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. 2017 Nov 30;8(1):e00841.
doi: 10.1002/brb3.841. eCollection 2018 Jan.

Maternal separation affects expression of stress response genes and increases vulnerability to ethanol consumption

Taciani de Almeida Magalhães  1 , Diego Correia  1 , Luana Martins de Carvalho  1 , Samara Damasceno  1 , Ana Lúcia Brunialti Godard  1
Affiliations

Maternal separation affects expression of stress response genes and increases vulnerability to ethanol consumption

Taciani de Almeida Magalhães et al. Brain Behav. .

Abstract

Introduction: Maternal separation is an early life stress event associated with behavioral alterations and ethanol consumption. We aimed to expand the current understanding on the molecular mechanisms mediating the impact of postnatal stress on ethanol consumption.

Methods: In the first experiment (T1), some of the pups were separated from their mothers for 6 hr daily (Maternal Separation group - MS), whereas the other pups remained in the cage with their respective mothers (Control group - C). In the second experiment (T2), mice from both groups were subjected to the model of free-choice between water and sucrose solution or between water and ethanol solution. Maternal behavior was assessed at the end of T1. At the end of both T1 and T2, pups were subjected to the light/dark box behavioral test and blood corticosterone concentrations were analyzed.

Results: Our maternal separation protocol led to intense maternal care and affected weight gain of the animals. The expression of stress response genes was altered with higher levels of Crh and Pomc being observed in the hypothalamus, and higher levels of Crhr1, Crhr2, Htr2a and lower levels of Nr3c1 and Htr1a being observed in the hippocampus after T1. At the end of T2, we observed higher levels of Avp and Pomc in the hypothalamus, and higher levels of Crhr1, Crhr2, Nr3c1, Slc6a4, Bdnf and lower levels of Htr1a in the hippocampus. Additionally, maternal separation increased vulnerability to ethanol consumption during adolescence and induced changes in anxiety/stress-related behavior after T2. Furthermore, voluntary ethanol consumption attenuated stress response and modified expression of reward system genes: enhancing Drd1 and Drd2, and reducing Gabbr2 in the striatum.

Conclusion: Maternal separation induced behavioral changes and alterations in the expression of key genes involved in HPA axis and in the serotonergic and reward systems that are likely to increase vulnerability to ethanol consumption in adolescence. We demonstrated, for the first time, that ethanol consumption masked stress response by reducing the activity of the HPA axis and the serotonergic system, therefore, suggesting that adolescent mice from the MS group probably consumed ethanol for stress relieving purposes.

Keywords: HPA axis; ethanol; gene expression; maternal separation; postnatal stress; reward system.

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Figures

Figure 1
Figure 1
Diagram summarizing the experimental design. White areas represent the experimental periods (T1 and T2) defined by the postnatal days (PND). The first experiment (T1) encompassed the maternal separation protocol whereby the mothers were separated from their offspring for 6 hours daily (group MS). Animals from the control group (C) remained with their mothers. Experiment 2 (T2) consisted of the free‐choice model, in which the mice had to choose between water and 0.5% sucrose solution (C+SUC and MS+SUC) or between water and 10% ethanol solution (C+EtOH and MS+EtOH). At the end of T1 and T2 blood corticosterone levels were determined and the mice subjected to the light/dark box behavioral test. All experimental groups were composed of seven animals (n = 7)
Figure 2
Figure 2
Analysis of weight gain (g) at the end of experiment 1 (T1) and experiment 2 (T2). (a) Weight gain (g) for groups MS and control (C) during T1. (b) Weight gain (g) for groups C+SUC, MS+SUC, C+EtOH and MS+EtOH during two weeks of T2. Experimental design is described in Figure 1. All experimental groups were composed of seven animals (n = 7). Data are expressed as mean and standard error. Two‐way ANOVA and Bonferroni's post hoc test correction were used to determine statistically significant differences between the groups in (a) and (b). *p < .05 C versus MS (T1)
Figure 3
Figure 3
Maternal care behavior analysis before (a) and after (b) maternal separation (Group MS) and cage sanitization (Group C). Experimental design is described in Figure 1. Tests were conducted with two mothers per cage (n = 6 females). Data are expressed as mean and standard error. Student's t‐test was used to determine statistically significant differences between the groups in (a) and (b).*p < .05 C vs MS (T1)
Figure 4
Figure 4
Light/dark box behavioral test at the end of experiment 1 (T1) and experiment 2 (T2). Proportion of time (%) spent in the light compartment following T1 (a) and T2 (d); Latency at the end of T1 (b) and T2 (e); and total number of transitions between the light and dark compartments following T1 (c) and T2 (f). Experimental design is described in Figure 1. All experimental groups were composed of seven animals (n = 7). Data are expressed as mean and standard error. Student's t‐test was used to analyze the differences between the groups C and MS in (a), (b) and (c) and between the groups C+SUC and MS+SUC and the groups C+EtOH and MS+EtOH in (d), (e) and (f). *p < .05 C+ SUC versus MS+ SUC (T2)
Figure 5
Figure 5
Plasma corticosterone concentration (ng/ml). Baseline values and concentrations for each group at the end of experiment 1 (T1) (a) and experiment 2 (T2) (b). Experimental design is described in Figure 1. All experimental groups were composed of seven animals (n = 7). Data are expressed as mean and standard error. One‐way ANOVA and Bonferroni's post hoc test correction were used to determine statistically significant differences between the baseline values and the groups C and MS in (a) and C+SUC, MS+SUC, C+EtOH and MS+EtOH in (b). *p < .05 baseline versus C and baseline versus MS in (a); and *p < .05 baseline versus C+SUC, baseline versus MS+SUC, baseline versus C+EtOH, and baseline versus MS+EtOH in (b)
Figure 6
Figure 6
Consumption of and preference for Sucrose and Ethanol at the end of experiment 2 (T2). Consumption (g/Kg) of Sucrose (SUC) (a) and Ethanol (EtOH) (b). Preference (%) for EtOH versus water (c) or SUC versus water (d). Experimental design is described in Figure 1. All experimental groups were composed of seven animals (n = 7). Data are expressed as mean and standard error. Two‐way ANOVA and Bonferroni's post hoc test correction were used to determine statistically significant differences between the groups C+SUC and MS+SUC in (a) and between the groups C+EtOH and MS+EtOH in (b). Student's t‐test was used to determine statistically significant differences between the groups in C+SUC and MS+SUC in (c) and between the groups C+EtOH and MS+EtOH in (d). *p < .05 C+EtOH versus MS+EtOH
Figure 7
Figure 7
Relative mRNA levels in the hypothalamus (a, b and c) and hippocampus (d, e, f, g, h, i and j) at the end of experiment 1 (T1). (a) Avp: Arginine vasopressin. (b) Crh: corticotrophin‐release hormone. (c) Pomc: pro‐opiomelanocortin‐alpha. (d) Crhr1: CRH receptor 1. (e) Crhr2: CRH receptor 2. (f) Nr3c1: glucocorticoid receptor. (g) Htr1a: serotonin receptor 5HT 1A. (h) Htr2a: serotonin receptor 5HT 2A. (i) Slc6a4: serotonin transporter. (j) Bdnf: Brain‐derived neurotrophic factor. Experimental design is described in Figure 1. All experimental groups were composed of seven animals (n = 7). Data are expressed as mean and standard error. The mRNA levels are expressed as arbitrary units. Student's t‐test was used to determine statistically significant differences between the groups C and MS. *p < .05 C versus MS
Figure 8
Figure 8
Relative mRNA levels in the hypothalamus (a, b and c) and hippocampus (d, e, f, g, h, i and j) at the end of experiment 2 (T2). (a) Avp: Arginine vasopressin. (b) Crh: corticotrophin‐release hormone. (c) Pomc: pro‐opiomelanocortin‐alpha. (d) Crhr1: CRH receptor 1. (e) Crhr2: CRH receptor 2. (f) Nr3c1: glucocorticoid receptor. (g) Htr1a: serotonin receptor 5HT 1A. (h) Htr2a: serotonin receptor 5HT 2A. (i) Slc6a4: serotonin transporter. (j) Bdnf: Brain‐derived neurotrophic factor. Experimental design is described in Figure 1. All experimental groups were composed of seven animals (n = 7). Data are expressed as mean and standard error. The mRNA levels are expressed as arbitrary units. Student's t‐test was used to determine statistically significant differences between the groups C+SUC and MS+SUC and between the groups C+EtOH and MS+EtOH. *p < .05 C+SUC versus MS+SUC and C+EtOH versus MS+EtOH
Figure 9
Figure 9
Relative mRNA levels in the striatum at the end of experiment 2 (T2). (a) Dat: dopamine transporter. (b) Drd1: dopamine receptor 1. (c) Drd2: dopamine receptor 2. (d) Gabbr1: GABAB receptor subunit 1. (e) Gabbr2: GABAB receptor subunit 2. Experimental design is described in Figure 1. All experimental groups were composed of seven animals (n = 7). Data are expressed as mean and standard error. The mRNA levels are expressed as arbitrary units. Student's t‐test was used to determine statistically significant differences between the groups C+SUC and MS+SUC and between the groups C+EtOH and MS+EtOH. *p < .05 C+EtOH versus MS+EtOH
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