Period 2 gene deletion abolishes beta-endorphin neuronal response to ethanol

Abstract

Background: Ethanol exposure during early life has been shown to permanently alter the circadian expression of clock regulatory genes and the beta-endorphin precursor proopiomelanocortin (POMC) gene in the hypothalamus. Ethanol also alters the stress- and immune-regulatory functions of beta-endorphin neurons in laboratory rodents. Our aim was to determine whether the circadian clock regulatory Per2 gene modulates the action of ethanol on beta-endorphin neurons in mice.

Methods: Per2 mutant (mPer2(Brdml)) and wild type (C57BL/6J) mice were used to determine the effect of Per2 mutation on ethanol-regulated beta-endorphin neuronal activity during neonatal period using an in vitro mediobasal hypothalamic (MBH) cell culture model and an in vivo milk formula feeding animal model. The beta-endorphin neuronal activity following acute and chronic ethanol treatments was evaluated by measuring the peptide released from cultured cells or peptide levels in the MBH tissues, using enzyme-linked immunosorbent assay (ELISA).

Results: Per2 mutant mice showed a higher basal level of beta-endorphin release from cultured MBH cells and a moderate increase in the peptide content in the MBH in comparison with control mice. However, unlike wild type mice, Per2 mutant mice showed no stimulatory or inhibitory beta-endorphin-secretory responses to acute and chronic ethanol challenges in vitro. Furthermore, Per2 mutant mice, but not wild type mice, failed to show the stimulatory and inhibitory responses of MBH beta-endorphin levels to acute and chronic ethanol challenges in vivo.

Conclusions: These results suggest for the first time that the Per2 gene may be critically involved in regulating beta-endorphin neuronal function. Furthermore, the data revealed an involvement of the Per2 gene in regulating beta-endorphin neuronal responses to ethanol.

Fig. 1. Effects of acute ethanol exposure on β-endorphin release from mediobasal hypothalamic cells of C57BL/6 mice and Per2 mutant mice in primary cultures

MBH cell cultures were treated with (25, 50, 100 mM) or without ethanol for 3 hrs (A) or 6 hrs (B). T = time of treatment. Data are mean ± SEM of six independent observations. *P <0.05, ** P <0.01, ***P <0.001, significantly different from controls of the same strain. ^a P< 0.05, significantly different between two strains at the same dose.

Fig. 2. Effects of chronic ethanol exposure on β-endorphin release in mediobasal hypothalamic cells of C57BL/6 mice and Per2 mutant mice

MBH cell cultures were treated with (25, 50, 100 mM) or without ethanol for 24 (A) to 48 hrs (B). T = time of treatment. Data are mean ± SEM of six independent observations. *P <0.05, ** P <0.01, ***P <0.001, significantly different from controls of the same strain. ^a P< 0.01, significantly different between two strains at the same dose.

Figure 3. Effects of acute ethanol administration on β-endorphin levels in mediobasal hypothalami of C57BL/6 and Per2 mutant mice

Postnatal rats were fed milk formula containing ethanol (AF) or no ethanol (PF) or left in the litter (AD) for 1 day. T = time of treatment. Data are mean ± SEM of six independent observations. *P <0.05, ** P <0.01, ***P <0.001, significantly different from controls of the same strain. ^a P< 0.01, significantly different between two strains at the same dose.

Figure 4. Effects of chronic ethanol administration on β-endorphin levels in mediobasal hypothalami of C57BL/6 and Per2 mutant mice

Postnatal rats were fed milk formula containing ethanol (AF) or no ethanol (PF) or left in the litter (AD) for 5 days. T = time of treatment. Data are mean ± SEM of six independent observations. *P <0.05, ** P <0.01, ***P <0.001, significantly different from controls of the same strain. ^a P< 0.01, significantly different between two strains at the same dose.