Psychopharmacology

, Volume 219, Issue 4, pp 1165–1177

Developmental differences in ethanol-induced sensitization using postweanling, adolescent, and adult Swiss mice

Authors

  • Caroline Quoilin
    • Centre de Neurosciences Cognitives et ComportementalesUniversité de Liège
  • Vincent Didone
    • Centre de Neurosciences Cognitives et ComportementalesUniversité de Liège
  • Ezio Tirelli
    • Centre de Neurosciences Cognitives et ComportementalesUniversité de Liège
    • Centre de Neurosciences Cognitives et ComportementalesUniversité de Liège
Original Investigation

DOI: 10.1007/s00213-011-2453-7

Cite this article as:
Quoilin, C., Didone, V., Tirelli, E. et al. Psychopharmacology (2012) 219: 1165. doi:10.1007/s00213-011-2453-7

Abstract

Rationale

The maturing adolescent brain has been suggested to be more sensitive than the adult brain to ethanol-induced neuroadaptations. In animal studies, sensitization to the stimulant effects of ethanol is used to study the vulnerability to chronic ethanol-induced neurobehavioral alterations.

Objectives

The aim of the present study was to systematically characterize age-dependent changes in the development and expression of the sensitization to the stimulant effects of a range of ethanol doses in female Swiss mice. Three ages were studied: 21-day-old mice (postweanlings), 35-day-old mice (adolescents), and 63-day-old mice (adults).

Methods

Postweanling, adolescent, and adult mice were daily injected with saline or various ethanol doses (1.5 to 4 g/kg) for 7 days. They were then tested for acute and sensitized locomotor activity.

Results

Postweanling and adolescent mice were more sensitive than adult mice to the acute stimulant effects of ethanol. In adult mice, daily injections of ethanol at doses between 2.5 and 4 g/kg led to significant sensitization. Higher ethanol doses (3.5 and 4 g/kg) were required to induce sensitization in postweanling and adolescent mice. However, younger mice showed ethanol sensitization of higher magnitude.

Conclusions

Young mice develop very strong ethanol sensitization at doses that mimic binge drinking in humans. These results might explain why early ethanol drinking during adolescence is related to a higher prevalence of subsequent alcohol disorders.

Keywords

EthanolAdolescenceLocomotor activityBehavioral sensitizationMice

Introduction

Adolescence is a critical and unique period of development. From a behavioral perspective, adolescents show an increase in the time spent in social interactions with peers (Csikszentmihalyi et al. 1977; Primus and Kellogg 1989) together with a rise in novelty seeking (Adriani et al. 1998; Arnett 1992), sometimes leading to risk-taking behaviors. At the same time, rapid brain maturational changes occur in various brain regions and especially in the prefrontal cortex and mesolimbic regions, which are part of the brain reward system associated with drug dependence (Chambers et al. 2003; Crews et al. 2007; Spear 2000).

These behavioral and neurobiological changes have been suggested to predispose adolescents to experiment with drugs and to be particularly vulnerable to the long-term neuroadaptations induced by drugs of abuse (Crews et al. 2007). Regarding alcohol, drinking behaviors generally begin during adolescence, and a number of teenagers adopt a pattern of alcohol consumption characterized by heavy binge drinking episodes—i.e., the consumption of high amounts of alcohol in a relatively short period of time. Moreover, clinical studies have shown that the age at first use of alcohol is a powerful predictor of later alcohol abuse and dependence (DeWit et al. 2000; Grant and Dawson 1997). A similar correlation between early alcohol exposure during adolescence and later increases in ethanol consumption was also described in animal research, especially in laboratory rodents (Acevedo et al. 2010; Bell et al. 2006b; Blizard et al. 2004).

Several explanations have been proposed to account for this correlation between an early initiation of alcohol consumption and enhanced risks of later abuse. One of them suggests that the adolescent brain shows a specific sensitivity to some of the acute effects of ethanol. More precisely, adolescents have been suggested to be less sensitive to the adverse and unpleasant effects of ethanol, which limit ethanol consumption in adults (Schuckit et al. 2000). In contrast, the positive and addictive effects of ethanol would be more potent in adolescents than in adults (Doremus-Fitzwater et al. 2010). As a consequence, adolescents might be encouraged to drink higher amounts of alcohol than adults. Empirical evidence in support of this explanation has been mainly provided by animal research. Regarding the adverse and unpleasant effects of alcohol, a lower sensitivity to the sedative (Little et al. 1996; Quoilin et al. 2010; Silveri and Spear 1998), ataxic (White et al. 2002), and hypothermic (Silveri and Spear 2000) effects of ethanol have been reported in adolescent relative to adult rats and mice. In contrast, adolescent rodents have been shown to be more sensitive than adults to the locomotor stimulant effects of ethanol (Quoilin et al. 2010; Stevenson et al. 2008) and to ethanol-induced social facilitation (Varlinskaya and Spear 2002).

It has also been suggested that the maturing adolescent brain is more sensitive than the adult brain to drug- and ethanol-induced neuroadaptations. This might lead to a higher vulnerability of adolescents to the long-term consequences of chronic alcohol consumption, which in turn would increase the likelihood of developing alcohol use disorders (Spear 2000). Locomotor sensitization, defined as a gradual increase in the locomotor stimulant effects following repeated administrations of a drug, has long been used as an animal model to study the neurobiological adaptations induced by repeated drug exposure in mice. Drug sensitization is a major component of addiction and is central to some contemporary theories of drug addiction (Robinson and Berridge 1993, 2001, 2003). Therefore, age-dependent changes in the susceptibility to drug sensitization have been investigated in an attempt to explain the higher vulnerability of the adolescent period to drug abuse.

In adult laboratory rodents, sensitization to the locomotor stimulant effects has been reported for various drugs, including ethanol (Broadbent et al. 2003, 2005; Camarini et al. 2011; Didone et al. 2008; Lessov and Phillips 1998). However, age-dependent changes in the susceptibility to develop sensitization to the locomotor stimulant effect of drugs were mainly studied using psychostimulants, such as cocaine and amphetamines. Against expectations, most of these studies showed a lack of or lower sensitization to the locomotor effects of psychostimulants in adolescent rats (Collins and Izenwasser 2002; Frantz et al. 2007; Kolta et al. 1990) and mice (Niculescu et al. 2005), although conflicting results, i.e., a higher sensitization in younger rodents, were also reported in a few studies (e.g., Camarini et al. 2008). In contrast to psychostimulants, age-dependent changes in ethanol-induced locomotor sensitization have been much less studied. Previous studies showed that adolescent Swiss mice, in contrast to adults, did not develop locomotor sensitization to ethanol or even show a behavioral tolerance to the locomotor stimulant effects of ethanol after repeated ethanol administrations (Carrara-Nascimento et al. 2011; Faria et al. 2008). In another study on DBA/2J mice, Stevenson et al. (2008) reported that adolescent mice were less sensitive than adult mice to ethanol-induced sensitization. However, these studies were limited to the comparison of two age groups (adults and adolescents) and low to moderate doses of ethanol (between 1.0 and 2.5 g/kg). As human adolescents typically adopt binge drinking behaviors, it is important to extend those studies on ethanol-induced sensitization to higher ethanol doses. This is especially important because our previous studies have shown that higher doses of ethanol, up to 3.5 g/kg, maintain their stimulant effects in adolescent mice in contrast to adult mice in which these doses are mainly depressant (Quoilin et al. 2010).

The aim of the present experiments was to systematically characterize the development and expression of sensitization to the stimulant effects of a range of ethanol doses (from 1.5 to 4 g/kg) in female Swiss mice. Female mice were chosen because they are generally reported to be more susceptible to the development of sensitization to the locomotor stimulant effects of ethanol (Phillips et al. 1997). In order to better characterize age-dependent changes in ethanol sensitization, three ages were studied: postweanling 21-day-old mice, adolescent 35-day-old mice, and young adult 63-day-old mice. Indeed adolescence in mice, which covers the period ranging from weaning to the entrance in adulthood, may be subdivided into shorter periods, each being associated with specific behavioral and neurobiological changes (Tirelli et al. 2003). As shown in previous experiments carried out in our laboratory (Quoilin et al. 2010), behavioral responses to ethanol gradually change during adolescence, with the youngest 21-day-old mice and adults exhibiting the greatest differences. We have therefore decided to study these two groups of 21- and 63-day-old mice, together with an intermediate age (35-day-old mice). In the first experiment, we tested whether the 2.5-g/kg ethanol dose, typically used in most adult sensitization studies, induces different profiles for the development and expression of sensitization in our three age groups of female Swiss mice. As young mice show much more potent ethanol-induced locomotor stimulation than adult mice (Quoilin et al. 2010), the lack of sensitization in young mice might be attributed to a ceiling effect. In order to test this hypothesis, experiment 2 tested the development and expression of sensitization with a lower ethanol dose. In a third experiment, high ethanol doses, modeling adolescent binge drinking in humans, were tested. Finally, blood alcohol concentrations were also measured in postweanling, adolescent, and adult female Swiss mice to ascertain that the behavioral effects obtained in the present experiments could not be attributed to pharmacokinetic differences.

Materials and methods

Subjects

For the whole study, 552 female Swiss mice bred in our colony were used. These mice were the progeny of breeders purchased from Janvier Laboratories (Le Genet St Isle, France). Pregnant mice were housed at four per cage and births were checked every day. At the time of birth (postnatal day 0), pups and their mothers were collectively gathered in order to group three litters with pups born on the same day (see Branchi (2009) for a review on communal nesting). Pups were weaned at postnatal day 21 and housed at two per cage at the start of the experiments in white polyethylene cages with pine sawdust bedding. Female mice were chosen for the present experiments in order to be consistent with the majority of ethanol sensitization studies and because female rodents generally show greater susceptibility to drug-induced sensitization. The animal room was maintained on a 12-h light/dark cycle (lights on at 8:00 a.m.) 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 a.m and 1:00 p.m. 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.

Drugs

Alcohol 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 (1.5, 2, 2.5, 3.5, and 4 g/kg) in order to avoid concentration-induced differences in absorption. Therefore, volumes of injections for each ethanol dose were respectively 0.01, 0.013, 0.016, 0.022, and 0.025 ml/g. 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.

Apparatus

The locomotor effects of ethanol were assessed with a videocamera system. The experimental environment consisted of transparent Plexiglas chambers (42 × 42 cm) partitioned into six compartments (7 × 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 × 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 units per second). Lighting was provided by four white neon-tubes (18 W) fixed on each leg of the frame, such that the light was not directed at the mice. These apparatus were used in two exemplars and each of them was individually located in a small, ventilated, and heated (21–23°C) white room (140 × 140 cm surface × 245 cm height). Videotapes were later scored by one or two trained observers blind to the drug treatment. The inter- and 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 (King and Chinchilli 2001). For the test sessions, videotape scoring was applied on the whole session (30 min) and was divided into six 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.

Procedure

General procedure

In all of the experiments, ethanol-induced locomotor sensitization was induced and tested with the same experimental protocol in postweanling, adolescent, and adult female mice. This experimental protocol is adapted from Didone et al. (2008) with further adaptations to young mice on the basis of the results from Stevenson et al. (2008). On the first day of the experimental procedure, when the mice were exactly 21, 35, and 63 days old, all of the mice were submitted to a habituation session. They were moved to the experimental room and left undisturbed for 30 min. The mice from all experimental groups were then i.p. injected with saline and immediately placed into the experimental apparatus where their locomotor activity was recorded for 30 min. The sensitization procedure started the next day with the same procedure except that the mice were injected for the first time with their assigned experimental treatment (0, 1.5, 2, 2.5, 3.5, or 4 g/kg ethanol) before placement in the experimental apparatus for 30 min. This first experimental session (acute session) was used to assess the acute effects of the various ethanol doses. The mice were then injected with their assigned treatment in their home cage for the next five consecutive days. Finally, the expression of sensitization was tested on day 8 (which corresponded to the seventh ethanol injection in the experimental groups). On this test day, mice from the experimental groups were injected with their assigned dose of ethanol and their locomotor activity was recorded for 30 min. The mice from the control groups were also injected for the first time with ethanol on the test day, such that the comparison between experimental and control mice allowed to test for the between-subject ethanol sensitization.

Experiment 1

The aim of the first experiment was to test for potential age differences in the development of behavioral sensitization after repeated administrations of 2.5 g/kg ethanol, a standard ethanol dose inducing significant locomotor sensitization in adult Swiss mice (Didone et al. 2008). Postweanling, adolescent, and adult mice were repeatedly administered with saline or 2.5 g/kg ethanol (n = 12/group), using the general procedure described above. On the sensitization test session, all mice were challenged with 2.5 g/kg ethanol in order to test for within-subject and between-subject ethanol sensitization.

Experiment 2

Experiment 2 was designed to challenge the ceiling effect explanation for the lack of ethanol sensitization in young mice. Postweanling, adolescent, and adult mice were randomly divided into three groups that were assigned to one of the following chronic treatment: saline (n = 24 / age), 1.5 g/kg ethanol (n = 12 / age), or 2.5 g/kg ethanol (n = 12 / age). All of the mice were then submitted to the general procedure described above and repeatedly injected with their assigned treatment. On the test day (day 8), the mice of the two ethanol groups were injected with their assigned dose of ethanol, while the saline control group was subdivided into two subgroups. The first subgroup was injected with 1.5 g/kg ethanol and served as a control group for the mice sensitized with 1.5 g/kg ethanol, while the second subgroup was injected with 2.5 g/kg ethanol and served as a control group for the mice sensitized with 2.5 g/kg ethanol. Locomotor activity was then recorded during 30 min.

To ensure that a ceiling effect did not mask the expression of sensitization in the mice repeatedly injected with 2.5 g/kg ethanol, an additional test session was performed on the next day (day 9) for the mice of this group. On day 9, these mice were injected with a lower 1.5 g/kg ethanol dose. This test session should unmask a possible ethanol sensitization in mice repeatedly injected with 2.5 g/kg ethanol. However, in order to have a proper control group for this part of the experiment, an additional group of mice (n = 12) was added to the experiment. The mice of this additional control group were daily injected with saline on days 1 to 8 and were tested with an acute 1.5 g/kg ethanol dose on the day 9 test session.

Experiment 3

Experiment 3 tested the development of sensitization to ethanol with higher ethanol doses (3.5 and 4 g/kg). Furthermore, a group repeatedly administered with an intermediate dose of ethanol (2 g/kg) was also added in experiment 3. Postweanling, adolescent, and adult female Swiss mice were randomly divided into six groups. The first three groups were assigned to one of the ethanol doses (2, 3.5, or 4 g/kg), while the other three groups served as their respective control and were repeatedly administered with saline. All of the mice were then submitted to the general procedure described above and repeatedly injected with their assigned treatment. On the test day (day 8), the mice of the three ethanol groups were injected with their assigned dose of ethanol, while the three control groups were injected with one of the corresponding ethanol doses (2, 3.5 or 4 g/kg). Locomotor activity was then recorded during 30 min.

As high ethanol doses such as 3.5 and 4 g/kg mainly induce depressant effects especially in adult mice, changes in locomotor activity between the acute session and the test session might be due to a tolerance to the depressant effects rather than a sensitization to the stimulant effects of ethanol. In order to test the sensitization to the stimulant effects of ethanol, the mice of the three ethanol groups were submitted to an additional test session on day 9. On day 9, all of these mice were injected with 2.5 g/kg and their locomotor activity was recorded during 30 min. However, in order to have a proper control group for between-subject ethanol sensitization, an additional group of mice (n = 12) was added to the experiment. The mice of this additional control group were daily injected with saline on days 1 to 8 and were tested with an acute 2.5 g/kg ethanol dose on the day 9 test session.

Experiment 4

In order to control for possible pharmacokinetic differences between mice of various ages, blood ethanol concentrations (BECs) were measured after chronic injections of 2.5 or 4 g/kg ethanol in postweanling, adolescent, and adult female mice (n = 8/group). Following the same schedule as described in the general procedure above, 21-, 35- and 63-day-old mice were repeatedly injected with 2.5 or 4 g/kg for 6 days. On the test day, mice were again injected with their assigned ethanol dose and decapitated 30 min after the injection. Blood samples were collected in heparinised capillaries, immediately centrifuged for 5 min, and stored at 4°C for later analysis on the same day. Five microliters of plasma from each sample was analyzed to determine BEC using the Analox AM1 alcohol analyzer (Analox Instruments, London, UK).

Statistical analysis

The statistical analyses were performed on the mean locomotor activity during the first 5 min of the session on the basis of previous studies in adult Swiss mice showing that the expression of sensitization was mainly observed during this time interval (Didone et al. 2008). Overall, data were analyzed with analyses of variance (ANOVA). The acute stimulant effects of ethanol in the different experiments were treated using two-way ANOVAs with ethanol dose (two, three, or four levels) and age (three levels) as between-subject factors. Ethanol-induced locomotor sensitization was tested in two different ways, either as a within-subject sensitization or as a between-subject sensitization.

Within-subject sensitization is defined as an increase in locomotor activity on the last ethanol session relative to the first ethanol session in the same subjects. In order to test for within-subject sensitization, the results for each subject were expressed as a percentage increase in locomotor activity between the first and the last ethanol sessions (i.e., locomotor activity counts on the last ethanol session divided by locomotor activity counts on the first ethanol session and multiplied by 100). One- or two-way ANOVAs were then computed on these percentage increases in locomotor activity using age as a between-subject factor (three levels) and ethanol dose as an additional between-subject factor in experiments 2 and 3. For the sake of parsimony, the results for the within-subject sensitization were collapsed for all three experiments in Fig. 7.

Between-subject sensitization is defined as a higher locomotor activity in the ethanol-treated group on the test session by comparison to the control group administered for the first time with the same ethanol dose on that test session. Because the aim of the present study was to test in each age group at which doses a between-subject sensitization develops, separate Student t-tests or two-way ANOVAs were computed for each age group separately and according to the specific experimental design of each experiment. In experiment 1, the results of the test session were analyzed with three t-tests, incorporating the sensitization treatment (saline or 2.5 g/kg of ethanol) as a between-subject factor. In experiments 2 and 3, two-way ANOVAs were performed, with the sensitization treatment (saline or ethanol) and dose (1.5 or 2.5 g/kg; 2, 3.5, or 4 g/kg) as between-subject factors. The blood ethanol concentrations were also analyzed with a two-way ANOVA incorporating age (three levels) and ethanol treatment (2.5 or 4 g/kg) as between-subject factors.

The ANOVAs were followed by Newman–Keuls post-hoc comparisons to assess between-group differences except in the second part of experiment 3 in which a one-tailed Dunnett test was used in order to identify which ethanol group had significantly higher levels of activity relative to the control group. When necessary, logarithmic transformations normalized raw data prior to the ANOVAs in order to meet the assumption of homogeneity of variances and normality. However, for the sake of clarity, means of the raw values are presented in graphs. Statistical significance was set at p < 0.05.

Results

Experiment 1

Acute effects of ethanol

The two-way ANOVA computed on the locomotor activity counts during the first 5 min of the acute session showed significant main effects of the treatment [F(1,66) = 11.21; p < 0.01] and age [F(2,66) = 4.37; p < 0.05], along with a significant interaction between these two factors [F(2,66) = 14.44; p < 0.00001]. As shown in Fig. 1, in which the acute stimulant effects of ethanol were collapsed for experiments 1, 2, and 3, these results confirmed higher locomotor stimulant effects at the dose of 2.5 g/kg in postweanling mice relative to both adolescent and adult mice (p < 0.01 for both comparisons).
https://static-content.springer.com/image/art%3A10.1007%2Fs00213-011-2453-7/MediaObjects/213_2011_2453_Fig1_HTML.gif
Fig. 1

Effects of the first ethanol injection (acute session) at various doses (0, 1.5, 2, 2.5, 3.5, or 4 g/kg) on locomotor activity in postweanling (21-day old), adolescent (35-day old), and adult (63-day old) mice. Data were collapsed across experiments 1 to 3 in order to show the dose–response curve for ethanol-induced locomotor activity. Data are expressed as mean ± SEM of locomotor activity scores during the first 5 min of the session

Ethanol-induced locomotor sensitization

As shown in Fig. 2, the three t-tests computed on the locomotor activity scores during the first 5 min of the sensitization session revealed between-subject sensitization in adolescent [t(22) = 2.86; p < 0.01] and adult [t(22) = 2.61; p < 0.05] mice, but not in postweanling mice [t(22) = 0.43; p = 0.67].
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Fig. 2

Between-subject sensitization after repeated injections of 2.5 g/kg ethanol in postweanling, adolescent, and adult mice. The results show the mean locomotor activity during the first 5 min of the test session after the injection of 2.5 g/kg ethanol in mice chronically treated with 2.5 g/kg ethanol (gray bars) or saline (white bars). Data are expressed as mean ± SEM of locomotor activity scores (n = 12/group). *p < 0.05 and **p < 0.01, significantly different from the respective control group that was repeatedly injected with saline during the sensitization phase

The within-subject sensitization confirmed that postweanling mice in contrast to adult mice did not develop sensitization to 2.5 g/kg ethanol. The one-way ANOVA performed on the percentage increase in locomotor activity between the first and last ethanol sessions revealed a significant effect of age [F(2,33) = 4.67; p < 0.05]. The increase in locomotor activity was significantly higher in adult mice relative to the other two groups (p < 0.05 for both comparisons; see the 2.5 g/kg dose in Fig. 7).

Experiment 2

Acute effects of ethanol

The two-way ANOVA computed on the locomotor activity counts during the first 5 min of the acute session indicated significant main effects of ethanol dose [F(2,168) = 51.70; p < 0.000001] and age [F(2,168) = 28.82; p < 0.000001] and a significant dose × age interaction [F(4,168) = 15.83; p < 0.000001]. The Newman–Keuls post-hoc comparisons performed on the interaction showed increases in locomotor activity in both postweanling and adolescent mice after the administration of 1.5 and 2.5 g/kg ethanol (p < 0.01 for all comparisons), whereas adult mice failed to show significant acute locomotor stimulant effects after the administration of 1.5 (p = 0.31) or 2.5 (p = 0.71) g/kg ethanol.

Ethanol-induced locomotor sensitization

Two-way ANOVAs were performed on the locomotor activity counts during the first 5 min of the sensitization day for each age separately to test for between-subject sensitization (Fig. 3a to c). There was no significant main effect of the chronic treatment (saline vs ethanol) in any of the age groups. However, there was a significant main effect of the ethanol dose (1.5 vs 2.5 g/kg) in postweanling [F(1,41) = 13.04; p < 0.001] and adolescent [F(1,44) = 6.20; p < 0.05] mice. This indicates that 2.5 g/kg ethanol was globally more stimulating than 1.5 g/kg ethanol in postweanling and adolescent mice (p < 0.001 and p < 0.05, respectively). Additionally, the treatment × dose interaction was significant in adult mice [F(1,44) = 6.82; p < 0.05], but not in postweanling [F(1,44) = 0.76; p = 0.39] and adolescent [F(1,44) = 3.15; p = 0.08] mice. Further between-mean comparison analyses demonstrated between-subject sensitization only for the 2.5-g/kg ethanol dose in adult mice in which the locomotor activity was significantly higher in the sensitized mice than in the control mice (p < 0.05). In contrast, both 1.5- and 2.5-g/kg ethanol doses failed to induce between-subject sensitization in postweanling and adolescent mice.
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Fig. 3

Between-subject sensitization after repeated injections of either 1.5 or 2.5 g/kg ethanol in postweanling, adolescent, and adult mice. ac For each age group, the locomotor activity during the first 5 min of the test session in mice injected with 1.5 or 2.5 g/kg of ethanol at the end of a chronic treatment with the same ethanol dose (gray bars) or saline (white bars). Data are expressed as mean ± SEM of locomotor activity scores (n = 12/group). *p < 0.05, significantly different from the respective control group that was repeatedly injected with saline during the sensitization phase

To test for within-subject sensitization, a two-way ANOVA was computed on the percentage increase in locomotor activity between the first and last ethanol sessions. The results showed a significant interaction between age and ethanol dose [F(2,66) = 3.28; p < 0.05]. Only adult mice repeatedly treated with 2.5 g/kg ethanol developed within-subject sensitization to the stimulant effects of ethanol (see the 1.5- and 2.5-g/kg doses in Fig. 7).

Additional test session (day 9)

On the second test day, postweanling, adolescent, and adult mice repeatedly injected with 2.5 g/kg ethanol were challenged with a lower 1.5 g/kg ethanol dose and compared to a control group injected with 1.5 g/kg ethanol for the first time. Three t-tests were computed on the locomotor activity counts during the first 5 min of the session for each age separately. These t-tests revealed no significant differences between sensitized and control mice for any of the age groups. These results show that postweanling mice do not express ethanol sensitization even when challenged with a lower ethanol dose.

Experiment 3

Acute effects of ethanol

The two-way ANOVA computed on the locomotor activity counts during the first 5 min of the acute session showed a significant main effect of ethanol dose [F(3,132) = 122.88; p < 0.000001] and a significant dose × age interaction [F(6,132) = 3.28; p < 0.01], whereas the main effect of age was not significant [F(2,132) = 2.10; p = 0.13]. The Newman–Keuls post-hoc comparisons confirmed different patterns of response to ethanol according to the age of the mice. The moderate ethanol dose (2 g/kg) was stimulating at all three ages (p < 0.0001; see Fig. 1). In adult mice, both 3.5 and 4 g/kg ethanol induced significant depressant effects (p < 0.001). In adolescent mice, only 4 g/kg ethanol induced statistically significant depressant effects (p < 0.05). Finally, in postweanling mice, although there was an apparent decrease in locomotor activity with both 3.5 and 4 g/kg ethanol, this reduction was not statistically significant (p = 0.78 for 3.5 g/kg; p = 0.18 for 4 g/kg).

Ethanol-induced locomotor sensitization

Three two-way ANOVAs were computed on the locomotor activity counts during the first 5 min of the sensitization session for each age to test for between-subject sensitization (Fig. 4a–c).
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Fig. 4

Between-subject sensitization after repeated injections of various ethanol doses (2, 3.5, or 4 g/kg) in postweanling, adolescent, and adult mice. ac For each age group, the locomotor activity during the first 5 min of the test session in mice injected with ethanol at the end of a chronic treatment with the same ethanol dose (gray bars) or saline (white bars). Data are expressed as mean ± SEM of locomotor activity scores (n = 12/group). *p < 0.05 and *p < 0.01, significantly different from the respective control group that was repeatedly injected with saline during the sensitization phase

In postweanling mice, the results revealed significant main effects for ethanol dose [F(2,66) = 36.43; p < 0.000001] and sensitization treatment [F(1,66) = 58.48; p < 0.05], along with a significant interaction between these factors [F(2,66) = 3.36; p < 0.05]. Further analyses confirmed significant between-subject sensitization in postweanling mice for the 3.5- and 4-g/kg (p < 0.05 and p < 0.01 respectively), but not for 2-g/kg (p = 0.53) ethanol dose.

In adolescent mice, there was a significant main effect for chronic treatment (saline vs ethanol) [F(1,66) = 5.04; p < 0.05] and a significant interaction of treatment × dose [F(2,66) = 14.43; p = 0.05]. As in postweanling mice, further analyses confirmed significant between-subject sensitization in adolescent mice for the 3.5- and 4-g/kg (p < 0.05), but not for 2-g/kg (p = 0.48) ethanol dose.

In adult mice, the repeated administration of the test doses failed to significantly alter locomotor activity relative to mice repeatedly treated with saline. There were no significant effects of treatment [F(1,66) = 3.60; p = 0.06] and its interaction with dose [F(2,66) = 0.80; p = 0.45].

To test for within-subject sensitization, a two-way ANOVA was performed on the percentage increase in locomotor activity between the first and last ethanol sessions. The results showed significant main effects for ethanol dose [F(2,98) = 24.82; p < 0.000001] and age [F(2,98) = 4.43; p < 0.05], along with a significant interaction between these factors [F(4,98) = 2.72; p < 0.05]. The Newman–Keuls post-hoc comparisons confirmed the absence of within-subject sensitization after the repeated administration of 2 g/kg ethanol in postweanling, adolescent, and adult mice (see the 2-g/kg dose in Fig. 7). However, with higher ethanol doses, especially 4 g/kg, postweanling mice developed higher within-subject sensitization than adult mice (p < 0.01; see the 4-g/kg dose in Fig. 7).

Additional test session (day 9)

On the second test day, postweanling, adolescent, and adult mice repeatedly injected with various ethanol doses (2, 3.5, and 4 g/kg) were challenged with a stimulant dose of ethanol (2.5 g/kg) in order to check whether sensitization to the locomotor stimulant effects of ethanol really developed. They were compared to postweanling, adolescent, and adult control mice injected with 2.5 g/kg ethanol for the first time. The three one-way ANOVAs revealed significant main effects for dose in postweanling [F(3,44) = 3.23; p < 0.05], adolescent [F(3,44) = 5.60; p < 0.01], and adult mice [F(3,44) = 4.46; p < 0.01]. As shown in Fig. 5 and confirmed by Dunnett t-tests, between-subject sensitization did not develop at the same doses for mice of different ages. In postweanling mice, significant between-subject ethanol sensitization was observed at 3.5- (p < 0.05) and 4-g/kg (p < 0.05) but not at 2-g/kg dose. In adolescent mice, there was a statistically significant between-subject ethanol sensitization only at 4-g/kg dose. For the 3.5-g/kg ethanol dose in adolescent mice, an increase in locomotor activity was apparent in Fig. 5, but it failed to reach statistical significance. Finally, in adult mice, all three ethanol doses induced significant between-subject ethanol sensitization (p < 0.01 for 2 g/kg; p < 0.05 for 3.5 g/kg; p < 0.01 for 4 g/kg).
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Fig. 5

Between-subject sensitization after repeated injections of various ethanol doses (2, 3.5, or 4 g/kg) in postweanling, adolescent, and adult mice. ac For each age group, the locomotor activity during the first 5 min of the test session in mice injected with 2.5 g/kg ethanol at the end of a chronic treatment with 2, 3.5, or 4 g/kg ethanol (gray bars) or saline (white bars). Data are expressed as mean ± SEM of locomotor activity scores (n = 12/group). The scale y-axis is different for the three age groups in order to correct for their differential acute response to ethanol. *p < 0.05, **p < 0.01, and ***p < 0.001—significantly different from the respective control group repeatedly injected with saline during the sensitization phase

Experiment 4

The two-way ANOVA computed on the BECs revealed a significant main effect of dose [F(1,36) = 1022.19; p < 0.000001], whereas the main effects of age [F(2,36) = 1.77; p = 0.18] and the dose × age interaction [F(2,36) = 1.59; p = 0.22] were not statistically significant. As shown in Fig. 6a, the effect of the dose was due to significantly higher BECs after the administration of a 4-g/kg dose relative to the lower 2.5-g/kg ethanol dose. In the right panel of Fig. 6, the corresponding locomotor activity of these groups at 30 min after injection is depicted for comparison. In contrast to BECs, analyses of locomotor activity revealed, in addition to a significant main effect dose [F(1,102) = 94.24; p < 0.000001], a significant main effect of age [F(2,102) = 3.88; p < 0.05]. Overall, these results show that the age-dependent differences in locomotor activity are not due to corresponding pharmacokinetic differences.
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Fig. 6

Blood ethanol concentrations and locomotor activity after the administration of 2.5 or 4 g/kg ethanol in postweanling, adolescent, and adult ethanol-sensitized mice. a BECs 30 min after an i.p. injection of 2.5 or 4 g/kg ethanol. Data are expressed as mean ± SEM of BECs. ***p < 0.001, significantly different from the 4-g/kg group. b Locomotor activity during 30 min of the sensitization test session in the same groups of mice. Data are expressed as mean ± SEM of locomotor activity

Discussion

Adolescence is a unique period of development, characterized by specific neurobiological and behavioral changes, including an increase in drug and alcohol use. This stage of development is also believed to be highly vulnerable to drug- and alcohol-induced neuroadaptations. In the present study, we have tested ethanol-induced locomotor sensitization in mice to assess vulnerability to ethanol-induced neuroadaptations at different ages. The results of the present study can be summarized as follows. Although young mice are much more sensitive to the acute stimulant effects of ethanol, they need higher ethanol doses than adult mice to develop locomotor sensitization to ethanol. However, with effective ethanol doses (at least 3.5 g/kg), younger mice develop very significant sensitization to the stimulant effects of ethanol, reaching higher levels of locomotor activity than that observed in adult mice (see Figs. 4 and 7).
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Fig. 7

Within-subject sensitization after repeated injections of various ethanol doses (0 to 4 g/kg) in postweanling, adolescent, and adult mice. Data were collapsed across experiments 1 to 3 and are expressed as mean ± SEM of percentage increase in locomotor activity between the first and last ethanol sessions. **p < 0.01 and ***p < 0.001, significantly different from 100%, the percentage denoting the absence of changes between the first and last ethanol sessions

First, the results of the present study confirm that postweanling and adolescent mice are more sensitive to the acute stimulant effects of ethanol than adult mice. As shown in Fig. 1, ethanol-induced locomotor stimulation was significantly higher in postweanling than in adult mice for all doses between 1.5 and 2.5 g/kg. Similar results have been found in other laboratories, in different strains of both male and female mice (Hefner and Holmes 2007; Quoilin et al. 2010; Stevenson et al. 2008). Therefore, this higher sensitivity of younger mice to the stimulant effects of ethanol seems to be a robust effect. In a previous study, we have shown that the higher sensitivity of younger mice to the stimulant effects of ethanol was characterized by an upward shift of the dose–response curve, i.e., the stimulant response to ethanol is higher in younger than adult mice (Quoilin et al. 2010). There was also an apparent shift to the right of this dose–response curve, but this effect was probably due to the higher sensitivity to ethanol-induced sedation in adult mice that truncates the right part of the curve for ethanol-induced locomotion in adult. The present results totally agree with these previously found differences between younger and adult mice in the dose–response curve for ethanol-induced locomotor activity. In humans, ethanol is also believed to be especially stimulating in adolescents, although there is little direct experimental evidence supporting this assertion. These higher stimulant effects of ethanol in adolescents may be of importance because of their potential involvement in risks for developing alcohol abuse and dependence. As stimulation is generally a desired effect of drugs, this higher sensitivity to the stimulant effects of ethanol could directly encourage alcohol drinking and therefore contribute to the propensity for alcohol abuse during adolescence. Results from both animal and human studies support this relationship between the stimulant effects of ethanol and the preference for alcohol drinking. For example, several animal studies have reported a positive correlation between ethanol-induced locomotor activity and ethanol preference (Agabio et al. 2001; Colombo et al. 1998; Rodd et al. 2004). In humans, it has been shown that heavy drinkers are more sensitive to the stimulant effects of ethanol relative to light drinkers (King et al. 2002). Finally, it has been hypothesized that the locomotor stimulant effects are directly related to the addictive properties of drugs due to a high homology between the brain regions underlying the stimulant and rewarding effects of drugs (Wise and Bozarth 1987).

Previous studies reported that adolescent mice are less sensitive to ethanol sensitization (Faria et al. 2008; Stevenson et al. 2008), which was in contrast with the general belief that adolescents are more vulnerable to ethanol-induced neuroadaptations. However, the reported lack of sensitization might be dependent on the specific methodological parameters of these studies, especially ethanol doses. In the two studies cited above, doses in the range between 1 and 2.5 g/kg were used and failed to induce locomotor sensitization in 28-day-old mice (Faria et al. 2008; Stevenson et al. 2008). In the present study, we have also found a lack of locomotor sensitization with repeated administrations of ethanol in the same range of doses (1.5 to 2.5 g/kg). In experiment 1, mice were daily injected with 2.5 g/kg ethanol, a standard ethanol dose for inducing locomotor sensitization in adults (Didone et al. 2008; Lessov et al. 2001). Although adult mice demonstrated both significant inter- and within-subject sensitization, postweanling mice did not show ethanol sensitization. With 2.5 g/kg ethanol, adolescent mice showed intermediate results with significant between-subject sensitization in experiment 1 but not in experiment 2. These results confirmed that adolescent female Swiss mice are less prone to sensitization than adult mice with the standard 2.5 g/kg ethanol dose. As postweanling mice also show a significantly higher response to the acute stimulant effects of ethanol, the lack of sensitization in young mice might be explained by a ceiling effect. As shown in Fig. 2, postweanling mice show higher stimulant effects on their first 2.5 g/kg ethanol injection than adult sensitized mice at their eighth ethanol injection. In order to test for the ceiling effect explanation, experiment 2 investigated for both the development of sensitization to a lower 1.5 g/kg ethanol dose and the expression of sensitization to a lower 1.5 g/kg ethanol dose in postweanling mice repeatedly injected with 2.5 g/kg ethanol. Together the results rule out the ceiling effect explanation. Postweanling mice did not develop ethanol sensitization with a lower ethanol dose and did not express locomotor sensitization when a reduced ethanol dose was injected on the test session.

Based on such results, previous studies concluded that adolescent mice are less sensitive to ethanol sensitization (Carrara-Nascimento et al. 2011; Stevenson et al. 2008) or globally develop behavioral tolerance to the stimulant effects of ethanol (Camarini et al. 2010; Faria et al. 2008). However, in order to have a full picture of ethanol-induced sensitization in adolescent mice, the present study also tested higher ethanol doses (3.5 and 4 g/kg). Indeed we had shown in adult mice that behavioral sensitization developed in all groups of mice repeatedly injected with ethanol doses above some threshold (around 1.5–2.5 g/kg), even when they were unable to express the stimulant effects of ethanol during the sensitization procedure due to the sedative effects of high ethanol doses (Didone et al. 2008). The results of the present study with high ethanol doses significantly alter the previous conclusion about ethanol-induced sensitization in adolescent mice. Although postweanling mice clearly require higher doses of ethanol to develop behavioral sensitization, the magnitude of the sensitized effects with such high ethanol doses is higher than in adults. These younger mice show both greater within-subject and between-subject sensitization at 3.5 and 4 g/kg (see Figs. 4 and 7). Furthermore, when challenged with a lower ethanol dose (2.5 g/kg), the mean locomotor activity of postweanling sensitized mice is more than twice that of adult sensitized mice (see Fig. 5). Relative to adult mice, postweanling mice therefore show two differences in ethanol-induced behavioral sensitization. First, there is a shift to the right of the dose–response curve for the threshold dose required to develop behavioral sensitization, with postweanling mice requiring higher ethanol doses. Secondly, when proper ethanol doses are used (3.5–4 g/kg), postweanling mice show sensitized locomotor behaviors of higher magnitude.

Two alternative explanations to these results should be discussed. First, with the higher ethanol doses, the increase in locomotor activity between the first and last ethanol injections might be due to a tolerance to its depressant effects rather than a sensitization to its stimulant effects. In order to test that hypothesis, the mice from experiment 3 were submitted to a loss of righting reflex (LORR) protocol 1 day after the last sensitization test. All mice were injected with 4 g/kg ethanol and the duration of their loss of righting reflex was recorded. The results (not shown) showed a significant age effect, with a lower duration of the LORR in postweanling mice relative to adult mice. However, there were no significant effects of the chronic treatment (saline vs ethanol) and no significant treatment × age interaction. These additional results indicate that the differences in ethanol-induced locomotor sensitization between postweanling and adult mice are unlikely to be confounded by differences in the development of a chronic tolerance to the sedative effects of ethanol. Pharmacokinetic differences between postweanling and adult mice might also explain the behavioral differences observed in the present study. In a previous study, we reported no differences in blood ethanol concentrations between postweanling, adolescent, and adult female Swiss mice after the administration of an acute dose of either 2.5 or 4 g/kg ethanol (Quoilin et al. 2010). In order to further explore this issue, we have measured in the present study the BECs of sensitized postweanling, adolescent, and adult mice after the repeated administration of either 2.5 or 4 g/kg ethanol. Our results revealed no differences in BECs between postweanling, adolescent, and adult mice, regardless of the tested dose, whereas there were statistically significant behavioral differences. These results, in agreement with previous studies (Carrara-Nascimento et al. 2011; Faria et al. 2008; Stevenson et al. 2008), confirm that pharmacokinetic factors do not account for the age-related differences in ethanol-induced locomotor sensitization.

As high ethanol doses (3.5 and 4 g/kg) were required to induce behavioral sensitization to ethanol in adolescent mice, one might wonder whether such doses are relevant to model human alcohol consumption. Human studies typically show that a significant number of adolescents adopt a pattern of drinking that is relevant to the high doses used in the present study. Indeed binge drinking, defined as consuming five or more drinks on the same occasion, is usual in adolescents, with 17.4% of people aged 12 to 20 showing this behavior pattern (Substance Abuse and Mental Health Services Administration 2010). Additionally, it is frequent to find adolescents occasionally drinking alcohol amounts up to doses inducing a deep sedation (Schöberl et al. 2008). It is also noteworthy that the psychoactive properties of acute ethanol in adolescents, with higher stimulant effects (Quoilin et al. 2010; Stevenson et al. 2008) and lower sedative effects (Cha et al. 2006; Hefner and Holmes 2007; Silveri and Spear 1998), make them more likely to drink higher amounts of alcohol. In agreement with this, previous studies also reported that adolescent rodents voluntarily consume more ethanol than adults (Bell et al. 2006a; Doremus et al. 2005; Moore et al. 2010; Truxell et al. 2007). In summary, some human adolescents often drink alcohol in frequency and amounts that should make them especially vulnerable to the phenomenon of sensitization described in the present study.

The higher inter- and within-subject sensitization reported in adolescent mice after the administration of high ethanol doses might be related to the observation of a negative correlation in humans between the age at first alcohol use and the likelihood of later dependence (DeWit et al. 2000; Grant and Dawson 1997). Indeed previous studies in adult mice have shown that ethanol-induced locomotor sensitization is a long-lasting phenomenon (Lessov and Phillips 1998). This means that the development of sensitization during adolescence could last into adulthood. Ethanol-induced sensitization is believed to be mediated by long-lasting brain modifications occurring during chronic exposure to ethanol. Sensitization is currently viewed as a major component of addiction and is central to some contemporary theories of drug addiction (Robinson and Berridge 1993, 2001, 2003; Vanderschuren and Pierce 2010). Accordingly, the neural regions underlying sensitization to the stimulant effects of abuse drugs—i.e., the mesolimbic dopaminergic system (see Vanderschuren and Kalivas 2000)—largely overlap those involved in the rewarding effects of these drugs (Wise and Bozarth 1987). Repeated ethanol exposures were also shown to durably increase the sensitivity of ventral tegmental area dopaminergic neurons to subsequent ethanol administration (Brodie et al. 1990; Brodie 2002; Ding et al. 2009; Rodd et al. 2005a,b). As changes in the mesocorticolimbic dopamine system have also been described during adolescence (Benes et al. 2000; Stanwood et al. 1997; Tarazi and Baldessarini 2000; Tarazi et al. 1998), the maturation of the brain dopaminergic system might also explain the differences between young and adult mice in ethanol-induced locomotor sensitization and in the acute sensitivity to the stimulant effects of ethanol. Additionally, adolescence is also characterized by a number of changes in the brain regions involved in drug reward. For example, the connections among reward-related subcortical regions, as well as the neurocircuitry connecting the prefrontal cortex and these regions, continue to develop during adolescence (see review in Doremus-Fitzwater et al. (2010)). There is also evidence that chronic ethanol administration during adolescence may alter adult brain functioning (Badanich et al. 2007; Sahr et al. 2004), which in turn could increase the reward value of ethanol and consequently the risks of alcohol-related problems in adulthood. However, further studies, especially with high ethanol doses, will be required to assess the repercussions in adulthood of an ethanol-induced sensitization that developed during adolescence. As current theories of addiction suggest that a high sensitivity to drug-induced locomotor sensitization might be a marker for an enhanced risk of later dependence (Robinson and Berridge 1993, 2001), the present results also support the idea that early alcohol abuse, before and during adolescence, might indeed increase the risks of later alcohol dependence.

Acknowledgements

This work was supported by grants from Fonds National de la Recherche Scientifique (FNRS) and 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.

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