Chronic ethanol exposure during adolescence alters the behavioral responsiveness to ethanol in adult mice

Behavioural Brain Research 229 (2012) 1–9

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Chronic ethanol exposure during adolescence alters the behavioral responsiveness to ethanol in adult mice Caroline Quoilin, Vincent Didone, Ezio Tirelli, Etienne Quertemont ∗ Département Psychologie, Cognition et Comportement, Université de Liège, Belgium

a r t i c l e

i n f o

Article history: Received 27 October 2011 Received in revised form 22 December 2011 Accepted 25 December 2011 Available online 31 December 2011 Keywords: Ethanol Adolescence Locomotor activity Behavioral sensitization Sedation Mice

a b s t r a c t Alcohol exposure during early adolescence is believed to durably alter the behavioral properties of ethanol, increasing the likelihood of later alcohol-related disorders. The aim of the present experiments was to characterize changes in the behavioral effects of ethanol in adult female Swiss mice after a chronic ethanol exposure during adolescence, extending from postnatal day 28 to postnatal day 42. After a chronic ethanol exposure during adolescence (daily injections of 0, 2.5 or 4 g/kg ethanol for 14 consecutive days), adult mice were tested at postnatal day 63. The locomotor stimulant effects of ethanol, together with ethanol sensitization were tested in experiment 1. In experiment 2, the sedative effects of ethanol were assessed with the loss of righting reflex procedure. Finally, in experiment 3, the anxiolytic effects of ethanol were tested with the light/dark box test. Adult mice chronically exposed to ethanol during adolescence showed a lower basal locomotor activity, but higher locomotor stimulant effects of ethanol than non-exposed mice. Additionally, these adult mice developed higher rates of ethanol sensitization after chronic re-exposure to ethanol in adulthood. Adult mice exposed to ethanol during adolescence also had a stronger tolerance to the sedative effects of high ethanol doses, although they showed no evidence of changes in the anxiolytic effects of ethanol. These results are in agreement with the thesis that chronic alcohol consumption during adolescence, especially in high amounts, increases the risk of later alcohol-related disorders. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The transition between childhood and adulthood is characterized by specific behavioral and neurobiological changes, which make adolescence a critical and unique period of development [1]. These features have been suggested to predispose adolescents to experiment with drugs, including alcohol [2,3]. Indeed, alcohol use often begins during early adolescence, with alcohol consumption frequently taking the form of binge drinking [4]. This behavioral pattern, defined as consuming five or more drinks on the same occasion, is reported by approximately one fifth of people aged 12–20 [5]. Moreover, binge drinking is especially prevalent during this developmental period, since it drops off after the entrance in adulthood [6,7]. Studies in laboratory rodents also confirmed this conclusion, with adolescents consuming up to twice more than their adult counterparts [8–11]. These observations are a matter of concern considering that early alcohol abuse may have serious consequences for the later

∗ Corresponding author at: Département Psychologie, Cognition et Comportement, Université de Liège, Boulevard du Rectorat 5/B32, 4000 Liège, Belgium. Tel.: +32 4 366 21 05; fax: +32 4 366 28 59. E-mail address: [email protected] (E. Quertemont). 0166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.12.039

development of alcohol-related disorders. In general, the age at first drug use has been suggested to be a major risk factor for subsequent drug abuse and dependence [12,13]. Regarding alcohol, clinical studies confirm that a first use of alcohol during early adolescence is associated with a higher probability of alcohol problems in adulthood [14]. More precisely, Grant and Dawson [15] have shown that the rate of lifetime dependence reaches 40% when the initiation of alcohol begins before the age of 14, while it is limited to 10% among individuals who start drinking after the age of 20. Finally, this correlation between early alcohol exposure and later increases in ethanol consumption has also been described in animal research, especially in laboratory rodents [10,16–19]. Several explanations have been proposed to account for this correlation. One of them suggests that the maturing adolescent brain could be more vulnerable to ethanol-induced neuroadaptations, due to the higher plasticity of the brain at this age [1,20–23]. In other words, adolescents could be more sensitive to the long-term consequences of chronic ethanol exposure, which in turn would increase the likelihood of later alcohol-related disorders [1]. In support of this explanation, clinical and preclinical studies have shown that ethanol consumption during adolescence may alter adult brain functioning, leading to neuropsychological functional deficits [24,25], as well as alterations in brain gene expression and neurochemistry [26–31].

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Through these neurobiological alterations, chronic ethanol exposure during adolescence also leads to behavioral changes later in life. Several studies have addressed this issue using rodent models of alcohol effects and have reported an altered response to alcohol in adults chronically exposed to ethanol during adolescence. For example, McBride et al. [32] show an easier acquisition of alcohol self-administration, as well as increases in craving-like behaviors and in potential for relapse in adult rats after alcohol exposure during adolescence. Diaz-Granados and Graham [33] also report attenuated ethanol-induced conditioned taste aversion in similar conditions. Moreover, adult rodents chronically exposed to alcohol during adolescence are more sensitive to ethanol-induced motor [34] and memory [35,36] impairments, whereas they are less sensitive to the sedative effects of ethanol [37,38]. These results indicate that adolescent ethanol exposure might durably alter its motivational properties, and especially the balance between its rewarding and aversive effects. In turn, these changes could enhance the likelihood to progress from episodic alcohol consumption to alcoholism. In mice, the motivational properties of ethanol are often indirectly investigated through the assessment of the acute locomotor stimulant effects of ethanol, along with the sensitization of these effects after repeated ethanol administrations. Indeed, ethanolinduced psychomotor stimulation is believed to be related to its addictive properties and is associated with its reinforcing effects, as evidenced by the high homology between brain regions underlying the stimulant and rewarding effects of alcohol [39]. Moreover, psychomotor sensitization, defined as a progressive and enduring increase in the stimulant effects of a drug following repeated administrations, is currently viewed as a major component of addiction in contemporary theories of drug abuse [40–42]. However, it remains unclear whether ethanol exposure during adolescence durably alters its stimulants properties and/or the development of ethanol sensitization in adulthood. The impact of adolescent ethanol exposure on other behavioral effects of ethanol in adults, such as its anxiolytic and sedative effects also needs to be further investigated. The anxiolytic effects of ethanol for example is among the most desired effects of ethanol consumption and is believed to contribute to alcohol abuse through a negative reinforcement mechanism [43,44]. Indeed, according to the “tension-reduction hypothesis”, elevated levels of anxiety increase the predisposition to develop alcohol use disorders [45]. In contrast, the sedative effects of ethanol are generally considered as a limiting factor for alcohol consumption [46]. Adolescence is defined as the developmental period corresponding to the transition from childhood to adulthood, during which youngsters acquire skills necessary to become independent [1]. In rodents, adolescence sometimes refers to the whole period ranging from weaning (postnatal day (P) 21) to the entrance in adulthood (P60), although it could be subdivided into shorter periods with specific behavioral and neurobiological changes [47]. However, some authors use a narrower definition of the prototypic adolescence period ranging approximately from P28 to P42 [1]. Accordingly, in the present experiments, the daily administration of ethanol between P28 and P42 was used as an animal model of chronic alcohol consumption during adolescence. In a recent study, we have shown that adolescent mice require higher ethanol doses than adult mice to develop sensitization to the stimulant effects of ethanol [48]. Therefore, in the present experiments, two different chronic ethanol treatments during adolescence are compared: the daily administration of either 2.5 g/kg ethanol, a typical moderate ethanol dose in mice, or 4 g/kg ethanol, a high ethanol dose. The aim of the present experiments was to characterize the changes in the sensitivity to some of the behavioral effects of ethanol in adult female Swiss mice after a chronic ethanol treatment during adolescence, extending from P28 to P42. Chronic

ethanol exposure consisted in daily injections of ethanol (2.5 or 4 g/kg) or saline for 14 consecutive days starting on P28. Acute behavioral responses to ethanol were then tested in adult mice at P63. In the first experiment, we tested whether ethanol exposure during adolescence altered the locomotor stimulant effects of ethanol and ethanol sensitization in adulthood. In experiments 2 and 3, a similar experimental protocol was used to test the effects of chronic ethanol administration during adolescence on the sedative and anxiolytic effects of ethanol in adulthood. 2. Material and methods 2.1. Subjects For the whole study, 132 female Swiss mice bred in our colony were used. These mice were bred from progenitors purchased from Janvier Laboratories (Le Genet St Isle, France). Pregnant mice were housed four per cage and births were checked every day. At the time of birth (postnatal day 0, P0), pups and their mothers were collectively gathered, in order to group three litters with pups born on the same day. Pups were weaned at P21 and housed two per cage at the start of the ethanol exposure in white polyethylene cages with pine sawdust bedding. Female mice were chosen for the present experiments in order to be consistent with previous ontogenetic studies of our laboratory [48,49]. The animal room was maintained on a 12-h light/dark cycle (lights on at 8:00 am) and kept at a constant temperature. Food (standard pellets, Carfil Quality BVDA, Oud-Turnhout, Belgium) and water were available ad libitum for the whole experiment. All experimental procedures were conducted during the light phase of the cycle between 9:00 am and 1:00 pm and were carried out according to the European Communities Council Directive (86/609/EEC) for care and use of laboratory animals. Protocols were reviewed and approved by the Animal Care Committee of the University of Liège. 2.2. Drugs Ethanol injections were 20% (v/v), diluted from 99.9% ethanol in an isotonic 0.9% saline solution. The same ethanol concentration was used for the different doses of ethanol (2, 2.5 and 4 g/kg) in order to avoid concentration-induced differences in absorption. Therefore, volumes of injections for each ethanol dose were respectively 0.013, 0.016 and 0.025 ml/g. The dose of 2 g/kg ethanol was only used in experiment 3 to test the anxiolytic effects of ethanol in adult mice. Vehicle control mice were injected with a saline solution (0.9%) in a volume of 0.01 ml/g. All injections were administered via the intraperitoneal (i.p.) route in all experiments. 2.3. Apparatus 2.3.1. Locomotor activity The locomotor effects of ethanol were assessed with a videocamera system. The experimental environment consisted of transparent Plexiglas chambers (42 cm × 42 cm) partitioned into 6 compartments (7 cm × 42 cm) and placed on a square platform of transparent glass held horizontally by a robust frame. The floor of each compartment was divided into six squares (7 cm × 7 cm) by black lines. A portable S-VHS video camera (Panasonic NS-MS1) was positioned directly underneath, such that the whole surface was covered, and allowed the simultaneous testing of six mice. A character generator (type Panasonic VW-CG2E) was connected to the control monitor and was used to indicate elapsed time (100/s). Lighting was provided by four white neon-tubes (18 W) fixed on each leg of the frame, such that the light was not directed to the mice. These apparatuses were used in two exemplars and each of them was individually located in a small, ventilated and heated (21–23 ◦ C) white room (140 cm × 140 cm surface × 245 cm height). Videotapes were later scored by one or two trained observers blind to the drug treatment. The interand intra-observer agreements (three observers) were estimated with Lin’s concordance correlation coefficients on a series of sample sessions, and were never lower than r = 0.966 [50]. For the test sessions, videotape scoring was applied on the whole session (30 min) and was divided into 6 intervals of 5 min. Locomotor activity was measured as the number of line crosses, a locomotor count being recorded each time a mouse crossed a line with four legs. 2.3.2. Light/dark box test The light/dark box test consists of a box with two compartments: a small, opaque, and closed dark compartment (16 cm × 16 cm × 16 cm) and a large, translucent, illuminated and opened white compartment (25 cm × 25 cm × 30 cm). The compartments are connected by an opening (5 cm × 5 cm) to allow mice to freely move from one compartment to the other. A spot light illuminated the apparatus to provide an aversive stimulus (250 lx; only visible from the white compartment). A camera (Logitech QuickCam Pro5000) was positioned 1 m above the apparatus and connected to a computer to record mouse behaviors during the test, and videos were later scored. The time spent in each compartment was used to assess the level of anxiety. Indeed, this apparatus provides an unfamiliar environment for mice where a natural conflict occurs between the tendency to explore a novel environment and

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2.4. Procedure 2.4.1. Chronic ethanol treatment during adolescence In all of the experiments, ethanol exposure during adolescence was carried out with the same protocol. At P28, female Swiss mice were housed two per cage, these conditions being kept until the end of the experiments. They were randomly divided into three groups: a high ethanol dose-exposed group (4 g/kg), a moderate ethanol dose-exposed group (2.5 g/kg), and a control group (saline). Mice were daily injected with their assigned treatment (i.p.) during 14 days in their home cages. Therefore, ethanol exposure extended from P28 to P41, which corresponds to the prototypic adolescence period in rodents [1]. In order to test for the consequences of ethanol exposure during adolescence on the subsequent sensitivity to the effects of ethanol at adulthood, mice were then tested for the behavioral effects of ethanol at P63. 2.4.2. Experiment 1 The aim of the first experiment was to test for potential alterations in the sensitivity to the stimulant effects of ethanol and their sensitization in adult mice after ethanol exposure during adolescence. At P62, 12 mice from each ethanol exposure groups were submitted to a habituation session. They were moved to the experimental room and left undisturbed for 30 min. Mice were then i.p. injected with saline and immediately placed into the experimental apparatus where their locomotor activity was recorded for 30 min. On the next day at P63, the same procedure was reproduced, except that mice were injected with 2.5 g/kg ethanol in order to assess their sensitivity to the locomotor stimulant effects of ethanol. All adult mice were then submitted to an ethanol sensitization procedure using an experimental protocol that was extensively validated in our laboratory [53]. On the next six consecutive days, these mice were daily injected with ethanol and their locomotor activity was recorded for 5 min in order to assess ethanol sensitization. At the end of this procedure, the expression of sensitization was tested. All mice were again injected with 2.5 g/kg ethanol, immediately placed into the experimental apparatus, and their locomotor activity was recorded for 30 min. Finally, the expression of ethanol sensitization to a higher ethanol dose (4 g/kg) was also tested following the same procedure on the next day. 2.4.3. Experiment 2 This experiment was designed to investigate the sensitivity to the sedative effects of ethanol in adult mice repeatedly exposed to saline or ethanol during adolescence. 36 mice (n = 12/exposure group) were tested for ethanol-induced loss of righting reflex (LORR). A lower sensitivity to the sedative effects of ethanol was defined as a shorter duration of LORR along with a recovery of the righting response at a higher blood ethanol concentration (BEC) [see Ref. [51]]. At P63, mice were i.p. injected with 4 g/kg ethanol before being replaced in their home cage. When they lost their righting reflex, they were placed into a V-shaped support (frame measuring 22 cm × 11 cm × 6 cm) until their reflex was recovered, the recovery being defined as the ability to right three times in 1 min. The duration of the LORR was recorded. Immediately after recovery of the righting reflex, mice were decapitated. Blood samples were collected in heparinized capillaries, centrifuged for 5 min and stored at 4 ◦ C for later analysis on the same day. Five microliter of plasma from each sample was analyzed to determine BEC using the Analox AM1 alcohol analyzer (Analox Instruments, London, UK). 2.4.4. Experiment 3 The third experiment was carried out in order to test for the consequences of ethanol exposure during adolescence on both anxiety-like behaviors and on anxiolytic effects of ethanol in adulthood. One week before testing, mice were housed in individual cages. At P63, 20 mice from each ethanol exposure group were subdivided into two sub-groups, which were injected with either saline (control group) or 2 g/kg ethanol. Anxiety was assessed in the dark/light box test. Five minutes after injection, mice were placed into the center of the white compartment. They were then allowed to freely explore the compartments for 5 min, and time spent in each of them was recorded. 2.5. Statistical analyses Locomotor counts, durations of the loss of righting reflex, blood ethanol concentrations, and percentages of time spent in the dark compartment were analyzed with analyses of variance (ANOVA) for mixed design. In experiment 1, the acute stimulant effects of ethanol were analyzed using a two-way ANOVA, with chronic treatment (three levels) as a between-subject factor, and post-injection intervals (six levels) as a within-subject factor. In order to test for within- and between-subject sensitizations, a three-way ANOVA was computed on the locomotor activity counts during the acute and sensitization sessions. Experimental session (two levels) and post-injection intervals (six levels) were defined as within-subject factors, while the chronic treatment (three levels) was defined as a between-subject factor. As basal locomotor activity was significantly different between ethanol exposure groups (see Section 3), a sensitization index was also

calculated for each mouse taking into account its basal activity using the following formula: (sensitized activity − acute activity)/basal activity. This score corresponds to a differential rate of increase from basal activity between the sensitization test session and the acute test session. A one-way ANOVA was computed on these scores of sensitization using chronic treatment (three levels) as a between-subject factor. Finally, the expression of sensitization induced by a high ethanol dose was treated with a one-way ANOVA with chronic treatment (three levels) as a between-subject factor. In experiment 2, one-way ANOVAs were performed on durations of the LORR and BECs, with chronic treatment (3 levels) as a between-subject factor. Finally, the percentages of time spent in the dark compartment in experiment 3 were analyzed using a two-way ANOVA, chronic treatment (3 levels) and test treatment (2 levels) being defined as between-subject factors. The ANOVAs were followed by Newman–Keuls post hoc comparisons to assess between-group differences. Statistical significance was set at p < 0.05.

3. Results 3.1. Experiment 1 On the habituation session, the two-way ANOVA performed on the locomotor activity counts during the whole 30 min of the session showed significant main effects of post-injection intervals [F(5,140) = 36.55; p < 0.01] and chronic treatment [F(2,140) = 5.65; p < 0.01], while the interaction between the two factors was not significant [F(10,140) = 0.60; p = 0.82]. The Newman–Keuls post hoc comparisons indicated that mice exposed to 2.5 and 4 g/kg at adolescence showed a lower basal locomotor activity relative to control mice all along the session (Fig. 1). The two-way ANOVA computed on the locomotor activity counts during the entire acute session revealed a significant main effect of post-injection intervals [F(5,140) = 39.05; p < 0.01] and a significant interaction between post-injection intervals and chronic treatment [F(10,140) = 4.25; p < 0.01], whereas the main effect of chronic treatment was not significant [F(2,140) = 2.16; p = 0.13]. The Newman–Keuls post hoc comparisons confirmed that time course of the locomotor activity during the test session was dependent upon the chronic treatment. As shown on Fig. 2, the significant interaction was due to significant differences in locomotor activity during the first 5 min of the session. At the beginning

Deltas (locomotor activity (counts/30 min)

the innate aversion to open field and brightly illuminated area. As the white, but not the dark, compartment provides stress-like conditions for mice, the percentage of time spent in the dark compartment is used as a measure of anxiety in mice.

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Adolescence ethanol exposure (g/kg) Fig. 1. Effects of ethanol exposure during adolescence (0, 2.5, 4 g/kg) on spontaneous locomotor activity in young adult female mice (habituation session of experiment 1). All mice were tested as adults (62-day-old mice) after a saline injection, but the groups differ in the chronic treatment that was administered during adolescence. Data are expressed as mean ± SEM of total locomotor activity scores during the whole 30-min habituation session (n = 12/group). *p < 0.05: significantly different from the control group.

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Fig. 2. Effects of ethanol exposure during adolescence (0, 2.5, 4 g/kg) on the acute stimulant effects of 2.5 g/kg ethanol in young adult female mice (acute session of experiment 1). All mice were tested as adults (63-day-old mice) after a 2.5 g/kg ethanol injection, but the groups differ in the chronic treatment that was administered during adolescence. Panel A depicts the time course of locomotor activity during the whole session in 6 intervals of 5 min. Panel B shows the locomotor activity during the first 5 min of the session. Data are expressed as mean ± SEM of locomotor activity scores in 5 min intervals (n = 12/group). ***p < 0.001: significantly different from the control group.

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Fig. 3, the development of ethanol sensitization during adulthood was characterized by overall higher stimulant effects of ethanol throughout the sensitization test session in all three groups. Additionally, ethanol-sensitized mice showed a much longer duration of the stimulant effects of ethanol during the sensitization test session (Fig. 3A) relative to the acute test session (Fig. 2A). This change in the time course of the stimulant effects of ethanol was especially striking in mice pre-exposed to 4 g/kg ethanol during adolescence. In order to better grasp differences in the development of ethanol sensitization in adulthood between mice exposed to different chronic treatments during adolescence, a sensitization index was also calculated for each mouse taking into account the basal activity. The one-way ANOVA computed on these sensitization scores indicated a statistically significant effect of the chronic treatment administered during adolescence [F(2,28) = 4.09; p < 0.05]. As shown on Fig. 4, mice pre-exposed to ethanol during adolescence had higher rates of ethanol sensitization in adulthood. This result is confirmed by the post hoc analyses (p < 0.05). Finally, in order to assess the expression of ethanol sensitization and/or tolerance after the administration of a higher dose of ethanol, all mice were administered 4 g/kg on the next day, and their locomotor activity was recorded during 30 min. The oneway ANOVA yielded a significant main effect of the adolescent chronic treatment [F(2,28) = 6.01; p < 0.01], with mice pre-treated

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of the session, the stimulant effects of ethanol were significantly enhanced in mice pre-exposed to ethanol during adolescence, with a significantly higher simulation in mice pre-treated with 4 g/kg ethanol, relative to the control mice (p < 0.01). Mice pre-treated with 2.5 g/kg ethanol during adolescence had intermediate results with a tendency toward statistical signification (p = 0.06). The three-way ANOVA computed on the time course of the locomotor activity counts for the adult acute and sensitization sessions showed significant main effects of session [F(1,140) = 49.23; p < 0.01], post-injection intervals within the sessions [F(5,140) = 17.83; p < 0.01] and adolescent chronic treatment [F(2,140) = 3.70; p < 0.05]. The significant main effect of adolescent chronic treatment was mainly due to the higher stimulant effects of ethanol in mice chronically treated with 4 g/kg ethanol during their adolescence relative to the other two groups. The significant main effect of session revealed the development of ethanol sensitization over repeated administrations of ethanol in adulthood. There were also significant first-order interactions for session × post-injection intervals [F(5,140) = 5.41; p < 0.01] and chronic treatment × postinjection intervals [F(10,140) = 3.79; p < 0.01], while the interaction between session and chronic treatment was not significant [F(10,140) = 1.98; p = 0,16]. Finally, the second-order interaction session × post-injection intervals × chronic treatment was close to statistical significance [F(10,140) = 1.72; p = 0.08]. As shown on

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Fig. 3. Effects of ethanol exposure during adolescence (0, 2.5, 4 g/kg) on the expression of ethanol sensitization after a chronic re-exposure to 2.5 g/kg ethanol in young adult female mice (sensitization session of experiment 1). All mice were tested as adults (70-day-old mice) after a 2.5 g/kg ethanol injection at the end of a chronic treatment of 7 daily ethanol injections. The groups differ only in the chronic treatment that was administered during adolescence. Panel A depicts the time course of locomotor activity during the whole session in 6 intervals of 5 min. Panel B shows the locomotor activity during the first 5 min of the session. Data are expressed as mean ± SEM of locomotor activity scores in 5 min intervals (n = 12/group). *p < 0.05: significantly different from the control group.

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Fig. 4. Effects of ethanol exposure during adolescence (0, 2.5, 4 g/kg) on the development of a sensitization to the stimulant effects of ethanol in adulthood (experiment 1). All groups of female mice were daily injected with 2.5 g/kg ethanol in adulthood, but the groups differ in the chronic treatment that was administered during adolescence. Data are expressed as mean ± SEM of a sensitization index (n = 12/group). *p < 0.05: significantly different from the control group.

with 4 g/kg of ethanol during adolescence showing a locomotor activity twice as high as the other two groups (p < 0.01; Fig. 5).

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Adolescence ethanol exposure (g/kg) Fig. 5. Effects of ethanol exposure during adolescence (0, 2.5, 4 g/kg) on locomotor activity after the administration of a high dose of ethanol in adulthood (final session of experiment 1). All female mice were tested as adults (71-day-old mice) after a 4 g/kg ethanol injection, but the groups differ in the chronic treatment that was administered during adolescence. Data are expressed as mean ± SEM of total locomotor activity scores during the whole 30-min test session (n = 12/group). **p < 0.01: significantly different from the control group.

for 2.5 g/kg; p < 0.01 for 4 g/kg; Fig. 6B). Overall, these data show that chronic ethanol administration during adolescence alters the sensitivity to the sedative effects of ethanol in adulthood.

3.2. Experiment 2 The one-way ANOVA computed on the durations of the loss of righting reflex displayed a significant effect of the adolescent chronic treatment [F(2,32) = 4.52; p < 0.05]. As shown on Fig. 6A, the mean duration of the loss of righting reflex after the administration of 4 g/kg ethanol was dependent upon the chronic treatment administered during adolescence, with mice repeatedly exposed to the higher dose of ethanol showing a significantly shorter duration of the LORR relative to control mice (p < 0.05). Furthermore, this altered sensitivity could not be attributed to differences in pharmacokinetics. Indeed, the one-way ANOVA performed on BECs at recovery of the righting reflex also revealed a significant effect of the chronic treatment [F(2,32) = 5.74; p < 0.01]. The Newman–Keuls post hoc comparisons indicated that alcohol pre-exposed mice regained their reflex at higher BECs than control mice (p < 0.05

3.3. Experiment 3 The two-way ANOVA performed on the percentages of time spent in the dark compartment revealed a significant main effect of the ethanol dose [F(1,52) = 27.83; p < 0.01], whereas the main effect of chronic adolescence treatment [F(2,52) = 0.44; p = 0.64] and the interaction between these factors did not reach statistical significance [F(2,52) = 0.17; p = 0.84]. The Newman–Keuls post hoc tests indicated that the injection of 2 g/kg immediately before the test session significantly reduced the time spent in the dark compartment in all three groups (Fig. 7). However, ethanol exposure during adolescence did not significantly affect the time spent in the dark compartment after either a saline or a 2 g/kg ethanol injection.

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Fig. 6. Effects of ethanol exposure during adolescence (0, 2.5, 4 g/kg) on the sedative effects of 4 g/kg ethanol in young adult female mice (experiment 2). All mice were tested as adults (63-day-old mice) after a 4 g/kg ethanol injection, but the groups differ in the chronic treatment that was administered during adolescence. Panel A depicts the duration of the loss of righting reflex (LORR). Panel B shows the blood ethanol concentrations (BECs) at recovery. Data are respectively expressed as mean ± SEM of duration of LORR and mean ± SEM of BECs (n = 12/group). *p < 0.05; **p < 0.01: significantly different from the control group.

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Ethanol tested dose (g/kg) Fig. 7. Effects of ethanol exposure during adolescence (0, 2.5, 4 g/kg) on the anxiolytic effects of ethanol and on anxiety-like behaviors in young adult female mice (experiment 3). On the test day, adult mice (63-day-old mice) were injected with either saline or 2 g/kg ethanol. In each condition, the groups differ in the chronic treatment that was administered during adolescence: saline (white bars), 2.5 g/kg ethanol (light gray bars) or 4 g/kg ethanol (dark gray bars). Data are expressed as mean ± SEM of the percentage of time spent in the dark compartment (n = 10/group). *p < 0.05; **p < 0.01: significantly different from the respective control group.

4. Discussion Because of their maturing brain, adolescents are believed to be more vulnerable to ethanol-induced long-lasting neuroadaptations, which in turn could enhance the risks of later alcohol-related disorders. According to this hypothesis, ethanol abuse during adolescence should induce enduring changes on both brain and behavior that will persist into adulthood. Among these changes, alterations in behavioral responses to ethanol consumption are of particular importance because of their relationship to alcohol abuse. The results of the present experiments confirm that alcohol exposure during adolescence durably alters the sensitivity to the behavioral effects of ethanol. Adult female mice chronically exposed to ethanol during adolescence showed a lower basal locomotor activity, enhanced locomotor stimulant effects after ethanol administration, higher rates of ethanol sensitization after chronic ethanol re-exposure and a stronger tolerance to the sedative effects of high ethanol doses. In contrast, we found no evidence of changes in the anxiolytic properties of ethanol. Adult female mice exposed to chronic ethanol during adolescence showed a significantly reduced locomotor activity throughout the 30 min habituation session (Fig. 1). This effect cannot be explained by changes in the levels of anxiety as the light/dark box test failed to show any effect of ethanol exposure during adolescence on anxiety-like behaviors. A conditioned compensatory response can also be ruled out as ethanol injections were administered in the home cages without exposure to the testing environment. One possible explanation is a reduced response to novelty in these mice chronically exposed to ethanol, although a previous study in rats did not report a reduced response to novelty in adolescent rats chronically administered ethanol and tested as adults [52]. Another possible explanation is a reduced spontaneous basal activity unrelated to the novelty of the testing environment. Reduced spontaneous activity may be indicative of several conditions, such as for example depression or more simply general health failing. However, these mice had a normal weight (data not shown) and did not show any other noticeable differences in behavior or in general health status, such that it is difficult to ascribe their reduced locomotor merely to general health failing. Clearly, further studies

will be required to solve this question and explain this unexpected result. One of the most striking results of the present study is enhanced stimulant effects of ethanol together with higher rates of ethanol sensitization in adult female mice chronically exposed to ethanol during adolescence. Higher acute stimulant effects of ethanol were observed during the first 5 min of the test session (Fig. 2), corresponding to the ascending limb of BECs. In previous studies, the acute locomotor stimulant effects of ethanol in male and female Swiss mice were similarly expressed 5–10 min after ethanol injection [53,54]. This increase in the locomotor stimulant effects of ethanol after exposure to ethanol during adolescence may have important implications. Indeed, it was hypothesized that the locomotor stimulant effects of drugs are directly related to their addictive properties. Animal and human studies confirm a relationship between alcohol consumption and/or abuse and stimulant properties of alcohol. In animal models, higher stimulant effects of ethanol were reported in rodents with a high preference for alcohol [55–57]. In humans, the stimulant effects of alcohol consumption are generally reported by consumers as a desirable effect. Furthermore, heavy alcohol drinkers are more sensitive to the stimulant effects of ethanol than light drinkers [58]. Therefore, the present results agree with the idea that alcohol consumption in early adolescence increases the risks of later abuse and dependence. Not only were adult female Swiss mice exposed to ethanol during adolescence more sensitive to the stimulant effects of ethanol, but they also showed higher rates of sensitization after repeated ethanol injections. This result is particularly striking, since it is generally more difficult to show significant increases from an already high basal level. In the present results, all adult mice, whatever the chronic treatment they have received during adolescence, developed a sensitization to the stimulant effects of ethanol. However, mice exposed to the highest dose of ethanol (4 g/kg) during adolescence had the strongest increase in the stimulant effects of ethanol when chronically re-exposed to ethanol as adults. On the test session, ethanol sensitization was primarily expressed by an enhanced duration of ethanol stimulant effects during the session. While ethanol stimulant effects occurred mainly during the first minutes of the acute session (Fig. 2A), these effects were detectable over the whole 30 min test session after repeated ethanol injections (Fig. 3A). Sensitization plays a prominent role in many contemporary theories of addiction and is seen as a marker of addiction [40,41]. The fact that adult female mice exposed to ethanol during adolescence show higher rates of ethanol sensitization therefore agrees with the thesis that exposure to chronic ethanol during adolescence increases the risks of alcohol dependence. Our results also revealed that adult female mice exposed to ethanol during adolescence developed a higher tolerance to the sedative effects of a high ethanol dose (4 g/kg). This was observed both using the loss of righting reflex procedure (Fig. 6) and a measure of locomotor activity (Fig. 5). Adult mice chronically exposed to a high dose of ethanol during adolescence had a shorter duration of ethanol-induced loss of righting reflex and a reduced hypolocomotor effect after the injection of 4 g/kg ethanol. Similar results were obtained in rats in previous studies [37,38]. As the sedative effects of ethanol are generally considered as a limiting factor in ethanol consumption [46], a higher tolerance to its sedative effects after ethanol exposure during adolescence might contribute to higher risks of alcohol abuse. In humans, it was shown that heavy drinkers and subjects at high-risks of alcoholism anticipate and feel less sedation when drinking alcohol [58,59]. As a consequence, a decreased sensitivity to the sedative effects of ethanol due to early alcohol exposure during adolescence could lead to a higher propensity of alcohol-related disorders. Our results also indicate that pharmacokinetic factors do not contribute significantly to this higher tolerance to the sedative effects of ethanol. Indeed,

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mice chronically treated with 4 g/kg ethanol during adolescence regained their righting reflex at higher BECs than control mice. More broadly, we had shown in a previous study that mice chronically exposed to daily ethanol injections (2.5 and 4 g/kg) do not develop a significant metabolic tolerance [48], such that it is very unlikely that the behavioral differences reported in the present experiments might be due to pharmacokinetic differences. In contrast to the stimulant and sedative effects, ethanol exposure during adolescence did not alter the anxiolytic effects of ethanol in adulthood. This is interesting for two reasons. First, these latter results show that adult female mice exposed to ethanol during adolescence are not characterized by non-specific changes in the sensitivity to all behavioral effects of ethanol. Instead, the differences are related to very specific effects, such as stimulant and sedative effects of ethanol. Additionally, the absence of differences in the anxiolytic effects of ethanol also confirms that a non-specific metabolic tolerance to ethanol does not explain the behavioral differences reported in the present study. An important result of the present study that should be highlighted is the need to administer high ethanol doses during adolescence in order to observe effects in adulthood. Indeed, most of the significant differences were obtained with the chronic administration of 4 g/kg ethanol during adolescence, whereas the effects of a chronic treatment with 2.5 g/kg ethanol most often failed to reach statistical significance. In a previous study, we showed that young adolescent and early postweanling mice required higher ethanol doses than adults to develop sensitization. However, with such high ethanol doses, younger mice developed ethanol sensitization of higher magnitude [48]. Ethanol doses that were previously shown to induce a very strong sensitization in young mice are equivalent to those leading to altered responses to ethanol in adulthood in the present study (4 g/kg). It is noteworthy that such a pattern of ethanol administration with high doses in a single binge is closed to the typical pattern of ethanol consumption in human adolescents. Indeed, it is frequent to find adolescents occasionally drinking alcohol amounts up to doses inducing a severe intoxication and a deep sedation [60]. In the light of the present results and those published by Quoilin et al. [48], we can also conclude that such a pattern of ethanol intoxication not only strengthens the development of ethanol sensitization during adolescence, but also leads to enduring effects in adulthood. In other words, the pattern of alcohol consumption of human adolescents makes them especially vulnerable to subsequent alcohol-related disorders. This conclusion is also supported by human epidemiological studies showing that age at first alcohol use is a powerful predictor of later alcohol abuse and dependence [14,15]. The present results were obtained on female mice in agreement with the ethanol sensitization studies in which female mice are often tested [53,61,62]. Although it is difficult to know whether similar results would be obtained with males, there is no reason to believe that the situation would be completely different in male mice. In a previous study, we showed that changes in the stimulant and sedative effects of ethanol during adolescence were similar in male and female Swiss mice [49], also suggesting that similar repercussions would be observed in adult male mice. There is no evidence that the estrous cycle of female mice has an impact on the stimulant effects of ethanol or on ethanol sensitization. Even if the estrous cycle had an impact on the behavioral measures of the present experiments, this would lead to an increase in intra-group variability, thereby reducing the statistical power of the study and not to a confounding variable with adolescent treatment. It is noteworthy that we did not find a higher variability in the stimulant effects of ethanol in female mice relative to male mice, suggesting that the estrous cycle is not a concern for this behavioral outcome. Although the present study was not aimed at unraveling the neurobiological mechanisms underlying the behavioral

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consequences of ethanol administration during adolescence, it is likely that the reported behavioral changes after ethanol exposure during adolescence are due to alterations in one of the major neurotransmitter systems involved in ethanol’s effects. During adolescence, the mesocorticolimbic dopamine system undergoes major changes [63–66]. For example, changes in extracellular dopamine levels, in dopamine receptor density, and in the number of dopamine fibers were reported during adolescence [see Ref. [67] for a review]. Changes have also been shown in GABAA and NMDA receptors [68–70]. Since these neurotransmitter systems are suggested to play a key role in mediating the stimulant and sedative effects of ethanol [71–74], alterations in the activity of these neurotransmitters by chronic ethanol administration are likely to interfere with the developmental neuroadaptations of adolescence. In agreement with this hypothesis, it was shown that chronic alcohol exposure during adolescence leads to higher levels of extracellular dopamine in adult rodents [26,75]. Other studies reported reduced phosphorylation of the NR2B subunit of the NMDA receptor in several brain areas after alcohol exposure during adolescence [30]. Finally, GABAA receptors alterations were reported following repeated ethanol administration in adult rodents [76–78], although similar effects were not tested in adolescent rodents. Together, alterations in dopamine, glutamate and GABA neurotransmitter systems might explain the present behavioral results, although further studies are clearly needed to confirm this hypothesis. In summary, when tested in adulthood, female Swiss mice chronically exposed to ethanol during adolescence show behavioral responses to ethanol that are typically associated with alcohol abuse and alcoholism in humans: higher stimulant effects, a stronger development of ethanol sensitization and reduced sedative effects. These results support the thesis that chronic alcohol consumption during adolescence, especially in high amounts leading to a strong intoxication, increases the risk of later alcoholrelated disorders.

Conflict of interest The authors declare no conflicts of interest.

Acknowledgments This work was supported by grants from the Fonds National de la Recherche Scientifique (FNRS) and the Fonds Spéciaux pour la Recherche de l’Université de Liège obtained by Etienne Quertemont. Caroline Quoilin is a research assistant under contract with the FNRS. The experiments reported in this article comply with the current laws of Belgium.

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