Adolescent alcohol disrupts development of noradrenergic neurons in the nucleus of the tractus solitarius and enhances stress behaviors in adulthood in mice in a sex specific manner

Abstract

Alcohol use disorders (AUDs) are common mental health issues worldwide and can lead to other chronic diseases. Stress is a major factor in the development and continuation of AUDs, and adolescent alcohol exposure can lead to enhanced stress-responsivity and increased risk for AUD development in adulthood. The exact mechanisms behind the interaction between adolescence, stress, and alcohol are not fully understood and require further research. In this regard, the nucleus of the tractus solitarius (NTS) provides dense norepinephrine projections to the extended amygdala, providing a key pathway for stress-related alcohol behaviors. While NTS norepinephrine neurons are known to be alcohol sensitive, whether adolescent alcohol disrupts NTS-norepinephrine neuron development and if this is related to altered stress-sensitivity and alcohol preference in adulthood has not previously been examined. Here, we exposed male and female C57Bl/6J mice to the commonly used adolescent intermittent ethanol (AIE) vapor model during postnatal day 28-42 and examined AIE effects on: 1) tyrosine hydroxylase (TH) mRNA expression in the NTS across various ages (postnatal day 21-90), 2) behavioral responses to acute stress in the light/dark box test in adulthood, 3) NTS TH neuron responses to acute stress and ethanol challenges in adulthood, and 4) ethanol conditioned place preference behavior in adulthood. Overall the findings indicate that AIE alters NTS TH mRNA expression and increases anxiety-like behaviors following acute stress exposure in a sex-dependent manner. These mRNA expression and behavioral changes occur in the absence of AIE-induced changes in NTS TH neuron sensitivity to either acute stress or acute alcohol exposure or changes to ethanol conditioned place preference.

Keywords: Adolescent alcohol; Alcohol use disorder; Development; Nucleus of the tractus solitarius; Stress.

Fig. 1.. AIE alters the timeline of NTS TH mRNA expression in a sex-specific manner.

Bar graph effect of AIE on NTS TH mRNA expression across ages in male and female mice. * indicates significant effect of sex, @ indicates significant overall effect of age, and # indicates significant Age by AIE interaction effect as determined by three-way ANOVA (p<0.05).

Fig. 2.. AIE induces sex-specific changes in anxiety-like behaviors in the light-dark box.

A-D) Summary data in male mice showing effects of AIE and stress on A) time in the light side B) time in the dark side C) transitions and D) latency to first enter the dark side. Male:Air- dark blue (Air-no stress, n=9; Air-stress, n=5) Male:AIE-light blue (AIE-no stress, n=5; AIE-stress, n=5). E-F) Summary data in female mice showing effects of AIE and stress on E) time in the light side F) time in the dark side G) transitions and H) latency to first enter the dark side. Female:Air-dark red (Air-no stress, n= 13; Air-stress, n=11), Female: AIE-light red (AIE-no stress, n=5, AIE-stress n=5). Significant differences between groups as determined by Tukey’s multiple comparison tests are indicated. *=p<0.05, **=p<0.01, ***= p<0.005. For panel F, * indicates a significant main effect of AIE vs Air as determined by two-way ANOVA, p<0.05

Fig. 3.. AIE increases stress induced NTS cfos expression in a sex-specific manner.

A-left) Example 4x fluorescent images of immunohistochemistry to label of cfos (green) and TH (red) in the NTS (outlined in gray dotted lines) in female Air mice with and without stress exposure. Scale bar: 200 um, V4= fourth ventricle. Box indicates area for 40x image shown at right. A-right) Example 40x fluorescent images of immunohistochemistry to label of cfos (green) and TH (red) in the NTS in all groups. Scale bar= 20 um. B-E) Summary data of overall cfos expression, cfos/TH colocalization, and cfos expression in non-TH cells in males (B-D) and females (E-G). * indicates significant difference between female AIE-stress and male Air-no stress mice as determined by three-way ANOVA with Tukey’s post hoc analysis, p<0.05. # indicates significant main effect of stress on cfos/TH colocalization in female mice as determined by within sex two-way ANOVA, p<0.05.

Fig. 4.. Acute EtOH exposure increases cfos expression in NTS similarly in adult male and female mice with and without AIE history

A) Example 4x and 40x fluorescent images of immunohistochemistry to label of cfos (green) and TH (red) in the NTS (outlined in gray dotted lines). Scale bars: 4x=200 um, 40x= 20 um. V4= fourth ventricle. B-E) Summary bar graphs showing effects of various EtOH concentrations (0-4 g/kg) on total cfos expression and cfos/TH colocalization in male and female mice with and without AIE history. Three-way ANOVA indicates a significant overall effect of EtOH concentration (p<0.05, see, main text) with no significant effects of sex, AIE, or any interaction effects for both total cfos expression and cfos/TH colocalization.

Fig. 5.. AIE does not alter CPP acquisition.

A) Line graph showing time spent in the EtOH paired chamber before and after CPP acquisition in male and female mice with and without AIE history. * indicates significant main effect of CPP acquisition on time in the EtOH paired side between pre-test and post-test as determined by three-way ANOVA (p<0.05). B) Individual data points from the group data in A. # indicates significant main effect of sex on time in the EtOH paired side between pre-test and post-test as determined by three-way ANOVA (p<0.05). N/group: Male Air (n=6), Male AIE (n=9), Female Air (n=7), and Female AIE (n=4).