CB1 receptor neutral antagonist treatment epigenetically increases neuropeptide Y expression and decreases alcohol drinking

Abstract

Alcohol consumption is mediated by several important neuromodulatory systems, including the endocannabinoid and neuropeptide Y (NPY) systems in the limbic brain circuitry. However, molecular mechanisms through which cannabinoid-1 (CB1) receptors regulate alcohol consumption are still unclear. Here, we investigated the role of the CB1 receptor-mediated downstream regulation of NPY via epigenetic mechanisms in the amygdala. Alcohol drinking behavior was measured in adult male C57BL/6J mice treated with a CB1 receptor neutral antagonist AM4113 using a two-bottle choice paradigm while anxiety-like behavior was assessed in the light-dark box (LDB) test. The CB1 receptor-mediated changes in the protein levels of phosphorylated cAMP-responsive element binding protein (pCREB), CREB binding protein (CBP), H3K9ac, H3K14ac and NPY, and the mRNA levels of Creb1, Cbp, and Npy were measured in amygdaloid brain structures. Npy-specific changes in the levels of acetylated histone (H3K9/14ac) and CBP in the amygdala were also measured. We found that the pharmacological blockade of CB1 receptors with AM4113 reduced alcohol consumption and, in an ethanol-naïve cohort, reduced anxiety-like behavior in the LDB test. Treatment with AM4113 also increased the mRNA levels of Creb1 and Cbp in the amygdala as well as the protein levels of pCREB, CBP, H3K9ac and H3K14ac in the central and medial nucleus of amygdala, but not in the basolateral amygdala. Additionally, AM4113 treatment increased occupancy of CBP and H3K9/14ac at the Npy gene promoter, leading to an increase in both mRNA and protein levels of NPY in the amygdala. These novel findings suggest that CB1 receptor-mediated CREB signaling plays an important role in the modulation of NPY function through an epigenetic mechanism and further support the potential use of CB1 receptor neutral antagonists for the treatment of alcohol use disorder.

Keywords: AM4113; Alcohol intake; Amygdala; Anxiety; CB1 receptor; CREB; Histone acetylation; NPY.

Figure 1.

Effect of AM4113 (1 mg/kg, i.p.) on alcohol drinking and anxiety-like behaviors in mice. (A) In a two-bottle free choice paradigm, AM4113 reduced consumption of 10% ethanol compared to vehicle treated group (*p<0.05; **p<0.01). (B) Bar graph shows the effect of AM4113 on mean alcohol intake (n=8-10 in each group). (C) In the light-dark box (LDB) exploration test, AM4113 treatment increased time spent exploring the light compartment compared to vehicle treatment. (*p<0.05). (D) AM4113 had no effect on general activity as entries to the dark compartment do not differ between AM4113-treated and vehicle-treated mice (n=11-12 in each group). Overall values are represented as mean ± SEM and individual values are shown with open circles.

Figure 2.

Alteration in the protein levels of phosphorylated CREB (pCREB) and CREB binding protein (CBP) and the mRNA levels of Creb1 and Cbp in amygdala of mice treated with AM4113 (1 mg/kg, i.p.) or vehicle. (A) Representative photomicrographs of high magnification (scale bar = 50 μm) show the gold immunolabeling of pCREB in the central (CeA), medial (MeA), and basolateral (BLA) nuclei of amygdala of AM4113 or vehicle treated mice. The locations of these amygdaloid areas are shown by box at low magnification (scale bar = 1mm) images of amygdala of AM4113 or vehicle treated mice. (B) Bar graph depicts pCREB protein levels in amygdaloid brain regions after AM4113 treatment compared to vehicle group (**p<0.01; ***p<0.001; n=5 in each group). (C) Representative photomicrographs of high magnification (scale bar = 50 μm) show the gold immunolabeling of CBP in CeA, MeA and BLA in mice treated with AM4113 or vehicle. The locations of these amygdaloid areas are shown by box at low magnification (scale bar = 1mm) images of amygdala of AM4113 or vehicle treated mice. (D) Bar graph depicts levels of CBP protein in CeA, MeA and BLA following AM4113 treatment compared to vehicle group (***p<0.001; n=6 in each group). (E) Bar graph depicts Creb1 and Cbp mRNA expression levels in the amygdala of mice treated with AM4113 or vehicle (*p<0.05, n=6-7 in each group). Individual values are shown with open circles and overall values are represented as mean ± SEM.

Figure 3.

Effect of AM4113 treatment on the global protein levels of acetylated histone H3K9 (H3K9ac) and acetylated histone H3K14 (H3K14ac). (A) Representative photomicrographs of high magnification (scale bar = 50 μm) of H3K9ac gold-immunolabeling in central (CeA), medial (MeA) and basolateral (BLA) nuclei of amygdala of mice treated with either vehicle or AM4113 (1 mg/kg, i.p.). The location of these amygdaloid areas are shown by box at low magnification (scale bar = 1mm) images of amygdala of AM4113 or vehicle treated mice. (B) Bar graph shows levels of H3K9ac in CeA, MeA and BLA of AM4113 and vehicle treated mice (***p<0.001; n=6 in each group). (C) Representative photomicrographs of high magnification (scale bar = 50 μm) of H3K14ac gold-immunolabeling in CeA, MeA and BLA in mice treated with either vehicle or AM4113 (1 mg/kg). The locations of these amygdaloid areas are shown by box at low magnification (scale bar = 1mm) images of amygdala of AM4113 or vehicle treated mice. (D) Bar graph shows levels of H3K14ac in CeA, MeA and BLA following administration of AM4113 compared to vehicle group (**p<0.01; n=6 in each group). Individual values are shown with open circles and overall values are represented as mean ± SEM.

Figure 4.

Effects of AM4113 treatment (1 mg/kg, i.p.) on NPY-specific changes in CBP and H3K9/14ac as well as NPY protein and mRNA expression in the amygdala. (A) Gene diagram showing three locations of primers (location 1-primer set 1; location 2-primer set 2; and location 3-primer set 3) used to measure occupancy of CBP and H3K9/14ac at NPY gene promoter in amygdala using chromatin immunoprecipitation (ChIP) assay. (B) A significant increase in the occupancy of H3K9/14ac at all three locations was observed following treatment with AM4113 compared to vehicle treated group (*p<0.05; **p<0.01; n=6 in each group). (C) A significant increase in the occupancy of CBP at locations 2 and 3 but not at location 1 of Npy promoter was observed following treatment with AM4113 compared to vehicle treated group (*p<0.05; ***p<0.001; n=8-9 in each group). (D) The measurement of Npy mRNA level revealed an increase in Npy mRNA levels in amygdala following treatment with AM4113 (**p<0.01; n=6-7 in each group). (E) Representative photomicrographs of high magnification (scale bar = 50 μm) of gold immunolabeling of NPY in central (CeA), medial (MeA), and basolateral (BLA) nuclei of amygdala of mice. The locations of these amygdaloid areas are shown by box at low magnification (scale bar = 1mm) images of amygdala of AM4113 or vehicle treated mice. (F) Bar graph shows NPY protein levels in CeA, MeA, and BLA of mice treated with AM4113 compared to vehicle treated group (*p<0.05; n=5 in each group). Individual values are shown with open circles and overall values are represented as mean ± SEM.

Figure 5.

Molecular mechanism of CB1 receptor-mediated attenuation of alcohol drinking and anxiety-like behavior. The blockade of CB1 receptors with neutral CB1 antagonist AM4113 leads to an increase in cAMP mediated CREB phosphorylation as these receptors are negatively coupled to adenylate cyclase (AC) and increase the levels of CREB binding protein (CBP). Direct or indirect activation of CBP/CREB signaling increases CBP and histone acetylation globally and at the Npy gene specifically leading to increased NPY expression in the amygdala. This CB1 receptor-mediated modulation of NPY expression through epigenetic modification via histone acetylation may be one of the important molecular mechanisms in the amygdala underlying regulation of alcohol consumption and anxiety-like behavior. eCB, endocannabinoid; CB1, Cannabinoid Receptor 1; cAMP, Cyclic adenosine monophosphate; PKA, Protein kinase A; CREB, cAMP-response element binding protein; pCREB, phosphorylated CREB; CBP, CREB binding protein; CRE, cAMP response element site; NPY, Neuropeptide Y.