Upregulation of dopamine D2 receptors in the nucleus accumbens indirect pathway increases locomotion but does not reduce alcohol consumption

Abstract

Brain imaging studies performed in humans have associated low striatal dopamine release and D2R binding with alcohol dependence. Conversely, high striatal D2R binding has been observed in unaffected members of alcoholic families suggesting that high D2R function may protect against alcohol dependence. A possible protective role of increased D2R levels in the striatum is further supported by preclinical studies in non-human primates and rodents. Here, we determined whether there is a causal relationship between D2R levels and alcohol intake. To this end, we upregulated D2R expression levels in the nucleus accumbens of the adult mouse, but selectively restricted the upregulation to the indirect striatal output pathway, which endogenously expresses D2Rs. After overexpression was established, mice were tested in two models of free-choice alcohol drinking: the continuous and intermittent access two-bottle choice models. As anticipated, we found that D2R upregulation leads to hyperactivity in the open field. Contrary to our expectation, D2R upregulation did not reduce alcohol intake during continuous or intermittent access or when alcohol drinking was tested in the context of aversive outcomes. These data argue against a protective role of accumbal indirect pathway D2Rs in alcohol consumption but emphasize their importance in promoting locomotor activity.

Figure 1

(a) AAV2/1 vector encoding a double-floxed inverted open reading frame (DIO) expressing D2R and the YFP variant mVenus under control of the synapsin promoter. ITR, inverted terminal repeat; IRES, internal ribosome entry site; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; bGH-pA, bovine growth hormone poly-A tail. (b) Schematic representation depicting bilateral injection site of AAV into NAc of D2-Cre mice. (c) mVenus expression in NAc 4 weeks post injection is primarily restricted to the core region. Scale=600 μm. (d) Co-localization of upregulated D2R (red) with mVenus (green) was observed in membrane-like regions and processes. Scale=50 μm. (e) Higher magnification image showing mVenus-labeled fibers in the ventral pallidum (VP), projection target of NAc indirect pathway. Scale=150 μm. (f) Double-labeling of Cre and mVenus shows that mVenus expression is restricted to Cre-positive cells in D2-Cre mice. Scale=50 μm. (g) Rare co-localization of tdTomato (D1-MSNs) and Venus (D2-MSNs overexpressing D2Rs) in the NAc of D2-Cre x D1-tdTomato mice 4 weeks after the AAV-D2R injection. (h) Choline acetyltransferase (ChAT), a marker for cholinergic interneurons rarely colocalizes with GFP-positive cells, in agreement with previous work in D2-Cre mice (Kravitz et al, 2010). Scale=50 μm. (i) Ligand binding analysis with the D2 receptor antagonist [³H]N-methyl-spiperone in NAc membranes shows significant upregulation in D2R_NAcInd mice.

Figure 2

(a) Distance traveled in 5-min bins over a 90-min period by mice expressing EGFP or D2Rs in indirect pathway NAc MSNs. (b) Mean total distance traveled over the 90-min test period. (c) Representative horizontal activity tracks for the initial 45 min. Inner box represents the center zone. (d) Center distance, calculated as a percentage of the total distance, was significantly increased. (e) D2R-OE_NAcInd mice also displayed increased rearing behavior. *Statistically significant.

Figure 3

(a) Ethanol consumption is increased as a function of ethanol concentration (v/v) in a continuous access, two-bottle choice procedure in both groups. Ethanol consumption is significantly higher in D2R-OE_NAcInd mice compared with controls expressing EGFP. Data for each ethanol concentration were obtained as an average of 2 days of drinking (p<0.005). (b) Both groups similarly adjust their preference of ethanol over water across the different ethanol concentrations. (c) Total fluid consumption (ethanol + water), while stable in both groups, is increased in D2R-OE_NAcInd mice (p<0.00001). (d) D2R overexpression results in lower body weights (p=0.066). (e–g) Dose-dependent consumption of sucrose, saccharin, and quinine alone when tested against water. Sucrose consumption is significantly increased in D2R-OE_NAcInd mice (p<0.005). (h–j) Preference for sucrose, saccharin, or quinine over water is not altered by D2R upregulation.

Figure 4

(a) Intermittent access (IA) to 15% ethanol vs water leads to a progressive escalation of ethanol intake over 10 sessions (20 intermittent days of ethanol exposure). Each session reflects the average consumption over 2 days of alcohol drinking in which alcohol bottle position was alternated to prevent side bias. D2R upregulation does not alter escalation of ethanol consumption. (b) The IA procedure effectively leads to a significant increase in ethanol consumption in both groups between the first and last session. (c) Although both groups prefer 15% ethanol over water, a reduction in preference was observed in the D2R-OE_NAcInd group compared with controls (p<0.05). (d) D2R upregulation also led to increased total fluid consumption (p<0.001).

Figure 5

(a) After 10 IA sessions of 15% ethanol, quinine was faded into the ethanol solution beginning with concentrations that were aversive in alcohol-naïve mice (0.03–0.06 mM) up to 0.6 mM. Both groups showed a slow, but comparable decrease in their alcohol consumption with increasing quinine concentrations. (b) Although D2R-OE_NAcInd maintained a lower preference for alcohol p<0.05 both groups were similarly resistant to reducing their preference with increasing quinine concentrations, eventually decreasing preference at the highest quinine dose. (c) Total fluid consumption was stable across quinine doses, but remained higher in the D2R-OE_NAcInd group (p<0.05) (F_(1,14)=8.13,).