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. 2018 Feb;42(2):338-351.
doi: 10.1111/acer.13568. Epub 2018 Jan 24.

Effects of Long-Term Alcohol Drinking on the Dopamine D2 Receptor: Gene Expression and Heteroreceptor Complexes in the Striatum in Rats

Kristin Feltmann  1 , Dasiel Oscar Borroto-Escuela  2 , Joëlle Rüegg  3   4 , Luca Pinton  2 , Thatiane de Oliveira Sergio  1 , Manuel Narváez  5 , Antonio Jimenez-Beristain  6 , Tomas J Ekström  3 , Kjell Fuxe  2 , Pia Steensland  1
Affiliations

Effects of Long-Term Alcohol Drinking on the Dopamine D2 Receptor: Gene Expression and Heteroreceptor Complexes in the Striatum in Rats

Kristin Feltmann et al. Alcohol Clin Exp Res. 2018 Feb.

Abstract

Background: Reduced dopamine D2 receptor (D2R) ligand binding has repeatedly been demonstrated in the striatum of humans with alcohol use disorder (AUD). The attenuated D2R binding has been suggested to reflect a reduced D2R density, which in turn has been proposed to drive craving and relapse. However, results from rodent studies addressing the effects of alcohol drinking on D2R density have been inconsistent.

Methods: A validated alcohol drinking model (intermittent access to 20% alcohol) in Wistar rats was used to study the effects of voluntary alcohol drinking (at least 12 weeks) on the D2R in the striatum compared to age-matched alcohol-naïve control rats. Reverse transcriptase quantitative PCR was used to quantify isoform-specific Drd2 gene expression levels. Using bisulfite pyrosequencing, DNA methylation levels of a regulatory region of the Drd2 gene were determined. In situ proximity ligation assay was used to measure densities of D2R receptor complexes: D2R-D2R, adenosine A2A receptor (A2AR)-D2R, and sigma1 receptor (sigma1R)-D2R.

Results: Long-term voluntary alcohol drinking significantly reduced mRNA levels of the long D2R isoform in the nucleus accumbens (NAc) but did not alter CpG methylation levels in the analyzed sequence of the Drd2 gene. Alcohol drinking also reduced the striatal density of D2R-D2R homoreceptor complexes, increased the density of A2AR-D2R heteroreceptor complexes in the NAc shell and the dorsal striatum, and decreased the density of sigma1R-D2R heteroreceptor complexes in the dorsal striatum.

Conclusions: The present results on long-term alcohol drinking might reflect reduced D2R levels through reductions in D2R-D2R homoreceptor complexes and gene expression. Furthermore, based on antagonistic interactions between A2AR and D2R, an increased density of A2AR-D2R heteroreceptor complexes might indicate a reduced affinity and signaling of the D2R population within the complex. Hence, both reduced striatal D2R levels and reduced D2R protomer affinity within the striatal A2AR-D2R complex might underlie reduced D2R radioligand binding in humans with AUD. This supports the hypothesis of a hypodopaminergic system in AUD and suggests the A2AR-D2R heteroreceptor complex as a potential novel treatment target.

Keywords: Alcohol Dependence; DNA Methylation; Epigenetics; Heteroreceptors and Homoreceptors; Receptor Dimers.

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Figures

Figure 1
Figure 1
Effects of long‐term voluntary alcohol drinking on striatal gene expression of the long isoform of the dopamine D2 receptor. Wistar rats were exposed to intermittent access to 20% ethanol (alcohol, n = 14) or 2 bottles of water (age‐matched alcohol‐naïve control, n = 8) for 5 months. Individual alcohol intake was determined as mean g/kg/24 h alcohol intake over the last 7 weeks. Effects of alcohol drinking on gene expression of the dopamine D2 receptor long isoform (D2L) in the nucleus accumbens (NAc) and dorsal striatum (STR) were quantified using reverse transcriptase quantitative PCR. Specific primers were used detecting exon–exon junctions of the D2L. Expression levels are quantified relative to the reference gene peptidylprolyl isomerase A (PPIA). In the NAc, 3 RNA samples (n = 2 alcohol, n = 1 control) could not be successfully converted into cDNA. Alcohol drinking significantly decreased D2L gene expression in the NAc (A, B), but not the dorsal STR (C, D) (*p < 0.05 compared to alcohol‐naïve controls, 2‐way ANOVA with Bonferroni post hoc (A, C); Pearson's correlation (B, D)).
Figure 2
Figure 2
Effects of long‐term voluntary alcohol drinking on the short isoform of the dopamine D2 receptor (D2S). Wistar rats were exposed to intermittent access to 20% ethanol (alcohol, n = 14) or 2 bottles of water (age‐matched alcohol‐naïve control, n = 8) for 5 months. Individual alcohol intake was determined as mean g/kg/24 h alcohol intake over the last 7 weeks. Effects of alcohol drinking on gene expression of the D2S in the nucleus accumbens (NAc) and dorsal striatum (STR) were quantified using reverse transcriptase quantitative PCR. Specific primers were used detecting exon–exon junctions of the D2S. Expression levels for each gene are quantified relative to the reference gene peptidylprolyl isomerase A (PPIA). In the NAc, 3 RNA samples (n = 2 alcohol, n = 1 control) could not be successfully converted into cDNA. There were no significant overall effects of drinking condition or interaction of drinking condition*brain region (NAc: A; dorsal STR: C) (2‐way ANOVA) on D2S gene expression. There was a significant correlation between individual alcohol intake and D2S expression levels in the NAc (B), but not the dorsal STR (D) (Pearson's correlation).
Figure 3
Figure 3
Effects of long‐term voluntary alcohol drinking on striatal gene expression of the adenosine A2A receptor. Wistar rats were exposed to intermittent access to 20% ethanol (alcohol, n = 14) or 2 bottles of water (age‐matched alcohol‐naïve control, n = 8) for 5 months. Individual alcohol intake was determined as mean g/kg/24 h alcohol intake over the last 7 weeks. Effects of alcohol drinking on adenosine A2A receptor gene expression in the nucleus accumbens (NAc) and dorsal striatum (STR) were quantified using reverse transcriptase quantitative PCR. A2A expression levels were quantified relative to the reference gene peptidylprolyl isomerase A (PPIA). In the NAc, 3 RNA samples (n = 2 alcohol, n = 1 control) could not be successfully converted into cDNA. Alcohol drinking did not affect Adora2a gene expression in the NAc (A, B) or the dorsal STR (C, D) (2‐way ANOVA with Bonferroni post hoc (A, C); Pearson's correlation (B, D)).
Figure 4
Figure 4
Effects of long‐term voluntary alcohol drinking on dopamine Drd2 gene methylation in the nucleus accumbens. Wistar rats were exposed to intermittent access to 20% ethanol (alcohol) or 2 bottles of water (age‐matched alcohol‐naïve control) for 5 months. Individual alcohol intake was determined as mean g/kg/24 h alcohol intake over the last 7 weeks. Individuals included in the CpG analysis are the same as the ones included in the expression analysis (n = 12 alcohol‐drinking, n = 7 alcohol‐naïve rats). Effects of alcohol drinking on methylation levels of a sequence within a CpG island in exon 1 of the Drd2 gene (see sequence above) in the NAc were quantified using bisulfite pyrosequencing. Alcohol‐drinking rats showed no changes in methylation levels of any CpG analyzed compared to alcohol‐naïve controls (unpaired Student's t‐test).
Figure 5
Figure 5
D2R‐D2R homoreceptor and A2AR‐D2R and sigma1R‐D2R heteroreceptor complexes in the striatum of alcohol‐naïve and alcohol‐drinking rats. These panels provide representative examples of the changes in the density of D2R‐D2R homoreceptor (A), the A2AR‐D2R (B), and sigma1R‐D2R (C) heteroreceptors complexes in the nucleus accumbens (NAc) shell (left panels), core (center panels), and the dorsal striatum (right panels) after at least 12 weeks of voluntary alcohol drinking in the intermittent access to 20% ethanol paradigm (lower panels) compared to control (upper panels). Using the in situ proximity ligation assay (in situ PLA) technique (Borroto‐Escuela et al., 2013; Fuxe et al., 2014a), together with confocal laser microscopy, receptor complexes were identified and visualized as red blobs (clusters, white arrows). PLA blobs were found in high densities per cell in a large number of nerve cells (picture: 25 × 25 μm, nuclei shown in blue by DAPI, 20 z‐layers) in frontal sections of the rat ventral and dorsal striatum (Bregma level: 1.00 mm). No specific PLA blobs were found in the anterior commissure and corpus callosum regions for either complex.
Figure 6
Figure 6
Effects of voluntary alcohol drinking on striatal density of the D2R‐D2R homoreceptor, A2AR‐D2R, and sigma1R‐ D2R heteroreceptor complexes. Rats were exposed to intermittent access to 20% ethanol (alcohol, 4.7 ± 0.4 g/kg/24 h) or 2 bottles of water (age‐matched alcohol‐naïve control), respectively, for at least 12 weeks. Effects of alcohol drinking on the density of D2R complexes in the nucleus accumbens (NAc) core and shell and the dorsal striatum (STR) were quantified using in situ proximity ligation assay (PLA). Data were analyzed using 2‐way ANOVA followed by Bonferroni post hoc with brain region and drinking condition as factors (n = 3 to 4, per group). There was a significant effect of alcohol drinking on D2R‐D2R homoreceptor complexes, but no drinking condition*brain region interaction (A). Alcohol drinking significantly increased the density of A2AR‐D2R heteroreceptor complexes in the NAc shell and the dorsal striatum, but not in the NAc core (B). Alcohol drinking significantly decreased the density of sigma1R‐D2R heteroreceptor complexes in the dorsal STR, but not in the NAc core or shell (C). Values represent group mean±SEM number of positive PLA dots per nuclei/sample field, for each rat given as mean values of 3 pictures (40 × 40 μm). **p < 0.01 compared to corresponding alcohol‐naïve controls.
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