Adenosinergic regulation of striatal clock gene expression and ethanol intake during constant light

Abstract

Circadian rhythm and sleep disruptions occur frequently in individuals with alcohol use disorders (AUD) and present significant barriers to treatment. Recently, a variant of adenosine transporter, equilibrative nucleoside transporter 1 (ENT1), was associated with the co-occurrence of sleep problems and AUD. We have previously shown that mice lacking ENT1 (ENT1 KO) have reduced adenosine levels in the striatum and drink more alcohol compared with wild types (WT). However, it is unknown whether ENT1 deletion disrupts circadian rhythms, which may contribute to alcohol preference in ENT1 KO mice. Here we used these mice to determine whether endogenous adenosine regulates circadian genetic and behavioral rhythms and influences alcohol intake during chronodisruption. We examined circadian locomotor activity in ENT1 KO vs WT littermates and found that ENT1 KO mice were both active earlier and hyperactive compared with WT mice at night. We used real-time PCR and immunohistochemistry to estimate striatal clock gene levels and found that PER2 expression in the striatum was blunted by ENT1 deletion or A2A receptor (A2AR) antagonism. Next, we exposed ENT1 KO and WT mice to constant light (LL) and found further elevation in ethanol intake in ENT1 KO, but not in WT mice, supporting the notion that circadian dysfunction may contribute to increased alcohol intake in ENT1 KO mice. Finally, we showed that A2AR agonist administration normalized PER1 and PER2 expression and circadian locomotor activity in ENT1 KO mice. Together, our results demonstrate that adenosine signaling regulates cellular and behavioral circadian timing and influences alcohol intake during chronodisruption.

Figure 1

Adenosine transporter equilibrative nucleoside transporter 1 (ENT1) regulates circadian locomotor activity. (a) Representative actograms from wild-type (WT; left side) and ENT1 KO mice (right side). (b) ENT1 KO mice had a positive phase angle of entrainment relative to WT, becoming active before lights were turned off (Zeitgeber Time 12, ZT12). (c) ENT1 KO mice had longer alpha (active phase) relative to WT. (d) Increased nighttime, but not daytime, activity duration in ENT1 KO mice compared with WT. (e) Higher nighttime, but not daytime, activity intensity in ENT1 KO mice compared with WT. *P<0.05 by unpaired, two-tailed t-test; n=12 per genotype. Data are expressed as mean±SEM.

Figure 2

Equilibrative nucleoside transporter 1 (ENT1) regulates Per2 expression in the striatum. (a) ENT1 KO mice showed a reduction in Per2 mRNA in the nucleus accumbens (NAc) during the normal, wild-type (WT) peak time (Zeitgeber Time 14, ZT14) compared with WT littermates ^#P<0.05 for main effect of genotype by two-way ANOVA; *P<0.05 by post hoc Tukey test; n=5 per genotype. (b) The normal WT peak in PER2 protein expression (ZT22) was also reduced in the NAc and caudate–putamen (CPu) of ENT1 KO mice; *P<0.05 by unpaired, two-tailed t-test; n=8 per genotype. (c) Representative confocal images showing reduced PER2 immunofluorescence at ZT22 in the NAc and CPu of WT (left) vs ENT1 KO mice (right). Colocalization of PER2 with NeuN indicated that PER2 was expressed in neurons. Scale bar, 100 μm. Data are expressed as mean±SEM.

Figure 3

Escalation in ethanol intake by equilibrative nucleoside transporter 1 (ENT1) KO mice during a constant light (LL) photocycle. (a) ENT1 KO mice showed increased ethanol consumption during LL compared with LD, whereas wild-type (WT) mice consumed the same amount of ethanol in LL and LD. (b) ENT1 KO mice also showed increased ethanol preference in LL compared with LD, whereas no such change occurred in WT. (c) Representative, double-plotted drinkograms showing circadian drinking patterns from WT and ENT1 KO mice during LD and LL (stars denote beginning of LL). ^#P<0.05 for main effect of genotype by two-way, repeated measures ANOVA; *P<0.05 by post hoc Tukey test; n=27 per genotype. Data are expressed as mean±SEM.

Figure 4

A2A receptor (A2AR)-mediated regulation of Per gene expression and circadian behavioral rhythms in equilibrative nucleoside transporter 1 (ENT1) KO mice. (a) Diagram depicting times of treatment with adenosine receptor ligands (Zeitgeber Time (ZT22); wild-type (WT) peak for PER protein expression in striatum) and killing (ZT14; WT peak for Per gene expression in striatum). (b) Three-day treatment of A1R antagonist DPCPX (6.0 mg/kg, i.p.) or A2AR antagonist ZM-241385 (20.0 mg/kg, i.p.) at ZT22 (late night) differentially affected clock gene expression in WT mice, with the former downregulating peak expression of Per1 and Per2, while the latter only reduced expression of Per2; *P<0.05 by one-way ANOVA; n=6 per treatment. (c) Three-day treatment of A2AR agonist CGS-21680 (2.0 mg/kg) upregulated Per1 only in ENT1 KO mice and Per2 in both WT and ENT1 KO mice compared with vehicle-treated WT controls; P<0.05 by unpaired, two-tailed t-test; n=6 per treatment. (d) Representative actograms from WT (left side) and ENT1 KO (right side) mice treated with vehicle (top) or A2AR agonist CGS-21680 (bottom) for 5 days. Stars denote daily treatment time at ZT22; for simplicity, only the first treatment day is starred. (e) Five-day treatment with CGS-21680 (1.0 mg/kg, i.p.) at ZT22 (late night) delayed activity onset in ENT1 KO, but not in WT mice. (f) Alpha was reduced by 5-day CGS-21680 treatment in both ENT1 KO and WT mice, but to a greater degree in the former; ^#P<0.05 for main effect of genotype by two-way ANOVA; *P<0.05 by post hoc Tukey test; n=5 per genotype × treatment. Data are expressed as mean±SEM.