Research reportNaloxone blocks ethanol-mediated appetitive conditioning and locomotor activation in adolescent rats
Research highlights
▶ Ethanol-induced appetitive second-order conditioning in adolescent rats. ▶ Ethanol induced motor behavioral stimulation in adolescent rats. ▶ Administration of naloxone inhibited the appetitive conditioning.▶ Naloxone dose-dependently blocked ethanol's motor activating effects. ▶ The opioid system mediates ethanol's reinforcing and motor activating effects.
Introduction
Until recently, research on underage drinking has been largely neglected [1], partially attributable to the belief that adolescent ethanol intake is a transient phenomenon that lessens as the subject engages in family and work-related duties [1]. Epidemiological and preclinical studies, however, show that the effects of adolescent drinking can be long lasting. Adolescent ethanol consumption is highly prevalent (with alcohol initiation in approximately 60% of teens aged 15–16 years in the United States [2]), and the risk of developing alcohol abuse and dependence is significantly higher in those who begin drinking before the age of 21 [3], [4].
The use of animal models has shed some light on the determinants of ethanol intake in adolescents. When compared with adult counterparts, adolescent rats are less sensitive to several effects of ethanol that presumably serve as deterrents to escalated ethanol use [5]. These effects include ethanol-induced locomotor impairment [6], sedation [7], and ethanol-induced hangover [8]. Adolescents are also more resistant than adults to the suppressive effects of high-dose ethanol on social behaviors [9]. However, adolescents are more sensitive than adults to the cognitive impairments associated with ethanol intoxication [10] and show increased social facilitation after low-dose ethanol [9].
A few studies have suggested that adolescents may show a distinctive response pattern to ethanol's appetitive and aversive motivational effects compared with older subjects. Ethanol-mediated conditioned place preference (CPP) is readily found in mice [11] and in genetically selected rats [12], [13], but is rare in heterogeneous, non-selected adult rats [14]. The expression of CPP by ethanol in heterogeneous adult rats has been reported only after very extensive training and pre-exposure procedures (15 days or more [15], [16], [17]), concurrent presentation of other reinforcers [18] or exposure to electric shock [19]. In contrast, Philpot et al. [20] observed ethanol-mediated CPP in rats after only four training trials conducted shortly after weaning on postnatal day 25 (PD25; 0.2 g/kg) and also during late adolescence on PD45 (0.5 and 1.0 g/kg). Adolescent and adult rats also appear to differ in their sensitivity to ethanol's aversive effects. Adult, but not adolescent, rats expressed conditioned taste aversion (CTA) after pairings of ethanol (1.0 and 1.5 g/kg) and a sapid stimulus [21]. Adolescents expressed CTA, albeit at a higher dose. These studies suggest that adolescents may be relatively insensitive to ethanol's aversive effects and more likely than adults to acquire ethanol-mediated appetitive learning.
Another approach for assessing ethanol's hedonic effects involves a second-order conditioning CPP procedure [22], [23], [24] that circumvents the traditional difficulty in establishing first-order CPP. In the second-order conditioning preparation, rats are given pairings of ethanol and a conditioned taste stimulus (CS1) infused intraorally (e.g., very small amounts of water or sucrose). In a second phase, the intraorally infused CS1 is paired with an environment (CS2) featuring distinctive cues (e.g., sandpaper flooring and striped wallpaper). Ethanol-mediated conditioning is indexed by measuring preference or aversion for the CS2 environment. Fourteen-day-old rats readily exhibited appetitive second-order conditioning when the intraoral CS1 was paired shortly (5–20 min post-administration) after intubation with 0.5 or 2.0 g/kg ethanol or during a late phase (30–45 min post-administration) of the intoxication induced by 0.5 g/kg ethanol [22], [23]. Second-order aversion, however, emerged when the CS1 predicted the late effects induced by 2.0 g/kg ethanol [22], [23]. A subsequent study [24] found age-related differences in the expression of ethanol-mediated second-order conditioning. Adolescent, but not adult, rats expressed second-order appetitive conditioning (0.5–2.0 g/kg). This outcome was unchanged if the CS1 predicted the initial or late stage of the ethanol post-administration interval. At higher doses (3.0–3.25 g/kg), ethanol induced conditioned aversion, which was similar in adolescents and adults [25]. One caveat of these studies [24], [25] was that the CS1 paired with ethanol (sucrose, 10%, v/v) had motivational properties of its own that may have interacted with ethanol's effects in the establishment of conditioning.
Ethanol-induced psychomotor stimulation represents yet another, albeit indirect, measure of ethanol's motivational effects. Humans at risk for developing alcohol problems show greater ethanol-induced tachycardia than individuals not at risk for the disorder [26]. Interestingly, adolescent, but not adult, rats self-administer sufficient oral ethanol to produce tachycardia [27]. Heterogeneous adult rats are also insensitive to the locomotor-activating effects of ethanol and, unlike mice [28], typically show locomotor depression after ethanol administration [29]. However, acute ethanol administration induces locomotor activation in heterogeneous infant rats [30], [31], [32], [33] and adult rats selectively bred to ingest large amounts of ethanol [34]. In a recent study, we observed ethanol-induced locomotor activation in adolescent rats (PD 28) after intubation with 2.5 g/kg, but not 0.5 g/kg, ethanol [35]. Moreover, females that had exhibited heightened sensitivity to ethanol's psychomotor effects on PD28 ingested significantly larger quantities of ethanol than counterparts less sensitive to ethanol-induced locomotor activation [35].
The adolescents’ idiosyncratic variation in reactivity to ethanol may put them at risk for alcohol-related problems [5]. More work is needed, however, to test the sensitivity of adolescents to ethanol's hedonic effects and the neurobiological mechanisms underlying ethanol reinforcement during this stage. The opioid neurotransmitter system is known to be involved in ethanol reinforcement and intake. Administration of opioid receptor antagonists inhibits CPP induced by ethanol in adult mice [36] and infant rats [37] and disrupts appetitive conditioning induced by ethanol in neonatal rats [38]. The psychomotor stimulant effects of ethanol also appear to be opioid-dependent. General opioid antagonism inhibits tachycardia produced by alcohol ingestion in healthy subjects [39] and dose-dependently reduces ethanol-induced locomotor activation in adult mice [28] and infant rats [32]. The involvement of the endogenous opioid system in mediating ethanol's motivational effects in adolescents has yet to be explored.
The present work assessed: (i) the sensitivity of adolescent, heterogeneous rats to ethanol's reinforcing and psychomotor stimulating effects and (ii) the participation of the opioid system in these effects of ethanol. Appetitive conditioning with ethanol was tested in Experiment 1 with a second-order conditioning procedure after administration of the general opioid antagonist naloxone. In addition to the important issue of testing the role of the endogenous opioid system, water instead of sucrose was used as the CS1 paired with ethanol's effects, which was different from our previous second-order conditioning study [24]. This procedural refinement removes potential confounding factors associated with the use of non-neutral sapid CSs and establishes the generality of the effect. Experiment 2 tested ethanol-induced locomotor activation after various doses of naloxone. In adult rats [40], naloxone can alter ethanol's pharmacokinetics. Therefore, we also measured blood alcohol levels (BALs) in adolescents following naloxone or vehicle injection.
Section snippets
Subjects
A total of 175 adolescent Wistar rats (89 males and 86 females) were used. These animals were 28–30 days old at the start of the experimental procedures, had a mean body weight of 123.04 ± 1.7 g (females, 113.49 ± 1.62 g; males, 131.94 ± 2.18 g), and were born and reared in the vivarium of the Center for Development and Behavioral Neuroscience at Binghamton University. The number of animals and litter representation across experiments were the following: Experiment 1 (44 males and 41 females,
CPP scores
Fig. 2 depicts the relative time spent in the CS2 compartment as a function of ethanol and naloxone treatment. Rats that experienced ethanol–CS1 pairings after administration of the naloxone vehicle (group 0.0 nal–ethanol) spent significantly more percent time in the CS2 compartment than any other group. This heightened preference for the CS2 compartment, indicating ethanol-mediated CPP, was inhibited in animals given naloxone (0.75, 1.5, or 2.5 mg/kg) prior to ethanol.
The ANOVA yielded a
Discussion
Analyzing the determinants of ethanol affinity during adolescence is important. Ethanol intake during this developmental stage can have long-lasting effects on future abuse of the drug. Grant and Dawson [4] found a four-fold increase in the likelihood of alcohol dependence in individuals who initiated alcohol use at age 14 compared with individuals who began drinking at age 20 or later. Adolescents exhibit a distinctive ethanol response pattern [5]. Among the several effects of ethanol,
Acknowledgments
This work was a collaborative project between the Research Foundation of SUNY Binghamton and Instituto Ferreyra and was supported by National Institute on Alcohol Abuse and Alcoholism grants AA011960, AA01309, and AA017823 to NES and AA018164 to MN and grants PIP CONICET 2010–2012 and PICT-PRH 246 (Argentina) to RMP. We are thankful to Teri Tanenhaus and Pouyan Rhamani for their technical assistance.
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