Involvement of the beta-endorphin neurons of the hypothalamic arcuate nucleus in ethanol-induced place preference conditioning in mice

Abstract

Background: Increasing evidence indicates that mu- and delta-opioid receptors are decisively involved in the retrieval of memories underlying conditioned effects of ethanol. The precise mechanism by which these receptors participate in such effects remains unclear. Given the important role of the proopiomelanocortin (POMc)-derived opioid peptide beta-endorphin, an endogenous mu- and delta-opioid receptor agonist, in some of the behavioral effects of ethanol, we hypothesized that beta-endorphin would also be involved in ethanol conditioning.

Methods: In this study, we treated female Swiss mice with estradiol valerate (EV), which induces a neurotoxic lesion of the beta-endorphin neurons of the hypothalamic arcuate nucleus (ArcN). These mice were compared to saline-treated controls to investigate the role of beta-endorphin in the acquisition, extinction, and reinstatement of ethanol (0 or 2 g/kg; intraperitoneally)-induced conditioned place preference (CPP).

Results: Immunohistochemical analyses confirmed a decreased number of POMc-containing neurons of the ArcN with EV treatment. EV did not affect the acquisition or reinstatement of ethanol-induced CPP, but facilitated its extinction. Behavioral sensitization to ethanol, seen during the conditioning days, was not present in EV-treated animals.

Conclusions: The present data suggest that ArcN beta-endorphins are involved in the retrieval of conditioned memories of ethanol and are implicated in the processes that underlie extinction of ethanol-cue associations. Results also reveal a dissociated neurobiology supporting behavioral sensitization to ethanol and its conditioning properties, as a beta-endorphin deficit affected sensitization to ethanol, while leaving acquisition and reinstatement of ethanol-induced CPP unaffected.

Figure 1

Representative image of inmunocytochemically labeled proopiomelanocortin (POMc) neurons in the hypothalamic arcuate nucleus (ArcN; which boundaries are delimited) and peri-ArcN region of vehicle (a,c) and estradiol valerate (EV)-treated (b,d) mice. Images are 10x (a,b) or 20x (c,d). Vehicle and EV groups (n = 6 per group) are animals treated with 0.2 ml of oil, or 2 mg of EV in 0.2 ml of oil (i.m.) per animal respectively. Cell counts revealed a 60% (approx.) decrease in the number of POMc-positve cells in EV-treated animals. 3V; third ventricle. Sections are Bregma -1.46 to -1.70 mm (approx.) according to Paxinos and Franklin (2003).

Figure 2

Effect of estradiol valerate (EV) treatment on the acquisition of ethanol-induced CPP. Mean ± SEM seconds per minute spent on grid during a floor choice testing of 60 minutes. Grid+ and Grid- conditioning subgroups: N values: Vehicle, n = 12, and EV, n = 10. Grid+; ethanol paired to grid floor, saline paired to hole floor. Grid-; ethanol paired to hole floor, saline paired to grid floor. Vehicle and EV groups refer to animals treated with a single i.m. injection of 0.2 ml of sesame oil, or 2 mg of EV in 0.2 ml of sesame oil per animal respectively (injection administered 8 weeks before initiation of ethanol (2 g/kg)-induced CPP). All animals were treated on alternate days with 2 g/kg of ethanol i.p. (CS+ floor) or saline (CS- floor). Preference for the ethanol-paired floor was obtained in both, vehicle- and EV-treated groups, as a main effect of conditioning group (* p < 0.01) was obtained regardless of previous treatment.

Figure 3

Effect of estradiol valerate (EV) treatment on the extinction of ethanol-induced CPP. Mean ± SEM seconds per minute spent on grid during a floor choice testing of 60 minutes (same animals as those shown in Fig. 2). After exhibiting ethanol-induced CPP, saline injections were paired with both, the CS+ and CS- floors (four sessions; two per floor). The day after the last extinction session, three consecutive (one per day) preference tests (tests 2, 3 and 4) were undertaken. Vehicle (V) and EV refer to animals treated with 0.2 ml of vehicle, or 2 mg of EV in 0.2 ml of vehicle (i.m.) per animal respectively. Preference for the ethanol-paired floor was only seen in control animals (*p < 0.01) on test 2. G+; Grid+, G-; Grid-.

Figure 4

Effect of a priming injection of ethanol (1 g/kg) on the reinstatement of ethanol-induced CPP in control and estradiol valerate (EV)-treated mice. Mean ± SEM seconds per minute spent on grid during a floor choice testing of 60 minutes. Vehicle and EV-treated animals (same animals shown in Fig. 2 and 3) showed a previously extinguished ethanol induced CPP. Five days after the last preference test (test 4), all animals received an i.p. injection of 1 g/kg ethanol immediately before being tested for preference. Preference for the ethanol-paired floor was obtained in both, vehicle and EV-treated groups (*a significant main effect of conditioning subgroup was found, but no effect of treatment with EV, p < 0.01). G+; Grid+, G-; Grid-.

Figure 5

Effect of naltrexone (0 or 2 mg/kg; i.p.) on the expression of ethanol-induced CPP. Mean ± SEM seconds per minute spent on grid during a floor choice testing of 60 minutes. Grid+ (G+) and Grid- (G-) conditioning subgroups, n = 12 per group. Grid+; ethanol paired to grid floor, saline paired to hole floor. Grid-; ethanol paired to hole floor, saline paired to grid floor. On the test day, naltrexone was injected 30 min prior to the choice test (*p < 0.01, difference between saline-treated G+ and Grid- groups.

Figure 6

Effect of estradiol valerate (EV) on behavioral sensitization to ethanol. Group means (± SEM) of locomotor activity performed during the CS+ and CS- pairing sessions (5 min) are presented (n = 20-24 per group, same animals shown in Figs. 2-4). Following the CPP procedure, animals received four i.p. saline/ethanol (2 g/kg) injections on alternate days, with a 2 day-break separating the first and second blocks of four pairing sessions. Vehicle and EV groups refer to animals treated with sesame oil or with 2 mg of EV in 0.2 ml of oil (i.m.) per animal respectively. CS+ data for vehicle, but not EV-treated mice showed a sensitized response to ethanol (*p < 0.05, comparing locomotor scores of the fourth session with ethanol in the control group to those of the first session).