Adolescent ethanol exposure is a risk-factor for later voluntary ethanol consumption.

Besides our individual biological genetic predisposition, several other factors might be responsible for a higher risk of developing substance use disorder later in life, especially early drug initiation during adolescence (Morales et al., 2020; Nawi et al., 2021). Interestingly, adolescence is a period of critical brain development that appears to be highly conserved across species in terms of its neurobehavioral and physiological features. Similar to the stages observed in humans, early (10–13 years), middle (14–17 years), and late adolescence/young adulthood [18–21 years (Christie and Viner, 2005; Backes and Bonnie, 2019)], in rodents, adolescence could be divided into early (postnatal day, PND 21–34), mid (PND 34-46) and late adolescence [PND 46-59 (Spear, 2004)]. Therefore, the use of rodents provides a great preclinical model (García-Fuster, 2021; Nieto et al., 2021) with face and construct validity (Spear, 2004) in which to study particular windows of adolescent vulnerability to psychopathology and therapeutic strategies (Adriani and Laviola, 2004).

In this context, our research group, among others [reviewed by Steinfeld and Torregrossa (2023)], aimed at characterizing adolescent windows of vulnerability during which the use of illicit drugs could be more harmful, both behaviorally and neurochemically (García-Cabrerizo et al., 2015; García-Cabrerizo and García-Fuster, 2016; García-Cabrerizo and García-Fuster, 2019; García-Fuster et al., 2017; Parsegian et al., 2022). In particular, a regimen of adolescent cocaine exposure known to induce psychomotor sensitization was more harmful during PND 33-39 than other earlier (PND 26-32) or later (PND 40-46) windows of adolescence (García-Cabrerizo et al., 2015), showing signs of increased negative affect (García-Cabrerizo and García-Fuster, 2019) and addictive-like behaviors (García-Fuster et al., 2017; Parsegian et al., 2022) when rats were re-exposed to cocaine in adulthood, as well as changes in neurochemical markers of neurotoxicity (García-Cabrerizo et al., 2015; García-Cabrerizo and García-Fuster, 2016; García-Fuster et al., 2017; Parsegian et al., 2022). Although all of these studies were done in male rats, we also recently reported that adolescent cocaine induced persistent negative affect in adult female rats (Bis-Humbert and García-Fuster, 2021). Therefore, this window and pattern of cocaine exposure during mid-adolescence is of high vulnerability to the long-term effects emerging in adulthood and caused by an early drug initiation.

Interestingly, cocaine is rarely consumed alone, since it is frequently combined with alcohol; out of the individuals who reported heavy alcohol use in the past month, 5% also consumed cocaine (Graziani et al., 2014). Both the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) as well as the National Center for Drug Abuse Statistics (NCDAS) in US, yearly report that alcohol is by far the most commonly consumed substance among teens and young adults. This data proves an earlier initiation and a higher use for alcohol, as compared to cocaine, and therefore suggests that alcohol initiation will be generally sooner, and that the combined use of both drugs would parallel the time at which cocaine’s use is started. The many prior studies evaluating the effects of adolescent alcohol consumption suggested that the behavioral irregularities (i.e., elevations in anxiety, disinhibition, impulsivity and risk-taking, decreased cognitive flexibility) and neural consequences of adolescent alcohol use may persist into adulthood [reviewed by Spear (2016); Spear (2018); Lees et al. (2020)], including an increased ethanol drinking in adulthood (Strong et al., 2010; Sherrill et al., 2011). However, studies evaluating the combined impact of alcohol and cocaine have been centered mainly in the cardiotoxic effects mediated by cocaethylene (the active metabolite produced by both drugs), so there is a need for novel data on the long-term impact of initiating alcohol alone and/or combined with cocaine during adolescence on later alcohol use disorder rates in adulthood.

Against this background, we characterized the changes induced by an even earlier start of adolescent ethanol exposure at PND 28, as compared to the one previously depicted for cocaine initiation (vulnerability window starting on PND 33), as well as their combined impact on the effects emerging in adulthood, and while including both sexes in the study (Towers et al., 2023; Becker and Koob, 2016), since most of the data summarized above was done in male rodents. Particularly, we assessed affective- (i.e., behavioral despair and/or stress-coping mechanisms, anxiety-related behaviors, anhedonic-like response) and/or addictive-like behaviors [i.e., voluntary consumption: 20% ethanol two-bottle choice paradigm (Colom-Rocha et al., 2023)]. Then, we selected the worst outcome induced by prior adolescent drug exposure, to test whether two therapeutical interventions (cannabidiol or ketamine) would prevent the increased ethanol consumption emerging in adulthood (Yardley and Ray, 2017). These options were chosen based on previous preclinical and/or clinical data suggesting an amelioration of ethanol-motivated behaviors both by cannabidiol (Viudez-Martínez et al., 2018; Viudez-Martínez et al., 2020; Nona et al., 2019; Maccioni et al., 2022; Tringali et al., 2023; Gasparyan et al., 2023) or ketamine [reviews by Worrell and Gould (2021); Goldfine et al. (2023); Kelson et al. (2023); Krystal et al. (2024)], although most of these studies were tested in either pups from pregnant rodents exposed to drugs during gestation, and/or in rodents that received drugs in adolescence and/or adulthood, but were tested at the same age-window of study, and therefore, data evaluating the possible preventive effects of these options following an early adolescent drug experience are missing. A pre-print version of this manuscript has been uploaded to Research Square (https://doi.org/10.21203/rs.3.rs-3943360/v1).

This study examined the long-term changes in affective-like responses and in ethanol voluntary consumption in adult male and female rats previously exposed to ethanol, cocaine or their combination during vulnerable windows of adolescence. The main results showed that exposing rats to these drugs in adolescence did not induce signs of increased negative affect during adulthood, although this conclusion is limited to the doses, regimens and methods of administration followed. However, when rats were also allowed to voluntarily consume ethanol in adulthood, some differences emerged depending on the drug they previously received in adolescence. In particular, adolescent ethanol exposure was a risk-factor for later developing an increased voluntary ethanol consumption in adulthood, both in male and female rats. This risk was similar when ethanol was combined with cocaine exposure in adolescence, since adolescent cocaine exposure alone did not affect ethanol intake in adulthood. Finally, rats exposed to ethanol in adolescence and pretreated in adulthood with either cannabidiol (and/or ketamine but just for females) reduced ethanol voluntary consumption. Overall, our data provided two therapeutical options capable of attenuating voluntary ethanol consumption rates in adulthood caused by a prior adolescent drug exposure.

This study initially hypothesized that the combined exposure of ethanol and cocaine in adolescence would show increased sings of negative affect in adult male and female rats. In this context, a recent review compared the behavioral phenotypes that emerge following different ethanol exposure models, and concluded that the complex outcomes from these studies highlighted the difficulties of assessing negative affective behaviors in rodent models designed for the study of alcohol use disorder (Bloch et al., 2022). In our case, the behavioral phenotyping was done through the sequential screening in different tests that try to capture different aspects of the symptomatology observed in humans, such as behavioral despair (García-Fuster et al., 2012) and/or stress-coping strategies (Molendijk and de Kloet, 2015; Commons et al., 2017) through FST, anxiety-like responses in EPM (García-Fuster et al., 2012) and NSF (Bis-Humbert et al., 2021; Ledesma-Corvi et al., 2022), and hedonic-like responses in SP (Jiménez-Romero et al., 2020). However, we found no evidence of a deteriorated affective-like state induced by any of the drugs administered in adolescence (ethanol, cocaine or their combination) in rats of both sexes. Note that adolescent drug exposure was administered by an experimenter in a non-contingent way so each experimental group received the same dose in terms of later being able to better understand the impact of a prior fixed drug exposure on ethanol voluntary intake in adulthood. Although this type of regimen did not inform about the motivation for the drug during adolescence, and might have impacted the lack of negative affect observed during drug abstinence, it is worth mentioning that this set of data aligned with our prior results suggesting that adolescent drug exposure alone may not be sufficient to induce negative affect in adulthood, since adult drug re-exposure was needed to observe a negative impact on behavior from an earlier adolescent exposure (García-Cabrerizo and García-Fuster, 2019; Bis-Humbert and García-Fuster, 2021; Bis-Humbert et al., 2021). Also, in this line of thought, adding stress could also be a possible factor that can reveal the phenotype, suggesting that adolescent early exposure to drugs increased susceptibility or vulnerability to later consumption. This idea reinforces the notion that one way to prevent substance use disorder later on relies on avoiding drug consumption early in life. Moreover, in line with the previously detected basal sex-differences for these particular tests of study (Hernández-Hernández et al., 2023; Ledesma-Corvi et al., 2023), the data reported a differential impact by sex in some tests, with a greater general exploratory-like behavior for females as observed in NSF, combined with a higher hedonic-like response in SP.

The next study evaluated the impact of the adolescent treatment on ethanol consumption later on in adulthood. Although no changes were observed when measuring affective-like responses, when exposing rats to a voluntary access to ethanol in adulthood [20% ethanol for three consecutive days per week, during 6 weeks; characterized in Colom-Rocha et al. (2023)], some differences in consumption emerged depending on the drug they previously received in adolescence. In particular, and in line with prior data (Strong et al., 2010; Sherrill et al., 2011) [reviewed by Spear (2016); Spear (2018); Lees et al. (2020)], adolescent ethanol exposure was a clear risk-factor for later developing an increased voluntary ethanol consumption in adulthood, both in male and female rats. In particular, previous adolescent ethanol exposure increased ethanol preference and voluntarily consumption in adulthood, being this effect of equal magnitude for both sexes. However, when correcting ethanol intake by weight to calculate the dose consumed per day (g/kg/24 h), female rats were exposed to higher doses than their male counterparts, in line with prior recent examples showing sex-specific drinking patterns in adult rodents (Colom-Rocha et al., 2023; Foo et al., 2023; McElroy et al., 2023). Moreover, the risk observed by ethanol combined with cocaine in adolescence was of a similar magnitude, or even a bit reduced, to the one induced just by ethanol, since adolescent cocaine exposure alone did not affect ethanol consumption in adulthood. Remarkably, the literature is full of data showing how adolescent ethanol exposure alters the rewarding effects of cocaine in adulthood (Hutchison and Riley, 2012; Mateos-García et al., 2015; Ledesma et al., 2017; Esteve-Arenys et al., 2017; Cantacorps et al., 2020), however, studies evaluating how adolescent cocaine impact later adult ethanol consumption are lacking. One example described the acute effects of cocaine stimulating ethanol intake in a two-bottle choice paradigm, but was done in adult male Sprague-Dawley rats (Colvin et al., 2022), Therefore, since adolescent cocaine exposure did not stimulate ethanol intake in adulthood, the effects observed by ethanol and cocaine combined in adolescence over the rate of voluntary ethanol consumption in adulthood might be driven exclusively by prior ethanol exposure in adolescence. These results reported that despite the fact that adolescent drug exposure did not induce any clear signs of negative affect, when adult rats were presented with a voluntary drug experience, the prior adolescent experience had clear consequences on the emerging addictive-like behaviors observed for both sexes. Although these negative effects were not observed for all drugs (lack of impact by adolescent cocaine), adolescent ethanol exposure increased the rates of ethanol consumption in adulthood. Interestingly, adolescent alcohol use was also proven a risk factor for adult alcohol and drug dependence in a clinical studies with twins (Grant et al., 2006). Therefore, since an early exposure to ethanol during adolescence induced long-term consequences for substance use disorders later on in adulthood, preventing an early-age initiation is postulated to be of vital importance to avoid future problems related to substance use disorders. However, for cases when adolescent drug exposure is already initiated, having access to therapeutical options that could prevent a later drug consumption when ethanol renders available in adulthood are of great relevance.

In this line of thought, our third study tested two therapeutical interventions (cannabidiol, ketamine) for preventing the increased ethanol consumption in adult rats following a prior adolescent ethanol exposure. Note that this experiment was done in the absence of ethanol-naive controls (i.e., all rats received ethanol in adolescence), since our prior experiment already characterized the long-term effects of ethanol vs vehicle exposure in adolescence, and the present goal was to ascertain whether the tested therapeutical options could reduce the voluntary drinking observed in adulthood and caused by a prior adolescent experience. In particular, the main results showed that rats exposed to ethanol in adolescence and pretreated in adulthood with cannabidiol (and/or ketamine but just for females) reduced ethanol voluntary consumption in adulthood. Interestingly, the rates of ethanol preference and/or consumption in adult male and female rats were similar to the ones observed in our prior study for the adolescent ethanol group, demonstrating that independent studies replicated and were therefore reliable. Moreover, both cannabidiol and ketamine showed antidepressant-like responses in FST in male rats exposed to ethanol in adolescence, in line with prior results in naïve rats (Bis-Humbert et al., 2020; Ledesma-Corvi et al., 2023), while rendered inefficacious or even deleterious for female rats (Ledesma-Corvi et al., 2022; Gálvez-Melero et al., 2023; Ledesma-Corvi et al., 2023). Prior data have already proven sex-differences in antidepressant-like responses, with clear drops in efficacy for female subjects (Kokras et al., 2011; LeGates et al., 2019). Clearly, the antidepressant-like response observed in FST in rats treated with adolescent ethanol did not parallel the response that these therapeutical options induced in terms of ethanol voluntary consumption rates (as detailed below), hence suggesting a broader therapeutical potential, other than their antidepressant-like action, both for cannabidiol and ketamine in adult rats of both sexes.

In accordance with this, for example, both male and female rats pretreated with cannabidiol showed decreased ethanol intake in adulthood as observed by a drop in preference (although only significant in male rats), a lower volume of ethanol consumed, and a lower overall dose than rats pretreated with vehicle. These results aligned with previous preclinical data suggesting an improvement of ethanol-motivated behaviors by cannabidiol [reviewed by Nona et al. (2019)]; see also (Viudez-Martínez et al., 2018; Viudez-Martínez et al., 2020; Maccioni et al., 2022; Tringali et al., 2023; Gasparyan et al., 2023)], including the cognitive deficits and neuroinflammation induced by early ethanol exposure (García-Baos et al., 2021). Particularly, prior studies have proven cannabidiol efficacy in pups from pregnant rodents exposed to drugs during gestation, directly in adolescence (immediate effects), and/or in rodents that received drugs in adulthood. For example, cannabidiol (30 mg/kg/day, i. p., for up to 4–6 weeks) repaired the behavioral and brain disturbances in offspring exposed to an animal model of fetal alcohol spectrum disorder (Gasparyan et al., 2023). When administered during adolescence, cannabidiol (40 mg/kg) before each drinking session reduced ethanol consumption and preference in male rats that underwent the intermittent 20% ethanol two-bottle choice paradigm (Tringali et al., 2023). Moreover, during adulthood, the administration of cannabidiol in male mice reduced the reinforcing properties, motivation and relapse for ethanol (Viudez-Martínez et al., 2018). Later on, a follow-up study from the same group described sex differences in the effects of cannabidiol on ethanol binge drinking in adult mice, proving, similarly to the results obtained here, that although female mice exhibited higher ethanol intake during each drinking in the dark session, cannabidiol reduced ethanol consumption for both sexes; these effects were observed after an acute (90 mg/kg; highest dose tested) and/or chronic administration [although with different dose-dependent efficacy for males and females; males: 30, 60 and 90 mg/kg; females: only with 90 mg/kg (Viudez-Martínez et al., 2020)]. In another study in adult male rats, cannabidiol (≥12.5 mg/kg) markedly reduced lever responding for ethanol and amount of self-administered ethanol in selectively bred Sardinian ethanol-preferring male rats, a validated animal model of excessive ethanol consumption (Maccioni et al., 2022). Against this pool of published data, our study is quite original and novel in that it proposed a different approach, since it evaluated the beneficial effects of cannabidiol on preventing the impact of early drug initiation during adolescence on voluntary ethanol consumption in adult male and female rats. Taken together, these findings, in conjunction with the prior published data, suggested that cannabidiol may be a great candidate for preventing the development of alcohol-use disorders, independently of when drug exposure was initiated (i.e., during prenatal period, adolescence, and/or adulthood), and independently of sex, if previously adjusting the specific sex-related conditions required for efficacy (i.e., dose, length of treatment, animal species, etc.).

Finally, the other therapeutical option tested, at the conditions used, was only capable of showing signs of improvement in female rats. Mainly, ketamine showed signs of efficacy by decreasing ethanol consumption in adulthood, but only in female rats exposed to ethanol in adolescence. In particular, it lowered the volume as well as the overall dose of ethanol consumed, as compared to rats pretreated with vehicle. In line with our results, some prior studies also showed a greater sensitivity to the ketamine treatment in females, although ketamine significantly reduced both ethanol intake and preference in a time- and dose-dependent manner in ethanol preferring adult rats of both sexes, while it did so at higher doses [effective doses: 7.5 mg/kg, and 10 mg/kg (Rezvani et al., 2017)] than the ones used in the present study. Another experiment also reported a better therapeutical response for females by reporting that ketamine reduced binge-like drinking behavior exclusively in female rats exposed to a drinking in the dark model and when ketamine was given prior to ethanol exposure (Crowley et al., 2019). Contrarily, other studies found a greater sensitivity for ketamine in male rodents, for example, while ketamine decreased ethanol consumption in male rats that self-administered high levels of ethanol, it increased ethanol consumption in female rats that showed low levels of ethanol self-administration (Strong et al., 2019). Also, adolescent exposure to ketamine significantly decreased preference for ethanol consumption in males, with a smaller reduction of ethanol consumption in females (Franco et al., 2020). In general, prior results with ketamine at the preclinical and clinical level reported some beneficial effects on ameliorating ethanol-motivated behaviors for both sexes [see recent reviews by Worrell and Gould (2021); Goldfine et al. (2023); Kelson et al. (2023); Krystal et al. (2024)], although with some differences in between sexes that should be further studied since they will likely play an important role in the future development of therapeutical options for alcohol use disorders. Overall, the use of ketamine for the treatment and/or prevention of alcohol use disorder is promising, with several ongoing clinical trials, but with still the need to further complete its efficacy validation and safety profile before recommending its broader clinical use [reviewed by Goldfine et al. (2023)].

These results provided two therapeutical options for preventing the effects of an early drug initiation during adolescence on ethanol voluntary consumption rates later on in adulthood. Thus, these results may be of relevance in view of possible future studies testing cannabidiol or ketamine in patients affected by alcohol use disorder, whose consumption started early in life during adolescence, and when sex-related differences might be affecting the treatment outcome.