Citation: Ledesma JC, Aguilar MA, Giménez-Gómez P, Miñarro J, Rodríguez-Arias M (2017) Adolescent but not adult ethanol binge drinking modulates cocaine withdrawal symptoms in mice. PLoS ONE 12(3): e0172956. https://doi.org/10.1371/journal.pone.0172956 Alcoholism medical symptoms.
Editor: Yael Abreu-Villaça, Universidade do Estado do Rio de Janeiro, BRAZIL
Copyright: © 2017 Ledesma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by grants from Ministerio de Economía y Competitividad (MINECO), Dirección General de Investigación, PSI2014-51847-R, Instituto de Salud Carlos III, Red de Trastornos Adictivos (RTA) RD12/0028/0005, Unión Europea, Fondos FEDER “una manera de hacer Europa”, Generalitat Valenciana, Conselleria de Educación, PROMETEOII/2014/063, and The European Foundation for Alcohol Research (ERAB), EA1308. Juan Carlos Ledesma was supported by a postdoctoral fellowship from the Conselleria d´Educació, Investigació, Cultura i Esport (APOSTD/2016/147), Generalitat Valenciana, Spain. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
2. Materials and methods
2.6. Study 3: Long-term effects of previous EtOH binge drinking after later cocaine withdrawal in adulthood on locomotor activity, elevated plus maze, prepulse inhibition, tail suspension, and object recognition
In order to further investigate whether adolescence rather than adulthood is a critical stage for the long-term deleterious effects of EtOH on the behavioral alterations evoked by chronic cocaine intake, in the present study both treatments were given during adulthood. Therefore, here we evaluated the long-term consequences of the previous administration of EtOH binge drinking on posterior cocaine withdrawal in adult mice, on the aforementioned behavioral paradigms. In this study we used adult mice that arrived at the animalarium on PND 55, and after the 13 days' acclimatization period they were given the EtOH treatment following the same protocol as in Study 1. Hence, mice received two injections of Sal or EtOH, separated by a 4-h interval, on PND 68, 69, 72, 73, 76, 77, 80 and 81. Three weeks thereafter, on PND 102, they were treated with the cocaine regime described above, that is, they received Sal or Coca 5 on PND 102 and 103, Coca 15 on PND 104, 105 and 106, and Coca 25 on PND 109, 110, 111, 112, and 113, resulting in the same experimental design as in Study 1.
Twenty-four hours after the last treatment, the behavioral experiments started in the same sequence as in the previous study: locomotor activity and elevated plus maze (PND 114), prepulse inhibition (PND 115–116), tail suspension (PND 118), and object recognition (PND 117–118).
An illustrative summary of the experimental procedures followed in the different studies is provided in Table 1.
None of our treatments had an effect on the locomotor activity of mice in any of the studies ( Fig 1 ).
3.1. Study 1: Long-term effects of adolescent EtOH binge drinking after cocaine withdrawal in adulthood on elevated plus maze, prepulse inhibition, tail suspension, and object recognition
Table 2 presents the results obtained from the different variables measured in the EPM for Study 1. In the case of the variable % time OA there is a significant effect for Treatment [F(1, 36) = 5.45, p<,0.05], and Interaction [F(1, 36) = 13.63, post hoc test showed that the % time OA spent by the Sal-Coca group was significantly lower than the Sal-Sal group (p<,0.01). Regarding the % entries OA, we found a significant effect for both Pretreatment [F(1, 36) = 7.89, p<,0.01], Treatment [F(1, 36) = 11.82, p<,0.01], and Interaction [F(1, 36) = 5.53, p<,0.05]. Post hoc analyses demonstrated that the % entries OA was lower in the Sal-Coca group with respect to the rest of the mice (p<,0.01). For CA entries, the ANOVA reflected an effect of Pre-treatment and Treatment [F(1, 36) = 6.40, p<,0.05] and [F(1, 36) = 7.28, p<,0.05], respectively. The Tukey HSD test indicated that the Sal-Coca group performed more CA entries than the rest of animals (p<,0.05).
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The results of the PPI experiment are displayed in Fig 2A and 2B ). The ANOVA of the data obtained with a 75 dB prepulse trials ( Fig 2A ) revealed a significant effect of Pretreatment [F(1, 36) = 30.52, p<,0.01], Treatment [F(1, 36) = 30.02, p<,0.01], and Interaction [F(1, 36) = 4.27, p<,0.05]. Pairwise comparisons demonstrated that the PPI of the EtOH-Coca group was significantly lower than the rest (p<,0.01). The ANOVA of the data obtained with 85-dB prepulse trials ( Fig 2B ) showed a significant effect for Pretreatment [F(1, 36) = 4.32, p<,0.01] and Treatment [F(1, 36) = 3.8, p<,0.05]. Post hoc analyses indicated that the PPI of the EtOH-Coca group was significantly diminished with respect to the other groups (p<,0.05).
For the TST ( Fig 3 ), the ANOVA indicated a significant effect of Pretreatment [F(1, 36) = 5.49, p<,0.05] and Treatment [F(1, 36) = 6.2, p<,0.05]. A Tukey HSD post hoc test indicated that the time spent immobile was higher in the EtOH-Coca group in comparison to the rest of the groups (p<,0.05).
The ANOVA of the data for the OR ( Fig 4 ) indicated a significant effect of Pretreatment [F(1, 36) = 60.06, p<,0.01], Treatment [F(1, 36) = 59.93, p<,0.01], and also Interaction [F(1, 36) = 5.82, p<,0.05]. Post hoc analyses displayed that, in comparison with the Sal-Sal group, administration of any of the pharmacological manipulations (EtOH and Coca alone or in combination) reduced the DI of the mice (p<,0.01). Moreover, the DI of the EtOH-Coca group was significantly lower than that of the rest of the groups (p<,0.01).
3.2. Study 2: Short-term effects of adolescent EtOH binge drinking on elevated plus maze, prepulse inhibition, tail suspension, and object recognition
Table 3 presents the results obtained from the different variables measured in the EPM for Study 2. The Student's T test revealed that the % time OA [F(1, 18) = 5.87, p<,0.05,] and % entries OA [F(1, 18) = 4.3, p<,0.05] were lower in the EtOH group. Moreover, the CA entries [F(1, 18) = 5.69, p<,0.05] were higher in this group compared to the Sal.
Regarding the PPI experiment, no differences were encountered between groups ( Fig 5 ).
The results for the TST experiment ( Fig 6A ) indicated that the time spent immobile was higher in mice previously treated with EtOH [F(1, 18) = 7.03, p<,0.05], and for the OR experiment ( Fig 6B ) we found that the DI of the EtOH group was significantly lower than that of the Sal group [F(1, 18) = 57.44, p<,0.01].
3.3. Study 3: Long-term effects of previous EtOH binge drinking after later cocaine withdrawal in adulthood on elevated plus maze, prepulse inhibition, tail suspension, and object recognition
No differences were encountered between experimental subgroups in the EPM, the PPI, and the TST for Study 3 (Figs 7 and 8, and Table 4 ). Nonetheless, for the OR experiment ( Fig 9 ) we found a significant effect of Pretreatment [F(1, 36) = 18.07, p<,0.01], Treatment [F(1, 36) = 5.51, p<,0.05], and Interaction [F(1, 36) = 6.03, p<,0.05]. The post hoc analyses revealed that the DI of the EtOH-Sal, the Sal-Coca, and the EtOH-Coca groups was lower than that of the Sal-Sal group (p<,0.01).
The current data represent the first approach towards assessing the long-lasting behavioral consequences of adolescent exposure to EtOH on cocaine withdrawal in adult mice. We used a murine model of pubertal binge administration of alcohol followed by a pattern of chronic binge cocaine intake in adulthood [ 42, 45 ], thereby imitating a pattern seen in many human polydrug users [ 1, 8 ]. The main important results of the present research are derived from Study 1, where we showed that this type of drug consumption in adolescent mice increases the vulnerability to develop some behavioral alterations in adulthood, which could be related to the high incidence of mental disorders in polydrug users. Thus, by using the PPI and the TST paradigms we found that an early history of EtOH ingestion enhances the risk of suffering from psychotic- and depressive-like symptoms, respectively, after withdrawal of chronic cocaine at an adult age. Furthermore, we found that the cognitive deficits in the OR task evoked by these drugs when given separately are boosted in mice exposed to EtOH during adolescence and, thereafter, to cocaine. In addition, we show that adolescent EtOH bingeing reduces the anxiogenic-like effects observed in the EPM after cocaine abstinence in adult mice. Adolescent animals tested just after the removal of EtOH binge also displayed anxiety- and depressive-like symptoms, as well as cognitive impairments compared to control mice (Study 2), thereby confirming the higher sensitivity to the effect of EtOH in this period of development. In contrast, when these drugs were given in the adult age we found only a deterioration in the OR task and, more importantly, we did not observe any potentiation when both treatments were combined during adulthood (Study 3). Overall, the present investigation supports the idea that adolescence is a critical stage for the deleterious effects of EtOH binge drinking, and that this kind of consumption may deteriorate the behavioral consequences of later cocaine withdrawal. An illustrative summary of the findings obtained across the different studies is presented in Table 5.
Behavioral effects of binge EtOH administration
Immediately after exposure to a chronic intermittent pattern of binge drinking during adolescence, mice showed an increase in anxiety and in immobility in the EPM and the TST, respectively. Cognitive deficits were also evident in the OR test. However, only cognitive deficits remained present three weeks after the last EtOH administration, either during adolescence or adulthood.
Adolescent mice treated with our binge EtOH regime displayed heightened anxiety scores in the EPM when tested immediately afterwards (Study 2, see Table 3 ). These data are in accordance with earlier reports also showing that adolescent binge EtOH treatment boosts anxiety-like behaviors in the EPM when measured the day after the end of alcohol administration in rodents [ 51 – 52 ]. However, we did not detect any long-term consequences of our EtOH treatment on the EPM in Study 1, when measured in adulthood ( Table 2 ). This data is in agreement with previous research showing similar results after exposure of adolescent mice to the same EtOH binge-treatment regime and tested three weeks thereafter [ 23 ]. Furthermore, in Study 2 we found that when adolescent animals were treated with EtOH and tested shortly afterwards in the TST (i.e., five days after the end of the binge drinking administration) their immobility scores where augmented with regard to the control group ( Fig 4A ). This supports the notion that clinical and pre-clinical evidence shows the high degree of comorbidity of alcoholism and depression. In fact, it has been proposed that symptoms of depression during EtOH abstinence increase the likelihood of relapse and indicate a worse prognosis in terms of treatment outcome [ 53 – 54 ].
In agreement with earlier reports, we observed that our EtOH treatment during adolescence induced both short- (i.e., when measured 5 days after its administration, Study 2) and long-term (i.e., when measured 37 days after its administration, Study 1) memory impairments in the OR task (Figs 3 and 4B respectively) [ 6, 42, 55 – 57 ]. In addition, in Study 3 we found the same long-lasting effects when EtOH was given during adulthood ( Fig 5 ). This indicates that previous EtOH treatment is able to induce memory deficits when given in both the adolescent and the adult periods, and that this effect is long-lasting in mice. Thus, it seems that memory is especially vulnerable to the deleterious effects of alcohol binge drinking.
Finally, in agreement with previous findings, EtOH binge drinking did not produce psychotic-like symptoms when administered during adolescence or adulthood, because the values of the PPI were not modified in the groups treated only with EtOH in any of the experiments [ 4 ].
Overall, we can conclude that EtOH binge drinking during adolescence, despite inducing a transient increase in anxiety and depressive-like symptoms during adolescence, only gave rise to long-lasting cognitive deficits that do not depend on the age at EtOH administration. Therefore, it seems that with the exception of memory disturbances, which may be detected both short- (Study 2) and long-term (Study 1 and 3) after EtOH administration and appear to be independent of the age of the animals, the anxiogenic- and depressive-like actions of binge drinking are only produced when EtOH is administered during adolescence, thus suggesting that these effects are age-dependent.
Behavioral effects of cocaine withdrawal
Withdrawal from cocaine without a previous history of EtOH exposure induced memory disturbances in the OR independently of the age at which cocaine was administered. Conversely, after cocaine withdrawal only mice exposed to this drug between PND 68–79 (Study 1) displayed anxiety-like effects in the EPM, thereby suggesting that the anxiogenic-like effects evoked by cocaine abstinence may be age-dependent. Irrespective of age, no psychotic- or depressive-like symptoms were manifested by mice after cocaine discontinuation in the PPI and the TST respectively.
In relation to the possible cognitive dysfunctions elicited by cocaine abstinence assessed by the OR procedure, we found that mice undergoing withdrawal performed this task more poorly than Sal-Sal control subjects in both Study 1 (adolescents) and Study 3 (adults) (Figs 3 and 5 respectively). This result has also been shown by other researchers, who observed that extended exposure to cocaine deteriorates recognition memory in rodents following a similar procedure to that of the current research [ 58 – 59 ].
In Study 1 we demonstrated that cocaine withdrawal evoked anxiety-like behaviors in the EPM ( Table 2 ). These results are in agreement with those of earlier studies in which it has been reported that cocaine discontinuation is accompanied by anxiety symptoms in rats [ 60 – 64 ]. However, in contrast to Study 1, the anxiety levels of mice under cocaine abstinence did not differ from those of the control group in Study 3 (data not shown). These discrepancies between the two studies may be due to the different age of drug exposure. In Study 1 cocaine was administered during PND 68–79, a period that corresponds to the beginning of the adult age (i.e., this age is considered as being “young adulthood or late adolescence”), in contrast, in Study 3 they were treated with cocaine from PND 102 to 113, an age that represents the height of adulthood in mice [ 41, 43 ]. It is well known that brain maturation in mammals occurs when the adult age is completely accomplished, as should be the case of the mice from Study 3. Consequently, it would be possible that these mice were less susceptible to the anxiogenic effects elicited by cocaine abstinence than those of Study 1 because they had a more developed brain. In agreement with this speculation, an earlier study carried out by Valzachi and colleagues [ 25 ] demonstrated that mice that were chronic cocaine-pretreated during adolescence exhibited a greater magnitude of cocaine withdrawal-induced anxiety-like behavior compared to adult males that received the same treatment conditions.
On the other hand, we found no effects of cocaine abstinence in the PPI procedure, irrespective of age (Studies 1 and 3), suggesting no significant psychotic-like actions of cocaine withdrawal in our experiments. This result is in the same line as previous studies showing that cocaine withdrawal did not modify the PPI of adult rats [ 65 – 67 ]. At present, data about the consequences of abstinence from cocaine in the PPI paradigm in rodents are scarce, and we have presented here the earliest investigation showing that cocaine-withdrawn mice did not manifest any psychotic-like symptoms, at least under our experimental conditions.
Similarly, regarding the TST results, we did not observe any significant alterations in the time that mice of any age remained immobile after cocaine abstinence, thereby indicating that drug discontinuation after its chronic intake did not induce any depressive-like effects. This is a very interesting result, because this is the first time that the consequences of cocaine withdrawal on the TST paradigm has been measured in mice. By using other behavioral paradigms that also model behavioral depression, such as the forced swim test and the sucrose preference test, different results had been reported in rodents. For example, in rats it has been shown that cocaine withdrawal is accompanied by depression-like symptoms [ 19, 68 – 69 ]. In contrast, other authors have found that abstinence from chronic cocaine administration did not induce any depression-like effects in mice, as in the present investigation [ 62 ]. Although methodological differences between studies may explain these divergent results, it could be possible that mice and rats have a different sensitivity to the depressive-like actions induced by cocaine withdrawal.
In summary, in relation to the possible behavioral disturbances evoked by cocaine cessation after its chronic administration during adulthood, we found that this treatment could induce anxiety-like effects depending on the age at which it is administered. Thus, it seems that young adult mice are more sensitive to the anxiogenic actions elicited by cocaine withdrawal than adult mice. No other difference was seen between the behavioral profile of younger and older abstinent mice, which exhibit similar cognitive dysfunctions in the OR but neither psychotic- nor depressive-like symptoms.
Behavioral effects of previous binge administration of EtOH on cocaine withdrawal
The most important and innovative data obtained in the present research came from the results derived from the mice treated with the combination of EtOH and cocaine. In this way, with the exception of the EPM experiment, in which we did not detect any alterations in the anxiety scores in these groups, we found that exposure to EtOH during adolescence elicited both psychotic- and depressive-like effects, and enhanced memory impairments in the PPI, TST, and OR respectively after cocaine cessation. None of these outcomes were seen when cocaine-withdrawn mice were previously treated with EtOH at the adult age. Therefore, it seems that adolescent EtOH binge drinking could increase the vulnerability of mice to suffer from different behavioral disturbances after later cocaine withdrawal.
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As can be observed in the EPM experiment in Study 1 ( Table 2 ), we found that the anxiety values obtained when adolescent mice were pretreated with EtOH and later with cocaine did not differ from those of the control mice. This was a very surprising result considering that, in this same study, mice only exposed to cocaine displayed enhanced anxiety values ( Table 2 ) and that in Study 2 we found that adolescent mice displayed short-term anxiogenic-like behaviors after binge administration of EtOH ( Table 3 ). Hence, it may be expected that previous exposure to binge administration of EtOH during adolescence will potentiate the later anxiogenesis evoked after cocaine cessation in adults. Nevertheless, taking into account the present results, an alternative explanation may be considered. It could be possible that mice exposed to EtOH during adolescence and to cocaine in adulthood displayed lower anxiety levels than those only exposed to cocaine because they have previously experienced the anxiety evoked by EtOH abstinence during adolescence, thus making them more resilient to the anxiogenic effects of posterior cocaine withdrawal. This could be interpreted as meaning that juvenile exposure to EtOH alleviates the anxiety symptoms associated with later cocaine abstinence. In that sense, it is widely accepted that there exists a correlation between anxiety and cocaine addiction, and that high anxiety is one of the factors that contribute to relapse because many cocaine-abstinent addicts consume the drug again to alleviate the anxiety induced by the withdrawal syndrome [ 25, 70 ]. So, one can speculate that EtOH consumption in adolescence protects against cocaine relapse by reducing anxiety levels after its abstinence in adulthood. However, another possibility is that if previous chronic EtOH administration and its removal is able to reduce the posterior anxiogenic effects elicited by cocaine cessation, it could maintain cycles of consumption and abstinence because subjects experience the aversive consequences of the abstinence syndrome to a lesser degree. Notwithstanding, future studies must be carried out to test this hypothesis.
Another meaningful result comes from the finding that when juvenile mice were pretreated with EtOH and later with cocaine (EtOH-Coca group from Study 1), their PPI was decreased ( Fig 1A and 1B ). This is an appealing result, because neither binge EtOH administration nor cocaine discontinuation affected the PPI of animals when given alone. Nevertheless, the combination of EtOH during adolescence and cocaine in adulthood acts synergistically to impair the suppression of the startling reflex. Interestingly, in Study 3 we observed that when these same treatments were both dispensed at the adult age it did not affect the PPI of animals. Therefore, the interaction between EtOH and Coca treatments on the PPI impairment is age-dependent. Hence, given that it has been established that the PPI is a suitable paradigm to infer psychotic symptoms in rodents [ 28, 32 ], we propose that adolescence is a critical period in which EtOH binge drinking could facilitate the posterior development of psychotic symptoms after chronic cocaine ingestion at the beginning of adulthood.
Similarly to what occurred with the PPI experiment, in Study 1 we also observed that when animals were previously exposed to EtOH during adolescence and later withdrawn from cocaine, they remained significantly more time immobile in the TST ( Fig 2 ). This suggests that a history of abuse of EtOH in adolescence and cocaine discontinuation after its chronic intake at an adult age could evoke some depressive-like symptoms in mice. In contrast, when both EtOH binge drinking and chronic cocaine dispensation were given in adulthood, the TST values were not altered (Study 3). Hence, it seems that adolescent exposure to EtOH bingeing followed by adult cocaine withdrawal is specifically susceptible to promote depression-like manifestations.
Finally, another very prominent result comes from the finding that we demonstrate how the combination of adolescent binge EtOH administration with later cocaine abstinence in adulthood boosted the detrimental effects that these two treatments may provoke in the OR when given separately. Thus, in Study 1 we reported that the DI of the EtOH-Coca group was not only lower than that of the control group, but also than those of the groups receiving EtOH or cocaine alone, which, as noted above, were already decreased with respect to the control group ( Fig 3 ). This enhancement of memory disruption was not observed in the EtOH-Coca group from Study 3, in which both treatments were given at the adult age ( Fig 5 ). This suggests that the combination of EtOH consumption during puberty and cocaine at the onset of adulthood potentiates the impairing effects of these substances on memory, thereby highlighting again the idea that heavy adolescent alcohol consumption could have a negative impact on the behavioral disturbances induced by later chronic cocaine intake.
In conclusion, the current results demonstrate for the first time the long-lasting consequences of juvenile exposure to EtOH on cocaine withdrawal in adult mice. Given that pubertal alcohol binge drinking followed by a later development of cocaine abuse is an increasingly common pattern of drug consumption in human beings, the results of the present research are particularly relevant, as they provide evidence of the augmented risk of developing several psychiatric illnesses in subjects with this pattern of drug intake. Nonetheless, this is a first behavioral approximation, and future studies will be required to establish the contribution of the different cerebral pathways and neurotransmitter systems involved in such effects, as well as to validate the hypotheses presented herein. In addition, given that the National Institute of Health has recommended that sex should be considered as a biological variable in all medical study design, and that it has been reported that there are sex-specific negative consequences of early adolescent ethanol exposure [ 71 ], it becomes essential to assess in the near future the possible differences between males and females in their response to our treatments. This will help to further our knowledge about the mechanisms involved in the psychopathologies shown by this kind of polydrug user, and to open up the way to the development of new pharmacological tools to prevent or palliate these disorders.