Antisense-induced downregulation of major circadian genes modulates the expression of histone deacetylase-2 (HDAC-2) and CREB-binding protein (CBP) in the medial shell region of nucleus accumbens of mice exposed to chronic excessive alcohol consumption.

Circadian genes in the medial accumbal shell (mNAcSh) region regulate binge alcohol consumption. Here, we investigated if antisense-induced knockdown of major circadian genes (Per1, Per2, and NPAS2) in the mNAcSh of mice exposed to intermittent access two-bottle choice (IA2BC) paradigm modulates the expression of histone deacetylase-2 (HDAC-2) and CREB-binding protein (CBP), key epigenetic modifiers associated with withdrawal-associated behaviors such as anxiety. Adult male C57BL/6J mice (N = 28), surgically implanted with bilateral guide cannulas above the mNAcSh, were chronically (4 weeks) exposed to alcohol (20% v/v) or saccharin (0.03%) via IA2BC paradigm. In the fourth week, a mixture of antisense (AS-ODNs; N = 14/group) or nonsense (NS-ODNs; N = 14/group) oligodeoxynucleotides against circadian genes were bilaterally infused into the mNAcSh. Subsequently, alcohol/saccharin consumption and preference were measured followed by euthanization of animals and verification of microinjection sites by visual inspection and the expression of HDAC-2 and CBP by using RT-PCR along with the verification of antisense-induced downregulation of circadian genes in the mNAcSh. As compared with NS-ODNs, AS-ODNs infusion significantly attenuated the alcohol-induced increase in HDAC-2 and reduction in CBP expression in the mNAcSh along with a significant reduction in alcohol consumption and preference. No significant effect was observed on either saccharin consumption or preference. Our results suggest that circadian genes in the mNAcSh may have a causal to play in mediating epigenetic changes observed after chronic alcohol consumption.

Antisense-induced knockdown of cAMP response element-binding protein downregulates Per1 gene expression in the shell region of nucleus accumbens resulting in reduced alcohol consumption in mice.

INTRODUCTION: We recently showed that circadian genes expressed in the shell region of nucleus accumbens (NAcSh) play a key role in alcohol consumption, though, the molecular mechanism of those effects is unclear. Because CREB-binding protein (CBP) promotes Per1 gene expression, we hypothesized that alcohol consumption would increase CBP expression in the NAcSh and antisense-induced knockdown of CBP would reduce Per1 expression and result in a reduction in alcohol consumption. METHODS: To test our hypothesis, we performed two experiments. The Drinking-in-the-dark (DID) paradigm was used to evaluate alcohol consumption in male C57BL/6J mice. In Experiment 1 we examined the effects of alcohol consumption on CBP gene expression in the NAcSh. Control animals were exposed to, sucrose [10% (w/v) taste and calorie] and water (consummatory behavior). In Experiment 2 examined the effects of CBP gene silencing on the expression of the Per1 gene in the NAcSh and alcohol consumption in mice exposed to alcohol using the DID paradigm. CBP gene silencing was achieved by local infusion of two doses of either CBP antisense oligodeoxynucleotides (AS-ODNs; Antisense group) or nonsense ODNs (NS-ODNs; Nonsense group) bilaterally microinjected into the NAcSh within 24 h before alcohol consumption on Day 4 of the DID paradigm. The microinfusion sites were verified by cresyl violet staining. RESULTS: Compared to sucrose, alcohol consumption, under the DID paradigm, significantly increased the expression of CBP in the NAcSh. Compared to Controls, bilateral infusion of CBP AS-ODNs significantly reduced the expression of Per1 in the NAcSh and alcohol consumption without affecting the amount of sucrose consumed. CONCLUSIONS: Our results suggest that CBP is an upstream regulator of Per1 expression in the NAcSh and may act via Per1 to modulate alcohol consumption.