Effects of acute stress, alcohol administration timing, and sex on BAC in rats

BAC levels were measured in plasma samples from rats of both sexes at two different time points, after the first and after the last administration of alcohol. We conducted a three-way ANOVA with the following factors: sex (F1), stress (F2), and time (F3). As shown in Fig. 1A, the analysis revealed a significant main effect of time [F [1,40] = 56.7, p < 0.001], indicating that BAC levels were higher after the final alcohol administration compared to the first day in both sexes. Additionally, a significant interaction between sex and stress [F [1,40] = 5.46, p = 0.025] was observed, where stressed male rats exhibited lower BAC levels than non-stressed male rats, an effect not seen in female rats.

Effects of acute stress, adolescent alcohol, and sex on body weight gain in rats

Another three-way ANOVA with sex (F1), stress (F2), and alcohol (F3) as factors was conducted to evaluate the effects and interactions of these factors on body weight gain (Fig. 1B). The analysis revealed only a significant main effect of sex [F [1,40] = 527.5, p < 0.001], showing that weight gain was greater in male rats compared to female rats.

Effects of acute stress, adolescent alcohol, and sex on stress-related hormonal levels in rats

We assessed the effects of acute stress and adolescent alcohol exposure on plasma ACTH and CORT levels in male and female rats. Sex (F1), stress (F2), and alcohol (F3) were considered as factors in the statistical analysis.

Plasma ACTH levels

As shown in Fig. 1C, while there were main effects of sex and stress on ACTH levels, a significant interaction between both factors was also found [F [1,40] = 7.33, p = 0.010]. Specifically, stressed female rats exhibited higher ACTH levels compared to non-stressed female rats; an effect not observed in male rats.

Plasma CORT levels

Regarding CORT levels, the analysis revealed main effects of sex and stress, along with a significant three-way interaction involving sex, stress, and alcohol [F [1,40] = 4.62, p = 0.038] (Fig. 1D).

To better understand the interactions between stress and alcohol, we analyzed the data separately by sex using two-way ANOVAs: (a) In male rats, there was a significant main effect of stress [F [1,20] = 11.59, p = 0.003], with stressed rats displaying higher CORT levels than non-stressed rats. (b) However, a significant interaction between stress and alcohol was observed in female rats [F [1,20] = 13.34, p = 0.002]. Post hoc comparisons revealed that stressed saline-treated rats had significantly higher CORT levels compared to non-stressed saline-treated female rats (p < 0.05). Additionally, alcohol-treated rats exhibited significantly lower CORT levels compared to saline-treated female rats within the stress subgroup (p < 0.05).

Fig. 1

Effects of acute stress, adolescent alcohol, and sex on BAC, body weight gain, and plasma ACTH and CORT levels. BAC at two different time-points of the intermittent alcohol exposure (A); total body weight gain (B); plasma ACTH levels (C); and plasma CORT levels (D) in male and female rats exposed to acute stress and alcohol exposure during adolescence. Bars represent the mean ± SEM (6 rats/subgroup). Data were analyzed using three-way ANOVA. Symbols are used to represent how significant interactions occur: (*) denotes p < 0.05, comparing stressed rats to non-stressed rats in males or females; (+) denotes p < 0.05, comparing rats to non-stressed, saline-treated rats in males or females

Effects of acute stress, adolescent alcohol, and sex on mRNA expression of CRH signaling-related genes in the brains of rats

We investigated the effects of acute stress and adolescent alcohol exposure on the mRNA expression of genes related to the CRH signaling system in the amygdala and hypothalamus of male and female rats. These molecular results were also analyzed using three-way ANOVAs with sex (F1), stress (F2), and alcohol (F3) as factors (Fig. 2).

AmygdalamRNA expression of Crh

The analysis of Crh expression in the amygdala revealed a significant main effect of alcohol [F [1,40] = 20.65, p < 0.001] (Fig. 2A), with alcohol-exposed rats showing higher mRNA levels of Crh compared to saline-treated rats. No other factors or interactions were associated with changes in Crh expression in the amygdala.

mRNA expression of Crhr1

There was a significant main effect of sex on Crhr1 levels [F [1,40] = 6.82, p = 0.013], with female rats exhibiting lower Crhr1 levels than male rats. Additionally, the analysis revealed a significant interaction between stress and alcohol [F [1,40] = 4.96, p = 0.032] (Fig. 2B). Specifically, alcohol-treated rats exhibited lower Crhr1 levels compared to saline-treated rats within the non-stress subgroup, whereas no significant differences within the stress subgroup.

mRNA expression of Crhr2

For Crhr2 expression in the amygdala, there was a significant main effect of stress on mRNA levels [F [1,40] = 18.90, p < 0.001], with stressed rats displaying lower Crhr2 levels than non-stressed rats. Furthermore, a significant interaction between sex and alcohol was observed [F [1,40] = 5.52, p = 0.024] (Fig. 2C). While alcohol-treated rats had lower Crhr2 levels compared to saline-treated rats in males, alcohol exposure did not affect this receptor in female rats.

HypothalamusmRNA expression of Crh

The analysis of Crh expression in the hypothalamus revealed significant main effects of sex and alcohol, along with a significant three-way interaction including sex, stress, and alcohol [F [1,40] = 5.94, p = 0.019] (Fig. 2D). To further explore this interaction, two-way ANOVAs with stress and alcohol were conducted separately for male and female rats: (a) In male rats, there were significant main effects of stress [F [1,20] = 9.03, p = 0.007] and alcohol [F [1,20] = 9.84, p = 0.005], with stressed and alcohol-treated rats displaying higher Crh levels than non-stressed and saline-treated rats, respectively. (b) In female rats, however, a significant interaction between stress and alcohol was observed [F [1,20] = 6.07, p = 0.023]. Post hoc comparisons revealed that alcohol-treated rats had higher Crh levels compared to saline-treated rats within the non-stress subgroup (p < 0.05). Additionally, stressed alcohol-treated rats exhibited lower Crh levels than non-stressed alcohol-treated rats (p < 0.05).

mRNA expression of Crhr1

There was a significant main effect of alcohol on mRNA levels of Crhr1 and a significant interaction between sex and alcohol [F [1,40] = 19.77, p < 0.001] (Fig. 2E). Specifically, alcohol-treated female rats exhibited lower Crhr1 compared to saline-treated females, whereas this effect was not statistically significant in male rats.

mRNA expression of Crhr2

Regarding Crhr2 expression in the hypothalamus, significant two-way interactions were observed (Fig. 2F). The interaction between sex and stress [F [1,40] = 6.42, p = 0.015] revealed higher Crhr2 levels in stressed female rats compared to non-stressed female rats, primarily among alcohol-treated females; however, no significant differences were observed in male rats. As for the interaction between stress and alcohol [F [1,40] = 9.55, p = 0.004], alcohol-treated rats exhibited significantly lower Crhr2 levels than saline-treated rats within the non-stress subgroup, but not within the stress subgroup.

Fig. 2

Effects of acute stress, adolescent alcohol, and sex on CRH signaling-related genes in the amygdala and hypothalamus. Crh expression in the amygdala (A); Crhr1 expression in the amygdala (B); Crhr2 expression in the amygdala (C); Crh expression in the hypothalamus (D); Crhr1 expression in the hypothalamus (E); and Crhr2 expression in the hypothalamus (F) of male and female rats exposed to acute stress and alcohol exposure during adolescence. Bars represent the mean ± SEM (6 rats/subgroup). Data were analyzed using three-way ANOVA. Symbols are used to represent how significant interactions occur: (*) denotes p < 0.05, comparing stressed rats to non-stressed rats in males or females; (**) denotes p < 0.05, comparing alcohol-treated rats to saline-treated rats in males or females; (***) denotes p < 0.05, comparing alcohol-treated rats to saline-treated rats in the stress or non-stress subgroup; (+) denotes p < 0.05, comparing rats to non-stressed, saline-treated rats in males or females; (+++) denotes p < 0.05, comparing rats to non-stressed, alcohol-treated rats in males or females

Effects of acute stress, adolescent alcohol, and sex on mRNA expression of NPY signaling-related genes in the brains of rats

The mRNA expression of genes related to the NPY signaling system were also analyzed using three-way ANOVAs with sex (F1), stress (F2), and alcohol (F3) as factors (Fig. 3).

AmygdalamRNA expression of Npy

Statistical analysis of Npy expression in the amygdala revealed significant two-way interactions (Fig. 3A). The interaction between sex and stress [F [1,40] = 16.24, p < 0.001] showed lower Npy levels in stressed male rats compared to non-stressed male rats; in contrast, stressed female rats exhibited higher Npy levels compared to non-stressed female rats. The interaction between sex and alcohol [F [1,40] = 16.20, p < 0.001] revealed lower Npy levels in alcohol-treated male rats compared to saline-treated male rats, whereas, alcohol-treated female rats had significantly higher Npy levels compared to saline-treated female rats. Regarding the interaction between stress and alcohol [F [1,40] = 7.93, p = 0.008], alcohol-treated rats exhibited lower Npy levels than saline-treated rats within the non-stress subgroup, while alcohol-treated rats showed higher Npy levels compared to their saline-treated counterparts within the stress subgroup. Taken together, these interactions together indicated the highest Npy levels in non-stressed, saline-treated male rats and stressed, alcohol-treated female rats.

mRNA expression of Npy1r

The analysis of Npy1r expression revealed a main effect of sex, along with notably significant two-way interactions between sex and other factors (Fig. 3B). The interaction between sex and stress [F [1,40] = 5.08, p = 0.030] showed lower Npy1r levels in stressed male rats compared to non-stressed male rats; however, no differences were observed in female rats. Similarly, the interaction between sex and alcohol [F [1,40] = 16.91, p < 0.001] revealed lower Npy1r levels in alcohol-treated male rats compared to saline-treated male rats, while no differences were observed in female rats.

mRNA expression of Npy2r

Regarding Npy2r in the amygdala, the three-way ANOVA revealed main effects of sex and stress, along with significant two-way interactions (Fig. 3C). The interaction between sex and stress [F [1,40] = 7.60, p = 0.009] showed lower Npy2r levels in stressed male rats compared to non-stressed male rats; however, no differences were observed in female rats. The interaction between sex and alcohol [F [1,40] = 5.86, p = 0.020] indicated that alcohol-treated male rats exhibited lower Npy2r levels compared to saline-treated male counterparts but no differences were observed in female rats. For the interaction between stress and alcohol [F [1,40] = 36.09, p < 0.001], alcohol-treated rats exhibited lower Npy2r levels compared to saline-treated rats within the non-stress subgroup, while alcohol-treated rats showed higher Npy1r levels than their saline-treated rats within the stress subgroup.

HypothalamusmRNA expression of Npy

The analysis of Npy expression in the hypothalamus revealed significant main effects of all three factors along with a significant three-way interaction [F [1,40] = 12.52, p = 0.001] (Fig. 3D). Two-way ANOVAs with stress and alcohol as factors were conducted separately by sex: (a) In male rats, a significant interaction between stress and alcohol was observed [F [1,20] = 9.89, p = 0.005]. Post hoc comparisons revealed higher Npy levels in alcohol-treated rats compared to saline-treated rats in the stress subgroup (p < 0.05), and lower Npy levels in stressed alcohol-treated rats compared to non-stressed alcohol-treated rats (p < 0.05). (b) In female rats, the analysis revealed significant main effects of stress [F [1,20] = 8.02, p = 0.010] and alcohol [F [1,20] = 23.64, p < 0.001], with stressed rats and alcohol-treated rats displaying lower Npy levels than non-stressed rats and saline-treated rats, respectively.

mRNA expression of Npy1r

Although there were significant main effects of stress and alcohol on Npy1r levels, a significant three-way interaction between sex, stress, and alcohol was observed [F [1,40] = 8.88, p = 0.005] (Fig. 3E). To further investigate, two-way ANOVAs with stress and alcohol were conducted separately in male and female rats: (a) In male rats, a significant main effect of alcohol was found [F [1,20] = 9.59, p = 0.006], with alcohol-treated rats displaying lower Npy1r levels compared to saline-treated rats. (b) In female rats, the analysis revealed a significant interaction between stress and alcohol [F [1,20] = 11.48, p = 0.003]. Post hoc comparisons showed lower Npy1r levels in alcohol-treated rats compared to saline-treated rats within both the non-stress (p < 0.05) and stress (p < 0.05) subgroups, as well as lower Npy1r levels in stressed rats compared to non-stressed rats (p < 0.05) within both the saline (p < 0.05) and alcohol (p < 0.05) subgroups.

mRNA expression of Npy2r

In this case, statistical analysis revealed a significant main effect of alcohol on Npy2r levels and a significant three-way interaction [F [1,40] = 23.85, p < 0.001] (Fig. 3F). Two-way ANOVAs with stress and alcohol as factors were conducted separately by sex: (a) In male rats, a significant interaction between stress and alcohol was found [F [1,20] = 10.16, p = 0.005]. Post hoc comparisons revealed lower Npy2r levels in alcohol-treated rats compared to saline-treated rats within the stress subgroup (p < 0.05), and higher Npy2r levels in stressed saline-treated rats compared to non-stressed saline-treated rats (p < 0.05). (b) In female rats, the analysis also revealed a significant interaction between stress and alcohol [F [1,20] = 15.18, p < 0.001]. Post hoc comparisons revealed lower Npy2r levels in alcohol-treated rats compared to saline-treated rats within the non-stress subgroup (p < 0.05), and higher Npy2r levels in stressed saline-treated rats compared to non-stressed saline-treated rats (p < 0.05).

Fig. 3

Effects of acute stress, adolescent alcohol, and sex on NPY signaling-related genes in the amygdala and hypothalamus. Npy expression in the amygdala (A); Npy1r expression in the amygdala (B); Npy2r expression in the amygdala (C); Npy expression in the hypothalamus (D); Npy1r expression in the hypothalamus (E); and Npy2r expression in the hypothalamus (F) of male and female rats exposed to acute stress and alcohol exposure during adolescence. Bars represent the mean ± SEM (6 rats/subgroup). Data were analyzed using three-way ANOVA. Symbols are used to represent how significant interactions occur: (*) denotes p < 0.05, comparing stressed rats to non-stressed rats in males or females; (**) denotes p < 0.05, comparing alcohol-treated rats to saline-treated rats in males or females; (***) denotes p < 0.05, comparing alcohol-treated rats to saline-treated rats in the stress or non-stress subgroup; (+) denotes p < 0.05, comparing rats to non-stressed, saline-treated rats in males or females; (++) denotes p < 0.05, comparing rats to stressed, saline-treated rats in males or females; (+++) denotes p < 0.05, comparing rats to non-stressed, alcohol-treated rats in males or females

Effects of acute stress, adolescent alcohol, and sex on mRNA expression of corticoid signaling-related genes in the brains of rats

Given that both POMC and corticoid receptors are key components in regulating the HPA axis, the mRNA expression of Pomc, Nr3c1, and Nr3c2 in the amygdala and hypothalamus were analyzed using three-way ANOVAs with sex (F1), stress (F2), and alcohol (F3) as factors (Fig. 4).

AmygdalamRNA expression of Pomc

The analysis of Pomc expression in the amygdala revealed significant main effects of sex [F [1,40] = 5.32, p = 0.026] and alcohol [F [1,40] = 30.71, p < 0.001], indicating higher Pomc levels in male rats compared to female rats, and lower Pomc levels in alcohol-treated rats compared to saline-treated rats (Fig. 4A).

mRNA expression of Nr3c1

The gene expression of the glucocorticoid receptor (Nr3c1) was significantly affected by all factors, with a significant three-way interaction [F [1,40] = 14.49, p < 0.001] (Fig. 4B). Two-way ANOVAs with stress and alcohol as factors were conducted separately in male and female rats: (a) In male rats, a significant interaction between stress and alcohol was observed [F [1,20] = 16.27, p < 0.001]. Post hoc comparisons revealed lower Nr3c1 levels in alcohol-treated rats compared to saline-treated rats within the non-stress subgroup (p < 0.05), and lower Nr3c1 levels in stressed saline-treated rats compared to non-stressed saline-treated rats (p < 0.05). (b) In female rats, the analysis revealed significant main effects of stress [F [1,20] = 38.35, p < 0.001] and alcohol [F [1,20] = 118.30, p < 0.001], with lower Nr3c1 levels observed in stressed rats compared to non-stressed rats, and in alcohol-treated rats compared to saline-treated rats.

mRNA expression of Nr3c2

Similar to Nr3c1, the analysis of Nr3c2 mRNA expression revealed main effects of all factors, along with a significant three-way interaction [F [1,40] = 13.38, p < 0.001] (Fig. 4C). Two-way ANOVAs with stress and alcohol as factors revealed sex differences: (a) In male rats, a significant interaction between stress and alcohol was observed [F [1,20] = 12.02, p = 0.002]. Post hoc comparisons revealed lower Nr3c2 levels in alcohol-treated rats compared to saline-treated rats within the non-stress subgroup (p < 0.05), and in stressed saline-treated rats compared to non-stressed saline-treated rats (p < 0.05). (b) In female rats, the analysis revealed significant a main effect of alcohol [F [1,20] = 85.45, p < 0.001], with lower Nr3c2 levels observed in alcohol-treated rats compared to saline-treated rats.

HypothalamusmRNA expression of Pomc

Statistical analysis of Pomc expression revealed a significant main effect of stress [F [1,40] = 7.95, p = 0.007], with stressed rats showing lower Pomc levels than non-stresses rats (Fig. 4D). Additionally, a significant interaction between sex and alcohol was observed [F [1,40] = 5.81, p = 0.021]. Specifically, alcohol-treated female rats exhibited lower Pomc levels compared to alcohol-treated rats in females, while no differences were observed in male rats.

mRNA expression of Nr3c1

The analysis of Nr3c1 expression in the hypothalamus revealed a significant main effect only of alcohol [F [1,40] = 7.18, p = 0.011] (Fig. 4E), indicating significantly lower Nr3c1 levels in alcohol-treated rats compared to saline-treated rats.

mRNA expression of Nr3c2

Regarding the mineralocorticoid receptor, an interaction between stress and alcohol was observed in Nr3c2 expression [F [1,40] = 24.45, p < 0.001] (Fig. 4F). Alcohol-treated rats had significantly higher Nr3c2 levels than saline-treated rats within the non-stress subgroup; in contrast, alcohol-treated rats exhibited lower Nr3c2 levels compared to saline-treated rats within the stress subgroup.

Fig. 4

Effects of acute stress, adolescent alcohol, and sex on corticoid signaling-related genes in the amygdala and hypothalamus. Pomc expression in the amygdala (A); Nr3c1 expression in the amygdala (B); Nr3c2 expression in the amygdala (C); Pomc expression in the hypothalamus (D); Nr3c1 expression in the hypothalamus (E); and Nr3c2 expression in the hypothalamus (F) of male and female rats exposed to acute stress and alcohol exposure during adolescence. Bars represent the mean ± SEM (6 rats/subgroup). Data were analyzed using three-way ANOVA. Symbols are used to represent how significant interactions occur: (*) denotes p < 0.05, comparing stressed rats to non-stressed rats in males or females; (**) denotes p < 0.05, comparing alcohol-treated rats to saline-treated rats in males or females; (***) denotes p < 0.05, comparing alcohol-treated rats to saline-treated rats in the stress or non-stress subgroup; (+) denotes p < 0.05, comparing rats to non-stressed, saline-treated rats in males or females

Effects of acute stress, adolescent alcohol, and sex on mRNA expression of opioid receptor genes in the brains of rats

The mRNA expression of key components of the opioid system was analyzed using three-way ANOVAs with sex (F1), stress (F2), and alcohol (F3) as factors (Fig. 5).

AmygdalamRNA expression of Oprm1

The analysis of Oprm1 expression in the amygdala revealed significant main effects of sex and alcohol, along with a significant three-way interaction [F [1,40] = 5.02, p = 0.031] (Fig. 5A). Two-way ANOVAs with stress and alcohol as factors were conducted in male and female rats to describe the three-way interaction: (a) In male rats, a significant interaction between stress and alcohol was observed [F [1,20] = 21.14, p < 0.001]. Post hoc comparisons showed lower Oprm1 levels in alcohol-treated rats compared to saline-treated rats within the non-stress subgroup (p < 0.05), and lower Oprm1 levels in stressed saline-treated rats compared to non-stressed saline-treated rats (p < 0.05). (b) In female rats, statistical analysis revealed no significant effects or interactions between stress and alcohol on Nr3c2 levels.

mRNA expression of Oprk1

Although significant main effects of sex, stress, and alcohol on Oprk1 expression were observed, a significant three-way interaction was also found [F [1,40] = 10.24, p = 0.003] (Fig. 5B). To further explore this interaction, two-way ANOVAs with stress and alcohol as factors were conducted separately by sex on Oprk1 levels: (a) In male rats, a significant interaction between stress and alcohol was observed [F [1,20] = 18.19, p < 0.001]. Post hoc comparisons revealed lower Oprk1 levels in alcohol-treated rats compared to saline-treated rats within the non-stress subgroup (p < 0.05), and lower Oprk1 levels in stressed saline-treated rats compared to non-stressed saline-treated rats (p < 0.05). (b) In female rats, statistical analysis revealed no significant effects or interactions between stress and alcohol on Oprk1 levels, similar to what was observed with Oprm1.

mRNA expression of Oprd1

Significant main effects of stress and alcohol on Oprd1 expression were observed; however, a significant interaction between the two factors was also found [F [1,40] = 7.65, p = 0.009] (Fig. 5C). Specifically, alcohol-treated rats had significantly lower Oprd1 levels than saline-treated rats within the non-stress subgroup, whereas no differences were found within the stress subgroup.

HypothalamusmRNA expression of Oprm1

Statistical analysis of Oprm1 expression in the hypothalamus revealed significant main effects of stress and alcohol, along with two-way interactions involving sex, stress, and alcohol (Fig. 5D). The interaction between sex and stress [F [1,40] = 6.45, p = 0.015] revealed lower Oprm1 levels in stressed female rats compared to non-stressed female rats; however, no differences were observed in male rats. The interaction between sex and alcohol [F [1,40] = 15.94, p < 0.001] showed significantly lower Oprm1 levels in alcohol-treated female rats compared to saline-treated female rats, with no differences observed in male rats. Finally, the interaction between stress and alcohol [F [1,40] = 6.72, p = 0.013] revealed that alcohol-treated rats exhibited lower Oprm1 levels than saline-treated rats within the non-stress subgroup, while this decrease did not reach significance within the stress subgroup.

mRNA expression of Oprk1

In addition to a significant main effect of alcohol on Oprk1 expression, a significant interaction between stress and alcohol was observed [F [1,40] = 9.02, p = 0.005] (Fig. 5E). Specifically, the interaction revealed higher Oprk1 levels in alcohol-treated rats compared to saline-treated rats within the stress subgroup, while no differences were observed within the non-stress subgroup.

mRNA expression of Oprd1

The analysis of Oprd1 expression revealed significant main effects of stress and alcohol, along with two-way interactions involving all factors (Fig. 5F). The interaction between sex and stress [F [1,40] = 5.68, p = 0.022] showed lower Oprd1 levels in stressed female rats compared to non-stressed female rats, with no differences in male rats. A second significant interaction between sex and alcohol [F [1,40] = 8.05, p = 0.007] showed significantly lower Oprd1 levels in alcohol-treated female rats compared to saline-treated female rats, while these lower Oprd1 levels did not reach significance in male rats.

Fig. 5

Effects of acute stress, adolescent alcohol, and sex on opioid receptor genes in the amygdala and hypothalamus. Oprm1 expression in the amygdala (A); Oprk1 expression in the amygdala (B); Oprd1 expression in the amygdala (C); Oprm1 expression in the hypothalamus (D); Oprk1 expression in the hypothalamus (E); and Oprd1 expression in the hypothalamus (F) of male and female rats exposed to acute stress and alcohol exposure during adolescence. Bars represent the mean ± SEM (6 rats/subgroup). Data were analyzed using three-way ANOVA. Symbols are used to represent how significant interactions occur: (*) denotes p < 0.05, comparing stressed rats to non-stressed rats in males or females; (**) denotes p < 0.05, comparing alcohol-treated rats to saline-treated rats in males or females; (***) denotes p < 0.05, comparing alcohol-treated rats to saline-treated rats in the stress or non-stress subgroup; (+) denotes p < 0.05, comparing rats to non-stressed, saline-treated rats in males or females

Effects of acute stress, adolescent alcohol, and sex on mRNA expression of opioid signaling-related genes in the brains of rats

The mRNA expression of other receptors associated with the opioid system was also analyzed using three-way ANOVAs.

AmygdalamRNA expression of Oprs1

Statistical analysis of Oprs1 expression revealed significant main effects of all factors, along with a significant three-way interaction [F [1,40] = 6.82, p = 0.013] (Fig. 6A). Once again, two-way ANOVAs with stress and alcohol as factors were conducted separately in male and female: (a) In male rats, a significant interaction between stress and alcohol was observed [F [1,20] = 11.24, p = 0.003]. Post hoc comparisons showed lower Oprs1 levels in alcohol-treated rats compared to saline-treated rats within the non-stress subgroup (p < 0.05), and lower Oprs1 levels in stressed saline-treated rats compared to their non-stressed saline-treated counterparts (p < 0.05). (b) In female rats, statistical analysis revealed no significant effects or interactions between stress and alcohol.

mRNA expression of Oprl1

The analysis of Oprl1 expression in the amygdala showed significant main effects of all factors, along with a significant three-way interaction [F [1,40] = 18.84, p < 0.001] (Fig. 6B). Two-way ANOVAs with stress and alcohol as factors in male and female rats revealed the following: (a) In male rats, a significant interaction between stress and alcohol was observed [F [1,20] = 41.05, p < 0.001]. Post hoc comparisons revealed lower Oprl1 levels in alcohol-treated rats compared to saline-treated rats within the non-stress subgroup (p < 0.05), and lower Oprl1 levels in stressed saline-treated rats compared to non-stressed saline-treated rats (p < 0.05). (b) In female rats, statistical analysis revealed a significant main effect of alcohol on Oprl1 levels [F [1,20] = 9.69, p = 0.006], with significantly lower Oprl1 levels in alcohol-treated rats compared to saline-treated rats.

HypothalamusmRNA expression of Oprs1

The analysis of Oprs1 expression showed a significant main effect of alcohol [F [1,40] = 43.97, p < 0.001], with alcohol-treated rats displaying higher Oprs1 levels than saline-treated rats (Fig. 6C).

mRNA expression of Oprl1

Regarding Oprl1 expression in the hypothalamus, statistical analysis revealed a significant main effect of sex and significant two-way interactions between sex and other factors (Fig. 6D). The interaction between sex and stress [F [1,40] = 6.50, p = 0.015] showed lower Oprl1 levels in stressed female rats compared to non-stressed female rats, with no differences in male rats. Another significant interaction between sex and alcohol [F [1,40] = 6.08, p = 0.018] revealed significantly higher Oprl1 levels in alcohol-treated male rats compared to saline-treated male rats, while no differences were observed in female rats.

Fig. 6

Effects of acute stress, adolescent alcohol, and sex on opioid signaling-related genes and vasopressin system genes in the amygdala and hypothalamus. Oprs1 expression in the amygdala (A); Oprl1 expression in the amygdala (B); Oprs1 expression in the hypothalamus (C); Oprl1 expression in the hypothalamus (D); Avp expression in the amygdala (E); Avpr1 expression in the amygdala (F); Avp expression in the hypothalamus (G); and Avpr1 expression in the hypothalamus (F) of male and female rats exposed to acute stress and alcohol exposure during adolescence. Bars represent the mean ± SEM (6 rats/subgroup). Data were analyzed using three-way ANOVA. Symbols are used to represent how significant interactions occur: (*) denotes p < 0.05, comparing stressed rats to non-stressed rats in males or females; (**) denotes p < 0.05, comparing alcohol-treated rats to saline-treated rats in males or females; (+) denotes p < 0.05, comparing rats to non-stressed, saline-treated rats in males or females; (++) denotes p < 0.05, comparing rats to stressed, saline-treated rats in males or females; (+++) denotes p < 0.05, comparing rats to non-stressed, alcohol-treated rats in males or females

Effects of acute stress, adolescent alcohol, and sex on mRNA expression of vasopressin system genes in the brains of rats

The mRNA expression of Avp and Avpr1a was analyzed using three-way ANOVAs with sex (F1), stress (F2), and alcohol (F3) as factors.

AmygdalamRNA expression of Avp

Statistical analysis of Avp expression in the amygdala showed significant main effects of stress and alcohol, along with a significant three-way interaction [F [1,40] = 27.10, p < 0.001] (Fig. 6E). To further explore the interaction, two-way ANOVAs with stress and alcohol as factors were conducted separately by sex: (a) In male rats, a significant interaction between stress and alcohol was observed [F [1,20] = 22.44, p < 0.001]. Post hoc comparisons revealed higher Avp levels in alcohol-treated rats compared to saline-treated rats within the non-stress subgroup (p < 0.05), and higher Avp levels in stressed saline-treated rats compared to non-stressed saline-treated rats (p < 0.05). (b) In female rats, a significant interaction between stress and alcohol was also observed [F [1,20] = 10.14, p = 0.005]. Post hoc comparisons revealed higher Avp levels in alcohol-treated rats compared to saline-treated rats within the stress subgroup (p < 0.05), and higher Avp levels in stressed alcohol-treated rats compared to non-stressed alcohol-treated rats (p < 0.05).

mRNA expression of Avpr1a

The analysis of Avpr1a expression revealed significant main effects of sex and stress, along with a significant three-way interaction [F [1,40] = 22.75, p < 0.001] (Fig. 6F). Two-way ANOVAs with stress and alcohol as factors was conducted in male and female rats: (a) In male rats, a significant interaction between stress and alcohol was observed [F [1,20] = 24.45, p < 0.001]. Post hoc comparisons revealed lower Avpr1 levels in alcohol-treated rats compared to saline-treated rats within the non-stress subgroup (p < 0.05), and lower Avpr1a levels in stressed saline-treated rats compared to non-stressed saline-treated rats (p < 0.05). (b) In female rats, statistical analysis revealed a significant main effect of stress on Avpr1a levels [F [1,20] = 4.81, p = 0.040], with significantly higher Avpr1a levels in stressed rats compared to non-stressed rats.

HypothalamusmRNA expression of Avp

The analysis of Avp expression revealed a significant three-way interaction [F [1,40] = 6.55, p = 0.014] (Fig. 6G). Two-way ANOVAs with stress and alcohol as factors were conducted separately by sex: (a) In male rats, statistical analysis showed no significant effects or interactions between stress and alcohol. (b) In female rats, a significant main effect of alcohol was observed [F [1,20] = 6.80, p = 0.017], with alcohol-treated rats exhibiting higher Avp levels than saline-treated rats.

mRNA expression of Avpr1a

Regarding Avpr1a expression in the hypothalamus, the three-way ANOVA revealed no significant main effects or interactions of the factors (Fig. 6H).