Determining the neuronal ensembles underlying sex-specific social impairments following adolescent intermittent ethanol exposure

Abstract

Binge drinking during adolescence can have behavioral and neurobiological consequences. We have previously found that adolescent intermittent ethanol (AIE) exposure produces sex-specific social alterations indexed via decreases of social investigation and/or social preference in rats. The prelimbic cortex (PrL) regulates social interaction, and alterations within the PrL resulting from AIE may contribute to social alterations. The current study sought to determine whether AIE-induced PrL dysfunction underlies decreases in social interaction evident in adulthood. We first examined social interaction-induced neuronal activation of the PrL and several other regions of interest (ROIs) implicated in social interaction. Adolescent male and female cFos-LacZ rats were exposed to water (control) or ethanol (4 g/kg, 25% v/v) via intragastric gavage every other day between postnatal day (P) 25 and 45 (total 11 exposures). Since cFos-LacZ rats express β-galactosidase (β-gal) as a proxy for Fos, activated cells that express of β-gal can be inactivated by Daun02. In most ROIs, expression of β-gal was elevated in socially tested adult rats relative to home cage controls, regardless of sex. However, decreased social interaction-induced β-gal expression in AIE-exposed rats relative to controls was evident only in the PrL of males. A separate cohort underwent PrL cannulation surgery in adulthood and was subjected to Daun02-induced inactivation. Inactivation of PrL ensembles previously activated by social interaction reduced social investigation in control males, with no changes evident in AIE-exposed males or females. These findings highlight the role of the PrL in male social investigation and suggest an AIE-associated dysfunction of the PrL that may contribute to reduced social investigation following adolescent ethanol exposure.

Keywords: Adolescent intermittent ethanol; Neuronal ensemble; Prelimbic cortex; Sex differences; Social interaction.

Figure 1.

The cFos-LacZ transgenic rat model, adolescent intermittent ethanol (AIE) exposure, and social testing in adulthood. (A) cFos-LacZ rats have a LacZ gene on a c-Fos promoter and express β-galactosidase (β-gal) in recently activated neurons. The inert prodrug Daun02 is converted into daunorubicin by β-gal and leads to genetic interference in these cells ultimately inactivating them. (B) Representative image of β-gal labeling (indigo blue) in coronal slice. (C) Timeline of Experiment 1: AIE or water exposure and behavioral testing in adulthood. (D) Timeline of Experiment 2: AIE or water exposure followed by cannulation surgery and social testing in adulthood. Scale bar, 20μm.

Figure 2.. AIE-induced changes of social responding and neuronal activation in the PrL.

Social investigation, social preference, and neuronal activation of the PrL indexed via β-gal+ cell counts in water- and AIE-exposed male (A, B, C, respectively) and female (D, E, F, respectively) rats taken from home cage, placed into the context, or exposed to a social partner. Significant differences between water and AIE exposure conditions are marked with asterisks (* p < 0.05 and *** p < 0.001); significant changes relative to the home cage condition are marked with number signs (# p < 0.001), significant changes relative to the context condition are marked with percent signs (% p < 0.001).

Figure 3.. AIE-induced changes in neuronal activation of IL, NAcSh, and CeA.

Neuronal activation of the IL, NAcSh, and CeA indexed via β-gal+ cell counts in water- and AIE-exposed male (A, B, C, respectively) and female (D, E, F, respectively) subjects taken from home, placed into the context, or exposed to a social partner. Significant differences between water and AIE exposure conditions are marked with asterisks (* p < 0.05); significant changes relative to the home cage condition are marked with number signs (# p < 0.05), significant changes relative to the context condition are marked with percent signs (% p < 0.001).

Figure 4.

Daun02 effects on social behavior and locomotor activity. Social investigation, social preference/avoidance, and locomotor activity of water- and AIE-exposed male (A, B, C, respectively) and female (D, E, F, respectively) rats. Significant differences between water and AIE exposure conditions are marked with asterisks (** p = 0.01) AIE vs water-exposed controls; significant differences between vehicle and Daun02 are marked with number signs (# p < 0.05).

Figure 5.

Daun02 effects on neuronal activation of the PrL indexed via β-gal expression. Representative image of PrL cannula placement (A) scale bar 1mm. Example images from the PrL of vehicle- (B) and Daun02-injected (C) subjects, scale bar 20μm. β-gal+ cell counts in the PrL of males (D) and females (E). Significant differences between vehicle and Daun02 are marked with number signs (# p < 0.05, ## p < 0.01).

Figure 6.

Neuronal activation of subcortical brain regions following Daun02 injection in the PrL. Neuronal activation indexed via β-gal expression of NAcC, NAcSh, and CeA in water- and AIE-exposed males (A, B, C, respectively) and females (D, E, F, respectively). Significant differences between vehicle and Daun02 are marked with number signs (# p < 0.05, ## p < 0.01).