dnCREB (HSV-dnCREB) in the DMS showed significantly increased ethanol seeking and decreased nose poking during the extinction period, whereas control mice with control (HSV-GFP) or inactive dnCREB in the DMS showed significantly decreased nose poking during the extinction. E, Mice injected with dnCREB (HSV-dnCREB) in the DLS showed habitual nose poking during the extinction, whereas mice injected with inactive dnCREB showed significantly decreased nose poking during the extinction (n = 8–12). F, Schematic representation of viral vector microinjection coordinates (Franklin and Paxinos, 2007) with HSV-GFP expression in coronal sections for DMS. HSV-GFP expression in the DMS is marked by arrows. The AP number on each illustration indicates millimeters from bregma. The placements of the injection needle are represented by circles for DMS and rhombuses for DLS. Scale bar, 200 μm. LV indicates lateral ventricles; LSN, lateral septa nuclei. All data are presented as mean ± SEM.

Figure 5.

A_2AR inhibition increases ethanol self-administration similar to that of ENT1^−/− mice. A, Ethanol consumption was increased in ENT1^+/+ mice with ZM241385 treatment, whereas no changes in ENT1^−/− mice were observed. B, Ethanol preference was also increased in ENT1^+/+ mice with 20 mg/kg ZM241385 treatment (n = 13). S indicates saline; 10, 10 mg/kg, i.p.; and 20, 20 mg/kg, i.p. ^#p < 0.05 for the main effect of genotype; *p < 0.05 for the main effect of each treatment. C, Taste preference for saccharin or quinine was not changed in response to ZM241385 treatment (20 mg/kg, i.p.) in either genotype (n = 13). D, Ethanol consumption was significantly decreased in ENT1^−/− mice with a 2 mg/kg concentration of A_2AR agonist (CGS21680), whereas ENT1^+/+ mice showed no response to treatment. E, Treatment with 2 mg/kg CGS21680 decreased ethanol preference in ENT1^−/− mice (n = 15–18). F, Taste preference for saccharin or quinine was not changed by CGS21680 treatment (2 mg/kg, i.p.) in either genotype. All data are presented as mean ± SEM.

Figure 6.

Schematic representation of the possible mechanism in the DMS for goal-directed ethanol seeking behaviors through A_2AR-CREB–mediated signaling.