In the present study, we aimed to assess the impact of early life stress, in the form of early maternal deprivation (MD, 24 h on postnatal day, pnd, 9), on voluntary alcohol intake in adolescent male and female
Epidemiological data indicate that adverse life events during the first few years of life can increase the risk for psychopathology, including drug addiction [
There is evidence about a relationship between stress and alcohol consumption (see [
In the present study we aimed to analyze the effects of MD on adolescent alcohol consumption, as well as addressing to which extent a previous history of early life stress (MD) could modulate the consequences of subsequent stressful life events (alcohol deprivation and alcohol deprivation combined with the concomitant exposure to restraint stress) on voluntary alcohol intake. Given the amount of evidence supporting sex differences in the consequences of MD, both male and female animals were employed in the present study.
Experiments were performed in agreement with the European Directive 2010/63/EU on the protection of animals used for scientific purposes and in compliance with the Spanish Royal Decree 1201/2005, October 21, 2005 (BOE n° 252), about protection of experimental animals. All efforts were made to minimize animal suffering and distress.
Experiments were carried out on the offspring of rats purchased from Harlan Laboratories (Rossdorf, Germany), which were mated (one male
Early MD was performed as previously described [
Once the rats became adolescents (pnd 28), rats were given free access to a bottle containing an ethanol solution (20% v/v) in addition to the water bottle. The ethanol solution (v/v) was prepared using 96° ethanol (Alcoholes Aroca S.L., Madrid, Spain) diluted in tap water. The positions of the bottles were changed daily to avoid position preference. Ethanol solution and water were changed every day. Measurements of alcohol and water intake were made daily during the recording periods, as well as body weight. Voluntary alcohol intake was recorded (1) during adolescence, from pnd 28 to pnd 50; (2) during a four-day period after one week of alcohol cessation, from pnd 57 to pnd 60; and (3) during a four-day period after a second period of alcohol cessation combined with exposure to restraint immobilization stress (30 min.
Schedule of experimental design. A total of ten litters were assigned to either of these two experimental groups: exposure to a single episode of early maternal deprivation (24 h on pnd 9, MD) or not deprived (control group, Co). All animals were given free access to alcohol (20%, v/v) and water in the two-bottle choice paradigm for (1) the whole adolescent period (from pnd 28 to pnd 50), for (2) four days (pnd 57 to pnd 60) after one week of alcohol cessation, and for (3) four days (pnd 67 to pnd 70) after a second period of alcohol cessation combined with 30 min. of restraint stress under white light conditions on the last three days of alcohol cessation.
Food intake was evaluated by placing a known amount of food in each cage and measuring the remaining amount after 24 hours. This procedure was performed during the adolescent period (voluntary alcohol consumption) and after the first and second alcohol cessation periods. Body weight was registered at pnd 9, pnd 16, and pnd 22 (before weaning) and daily thereafter during the periods of voluntary alcohol consumption, that is, adolescence, and following alcohol cessation. Body weight (BW) gain (in grams) was analyzed and calculated as the difference in BW referenced to pnd 9 during the preweaning period, pnd 28 during adolescence, and pnd 50 during the two periods of alcohol cessation. Additionally, growth rate (GR) was calculated as the percentage of BW gain divided by the reference BW employed during each period: GR (%) = (BW gain/
Data were analyzed using a two-way analysis of variance (ANOVA), considering sex (male or female) and neonatal condition (Co or MD) as independent factors. Shapiro-Wilk and Levene tests were used to confirm normality and homoscedasticity of the data. Repeated measures ANOVA was employed when appropriate, that is, BW gain and growth rate. Tukey post hoc comparisons were performed only for significant interactions, as well as additional one-way ANOVAs. Significance level was set at
No significant differences in alcohol intake were found during voluntary alcohol consumption at adolescence (control males:
Voluntary alcohol intake during adolescence
Following one-week cessation, no significant differences were found on daily voluntary alcohol consumption (data not shown). However, a significant effect of the neonatal condition was found during the first 2 h of alcohol exposure (
Voluntary alcohol intake following one week of alcohol cessation (a) and following a second week of alcohol cessation combined with restraint stress (b)
Following a second period of alcohol cessation combined with restraint stress during the last three days, no significant differences were found on daily voluntary alcohol consumption (data not shown). However, a significant effect of the neonatal condition was found during the first 2 h of alcohol exposure (
During the preweaning period, a significant effect of the neonatal condition was found when analyzing BW gain (
Metabolic parameters during the preweaning period.
Males | Females | |||||||
---|---|---|---|---|---|---|---|---|
Control | MD | Control | MD | |||||
BW gain | Growth rate | BW gain | Growth rate | BW gain | Growth rate | BW gain | Growth rate | |
pnd 10 | 1.9 ± 0.1 | 9.0 ± 0.8 | −0.8 ± 0.1B | −4.3 ± 0. 3B | 1.7 ± 0.2 | 8.4 ± 1.2 | −0.9 ± 0.1B | −4.6 ± 0.4B |
pnd 16 | 15.0 ± 0.3 | 70.3 ± 4.2 | 9.0 ± 0.6B | 46.5 ± 4.8B | 13.6 ± 1.4 | 66.5 ± 8.0 | 8.8 ± 0.7B | 46.8 ± 5.7B |
pnd 22 | 37.3 ± 0.9 | 175.0 ± 9.7 | 27.9 ± 1.1B | 143.0 ± 5.4B | 34.0 ± 1.6 | 165.3 ± 10.4 | 26.7 ± 0.8B | 140.5 ± 8.3B |
Body weight gain (g) and growth rate (%) on different days during the preweaning period. Reference day was considered postnatal day, pnd, 9. Rats were exposed to a single episode of early maternal deprivation (24 h on pnd 9, MD) or not deprived (control group, Co). Data are expressed as mean ± S.E.M and were analyzed by a repeated measures two-way ANOVA,
During adolescence, when animals had free access to alcohol, BW gain (Figure
Metabolic parameters during adolescence in response to voluntary alcohol intake. (a) Body weight (BW) gain (g), (b) growth rate (%), and (c) accumulated food intake (g). Rats were exposed to a single episode of early maternal deprivation (24 h on pnd 9, MD) or not deprived (control group, Co) and exposed during adolescence, from postnatal day (pnd) 28 to pnd 50, to voluntary alcohol intake (20% v/v, in a two-bottle choice paradigm). Data are presented as mean ± S.E.M and were analyzed by a repeated measures two-way ANOVA (BW gain and growth rate) or by a two-way ANOVA (accumulated food intake).
A significant effect of sex on BW gain was found after the first (
Metabolic parameters following stressful events in young adult rats in response to voluntary alcohol intake. ((a), (c)) Growth rate (%) and ((b), (d)) accumulated food intake (g). Rats were exposed to a single episode of early maternal deprivation (24 h on pnd 9, MD) or not deprived (control group, Co) and exposed to voluntary alcohol intake (20% v/v, in a two-bottle choice paradigm) during adolescence and following one week of alcohol cessation (from pnd 57 to pnd 60, upper panels) and after a second period of alcohol cessation combined with exposure to restraint immobilization stress (30 min.
Early life stress, in the form of a single 24-hour episode of MD at pnd 9, did not affect baseline voluntary alcohol intake during adolescence in rats of both sexes. However, MD seemed to exacerbate the effects of subsequent stress exposure on alcohol intake. Animals exposed to neonatal MD, compared to control non-MD animals, showed higher levels of alcohol intake following a short period of alcohol cessation as well as after the combination of an additional period of alcohol cessation concomitant to restraint stress exposure.
Under the present experimental conditions, MD,
Early life stress has been associated with alterations in the development and establishment of several neurotransmitter circuitries involved in the stress response. As an example, MD has been reported to alter the development of the dopaminergic, serotonergic, and glutamatergic systems, known to be involved in alcohol effects and vulnerability [
In humans, epidemiological studies indicate higher rates of alcohol abuse and dependence in men compared to woman [
As in previous studies from our group, MD animals showed a significantly diminished BW gain until weaning when compared to Co animals [
Early maternal deprivation did not affect baseline voluntary alcohol consumption during adolescence but enhanced the levels of voluntary alcohol intake following exposure to stressful events later in life. Moreover, this episode of early life stress seemed to alter energy balance and metabolism since somatic growth and food intake were persistently modified in these animals. Future research is needed in order to clarify the impact of metabolic changes, such as altered leptin levels, on the susceptibility to increase alcohol intake in response to stress.
The authors have no conflict of interests.
María-Paz Viveros, Eva M. Marco, and Jose Antonio López-Moreno were responsible for the design of the experiments performed. Sara Peñasco and Eva M. Marco contributed to the acquisition of animal data. Sara Peñasco, Virginia Mela, Eva M. Marco, and María-Paz Viveros assisted with data analysis and interpretation of findings. María-Paz Viveros and Eva M. Marco drafted the paper. All authors critically reviewed content and approved final version for publication.
The authors would like to thank María del Rincón for her assistance in the acquisition of animal data. They acknowledge the Instituto de Salud “Carlos III” (FIS), RETICS, Red de Trastornos Adictivos (RD2012/0028/0021; RD12/0028/0015), GRUPOS UCM-BSCH: Reference, UCM 951579, and the European Foundation for Alcohol Research (EA 12 21) for funding.