Some characteristics of the adolescence (impulsivity, risky behavior, seeking of new experiences) have been related to delayed maturation of prefrontal cortex and neurotransmitter systems as well as late development of behavioral inhibitory systems, which may render adolescents especially vulnerable to taking drugs of abuse and developing addiction (Spear, 2000; Chambers et al., 2003). Ethanol exposure during adolescence can cause dramatic neurobehavioral and neurotoxicological effects compared to exposure during adulthood, as described in humans (Grant and Dawson, 1997; De Wit et al., 2000; Ehlers et al., 2006) and rodents (Crews et al., 2000; Faria et al., 2008; Walker and Ehlers, 2009; Guerri and Pascual, 2010; Soares-Simi et al., 2013; Carrara-Nascimento et al., 2014).

The main route of ethanol elimination is the liver metabolism, where it is converted into acetaldehyde by alcohol dehydrogenase and subsequently to acetate by aldehyde dehydrogenase (ALDH). Those enzymes are responsible for the elimination of alcohol in concentrations below 20 mmol/L (Li, 1977; Lieber, 1986). The efficacy of ethanol metabolism increases with age following its systemic administration, since the blood ethanol concentration remains higher for longer time in younger rats compared to older ones (Kelly et al., 1987). In fact, liver alcohol dehydrogenase efficiency and ALDH activity varies with age (Collins et al., 1975; Hollstedt et al., 1977). Recent data of our group suggested that adult but not adolescent mice developed metabolic tolerance to increases in blood ethanol concentration induced by chronic intermittent ethanol exposure (Carrara-Nascimento et al., 2013), suggesting that the age of exposure to ethanol may also influence ethanol metabolism.

The activity of ALDH may exert some influence on ethanol consumption, since accumulation of acetaldehyde in the peripheral system induces aversive effects when accumulated in the blood (Quertemont, 2004). As an example, high alcohol-drinkers show faster acetaldehyde metabolism and are less vulnerable to its aversive effects, such as flushing, headache, tachycardia, dizziness and nausea (Quintanilla et al., 2006).

Animal models that promote motivation for alcohol seeking/intake include alcohol withdrawal periods since periods of abstinence lead to progressive increases in alcohol consumption that ultimately results in the relief of the abstinence-induced withdrawal symptoms (the so called alcohol deprivation effect—ADE; Spanagel and Hölter, 1999). Drinking in the dark (DID) paradigm is considered a binge-like model since it promotes high levels of blood ethanol concentration (Rhodes et al., 2005). Another procedure to promote increase in ethanol consumption is to preexpose the animals to the drug (Lessov et al., 2001; Camarini and Hodge, 2004; Carrara-Nascimento et al., 2014).

In the present study, we designed a protocol that includes some aspects of human alcohol addiction, such as age of first contact with ethanol, ADE and DID. We hypothesized that mice exposed to ethanol during adolescence would have higher ethanol consumption later in life. We also assessed whether these differences in ethanol consumption pattern might be related to ALDH activity in the liver.

The current study proposes an experimental protocol that includes risk factors for addiction (adolescence period), incentive salience (behavioral sensitization) and induction of binge-like consumption (withdrawal and reexposures during dark period) to resemble some of the aspects of the addiction in humans. We showed that both adolescent and adult mice treated with 2.0 g/kg ethanol (Adolescent-EtOH and Adult-EtOH) displayed ethanol behavioral sensitization and that adolescents were less sensitive than adults, which is in agreement with studies from our laboratory and others (Stevenson et al., 2008; Quoilin et al., 2012; Soares-Simi et al., 2013; Carrara-Nascimento et al., 2014). The most striking result is that Adolescent-EtOH but not Adult-EtOH displayed escalated amounts of 4% ethanol intake during acquisition and maintained higher ethanol intake than Adult-EtOH after repeated withdrawals and reexposures, even when ethanol solution was adulterated with quinine or when sucrose was reduced to 0%. Age-related differences in 15% ethanol intake emerged only during the last reexposure (reexposure 6) after repeated withdrawals. In all phases, Adult-EtOH mice displayed lower 4% ethanol intake compared to the other groups.

Repeated cycles of withdrawals and reexposures have been used to increase ethanol consumption. In our previous study (Carrara-Nascimento et al., 2014), using behavioral sensitization paradigm as pretreatment exposure, we demonstrated that ethanol pretreated mice showed higher 10% ethanol intake when compared to SAL pretreated mice, regardless of age of preexposure or behavioral sensitization magnitude. Adolescent and adult mice exposed to chronic ethanol vapor chamber also increased ethanol intake similarly in a two-bottle choice test (Carrara-Nascimento et al., 2013).

In the current study, using three-bottle choice test, age-differences in ethanol-pretreated mice emerged when sucrose concentration was 6% in the 4% ethanol solution, in that Adolescent-EtOH consumed more sweetened 4% ethanol solution than Adult-EtOH. We could explain the gradual divergence between those groups based on the facts that: (1) low ethanol concentration may be perceived as more palatable than high concentration; (2) adolescent animals consume more sucrose than adults; (Anderson et al., 2010); and (3) adolescents present higher sensitivity to the hedonic properties of sucrose than adults (Wilmouth and Spear, 2009) and thus, they would increase their consumption because of the appetitive taste of both ethanol and sucrose. However, these explanations do not take in consideration the lack of difference in ethanol intake between Adolescent-SAL and Adult-SAL. Moreover, Maldonado et al. (2008) demonstrated that adolescent rats consumed more ethanol than adults using sweetened alcohol solutions and concluded that sucrose was not relevant to the age difference found. In the present study, the age differences in ethanol pretreated mice were maintained even when sucrose was completely faded out, suggesting that the behavioral sensitization during adolescence or adulthood may account for the age-differences in voluntary ethanol consumption. We may suggest that previous behavioral sensitization decreased ethanol intake in adult but not in adolescent mice. This might be because of the ontogeny of the dopaminergic system with an inverted U-shaped format in brain regions involved in motivation and rewarding (McCutcheon and Marinelli, 2009). Functional characteristics of the dopaminergic system during development have been implicated in distinct patterns of behavioral response to drugs between younger and older animals, such as sensitization and/or consumption (Doremus et al., 2005; Frantz et al., 2007; Camarini et al., 2008; Faria et al., 2008; Valzachi et al., 2013; Camarini and Pautassi, 2016).

Although we have not found differences to the acute simulant effects of ethanol between adolescent and adult mice in the present and previous studies (Faria et al., 2008; Carrara-Nascimento et al., 2011, 2013; Soares-Simi et al., 2013), adolescents showed lower levels of locomotor sensitization to ethanol than adults when receiving low doses of ethanol (Faria et al., 2008; Stevenson et al., 2008). Moreover, it has been demonstrated that female adolescent mice need higher ethanol doses (i.e., 4.0 g/kg) than adults to develop ethanol locomotor sensitization (Quoilin et al., 2012), suggesting ontogenic differences in ethanol-induced behavioral sensitization. However, we cannot discard the hypothesis that the lower activity in adolescents compared to adults is in fact a process of tolerance to the hypolocomotor (sedative) effect of ethanol rather than locomotor sensitization. Although Phillips et al. (1996) have shown that sensitization does not result from tolerance to the sedative effects of ethanol in BXD/Ty recombinant inbred strains, this hypothesis should be further investigated in adolescent mice.

In the present study, sensitized adolescent mice displayed an ethanol consumption pattern that differed from the sensitized adult mice, in that they showed a gradual increase in consumption of 4% ethanol solution. It seems to have an interaction between low sensitivity to behavioral sensitization and consumption of ethanol solution at low concentration. In other words, sensitized adolescent mice drunk ethanol at low concentrations in stimulant doses to reach the appetitive effects of ethanol. The availability of ethanol solution at low concentration allowed those mice to control the ethanol self-administration to reach those effects. Taking this into account, it is likely that the low predisposition to behavioral sensitization in adolescents, in fact, yield animals more prone to escalate alcohol consumption at low concentration.

It has been demonstrated that adolescent rodents are not efficient to titrate their ethanol consumption as adults (Maldonado et al., 2008). Rodents learn to titrate ethanol intake based on their previous experiences with ethanol, likely mediated by postingestional effects of ethanol (Samson et al., 2002; Czachowski et al., 2006). In the present study, preexposure to ethanol during adolescence or adulthood differentially impacted the ability of animals to titrate their ethanol consumption, in that Adult-EtOH consumed less ethanol than Adolescent-EtOH. The statistical analysis that considered all experimental phases revealed that Adult-EtOH displayed lower 4% ethanol intake compared to all the other groups. Moreover, Adult-EtOH mice also drank less 15% ethanol than Adolescent-EtOH during the last phase of the experiment (0% sucrose). Thus, we suggest that previous behavioral sensitization in adult but not in adolescent mice exerted a protective effect in adult mice towards increased ethanol intake in a model of three-bottle choice using sucrose fading procedure. Using a different protocol, we have demonstrated that preexposure to ethanol increased ethanol intake, regardless of age (Carrara-Nascimento et al., 2014). It is noteworthy that in the latter study, the protocol included only one abstinence phase.

A hypothesis to explain the steady escalation of 4% ethanol solution is through pharmacological sensitization (Zernig et al., 2007). It is likely that those mice showed a rapid escalation to reach a state of greater sensitivity to ethanol-induced sensitization, and desired stimulation levels. They also showed persisted ethanol intake throughout the five reexposure periods and higher consumption than Adult-EtOH, confirming the important role of ethanol exposure during adolescence to induce use disorders later on adulthood. It is interesting to note that when more chronic ethanol reexposures were introduced, a significant higher increase in 15% ethanol intake in Adolescent-EtOH group compared to Adult-EtOH group also appeared, suggesting an influence of high ethanol consumption at higher concentrations in the timeline of ethanol exposure. Rodent preferences usually shift to the highest ethanol concentrations after withdrawals in three-bottle choice tests with multiple ethanol concentrations (Hölter et al., 1998; Rodd-Henricks et al., 2001; Ribeiro et al., 2012).

Even though quinine has decreased 4% ethanol consumption in both Adolescent-EtOH and Adult-EtOH mice, the age-differences were maintained, showing that motivation levels to drink were equally preserved. Interestingly, aversion to quinine taste was minimized in 15% ethanol solution. This suggests a great motivation to drink even under aversive taste or, alternatively, quinine bitter tasting was masked by the high ethanol concentration.

ADE exerted a greater effect in younger mice, since consumption of 4% ethanol increased during reexposure 2 in Adolescent-EtOH and Adolescent SAL, which was tolerated in Adolescent-SAL but not in Adolescent-EtOH. Tolerance was also observed to 15% ethanol consumption in both Adolescent-SAL and Adult-SAL. A marked difference between mice pretreated with ethanol or SAL is that non-pretreated mice, regardless of age, showed increased 15% ethanol intake during the last acquisition period and developed tolerance during subsequent reexposures. Moreover, the initial longer ADE induced a greater effect on 15% ethanol consumption in non-pretreated mice compared to sensitized mice (Adolescent-EtOH and Adult-EtOH). Although speculative, these data suggest that behavioral sensitization is not necessarily related to increased ethanol consumption. Discordant results have been reported on the correlation between behavioral sensitization and ethanol consumption. Abrahao et al. (2013) demonstrated an association between locomotor sensitization and ethanol drinking in Swiss mice. The difference between our study and theirs is the number of ethanol bottles during the test, ethanol concentration, and more important, the classification of mice receiving ethanol into “sensitized” and “nonsensitized” in their study. Other important difference in this study from ours is that during the initial phase of the self-administration protocol, animals were given forced exposure to the ethanol solution before having access to the two-bottle choice (Abrahao et al., 2013). Ribeiro et al. (2008) did not find a correlation between these two parameters. Fabio et al. (2014) showed an enhancement of ethanol consumption in adolescent, but not in adult mice, preexposed to binge ethanol intoxication, regardless development of behavioral sensitization.

Regardless of the previous treatment, we found a significant effect of age on ALDH activity, with older mice showing higher activity compared to younger ones, suggesting decreased rate of alcohol metabolism in younger mice. In support of our results, Collins et al. (1975) demonstrated age-differences in ALDH activity between mice from PND = 50–60 and PND = 95–110. We also found that exposure to ethanol during adolescence did not alter ALDH activity on adulthood, since there were no significant differences in ALDH activity between Adolescent-EtOH and Adolescent-SAL. This is a caveat in our study because all mice were exposed to ethanol during voluntary consumption. Although we lack this control, ethanol self-administration studies in rats reported no differences in the ALDH activity between those that were given ethanol compared to their controls (Amir, 1978). In humans, chronic exposure to alcohol increases acetaldehyde in the blood and decreases ALDH activity in the liver (Jenkins and Peters, 1980; Palmer and Jenkins, 1982). Interestingly, reduction in this enzyme activity is related to liver damage or excessive alcohol consumption. Aldehydes have an important role on cell signaling for apoptosis and in the pathophysiology of alcoholism (Kruman et al., 1997; Hayes et al., 2000).

In conclusion, preexposure to ethanol during adolescence may have altered ethanol-induced stimulation threshold. Behavioral sensitization during adolescence or adulthood induced different patterns of ethanol consumption, in that adult but not adolescent preexposed mice showed lower ethanol consumption, without affecting ALDH activity.

PFC-N and RC conceived the experiments. PFC-N and LBH conducted the behavioral experiments. PFC-N and MBC performed the aldehyde dehydrogenase activity assay. PFC-N, LBH, TM and RC analyzed the data and participated in drafting the article. RC is responsible for funding and revising the final version.

The authors thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; São Paulo Research Foundation) for the research funding (grant # 2012/10260-7) and for scholarship to PFC-N (grant # 2012/17228-1) and CNPq (National Council for Scientific and Technological Development) for the research funding (grant # 470070/2012-9). RC and TM are research fellows of CNPq.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.