Adolescent Intermittent Ethanol Drives Modest Neuroinflammation but Does Not Escalate Drinking in Male Rats

Abstract

During adolescence, the brain is highly susceptible to alcohol-induced damage and subsequent neuroimmune responses, effects which may enhance development of an alcohol use disorder (AUD). Neuroimmune reactions are implicated in adolescent alcohol exposure escalating adulthood drinking. Therefore, we investigated whether intermittent alcohol exposure in male, adolescent rats (AIE) escalated adult drinking via two-bottle choice (2BC). We also examined the influence of housing environment across three groups: standard (group-housed with enrichment during 2BC), impoverished (group-housed without enrichment during 2BC), or isolation (single-housed without bedding or enrichment throughout). In the standard group immediately after AIE/saline and after 2BC, we also examined the expression of microglial marker, Iba1, reactive astrocyte marker, vimentin, and neuronal cell death dye, FluoroJade B (FJB). We did not observe an escalation of adulthood drinking following AIE, regardless of housing condition. Further, only a modest neuroimmune response occurred after AIE in the standard group: no significant microglial reactivity or neuronal cell death was apparent using this model, although some astrocyte reactivity was detected in adolescence following AIE that resolved by adulthood. These data suggest that the lack of neuroimmune response in adolescence in this model may underlie the lack of escalation of alcohol drinking, which could not be modified through isolation stress.

Keywords: adolescence; astrocytes; ethanol; microglia; social isolation; stress.

Figure 1

Timeline of experiments. Rats in the standard group were housed normally with environmental enrichment (EE), impoverished rats were housed without EE but otherwise normally, and rats in the isolation group were singly housed without EE, bedding, or visual access to other cages. All rats were singly housed during two-bottle choice (2BC). AIE = adolescent intermittent ethanol, AIS = adolescent intermittent saline, ADE = alcohol deprivation effect testing, IP group = examined in adolescence after AIE/AIS injections, IP+2BC group = examined in adulthood after AIS/AIE injections and 2BC. Image created with BioRender.com.

Figure 2

Body weight and blood ethanol concentration (BEC). (A) All rats gained weight across adolescence during adolescent intermittent ethanol exposure (AIE). (B) In adulthood, body weight was significantly higher in the isolation group compared to the standard or impoverished groups. (C) BEC was slightly lower in the isolation group compared to impoverished. * p < 0.05, ** p < 0.01.

Figure 3

Two-bottle choice (2BC) drinking in adulthood. AIE did not influence ethanol intake or preference in the standard groups (A,B), the impoverished groups (C,D), or the isolation groups (E,F). Ethanol intake (G) and preference (H) were similar across experiments, shown here collapsed across AIE/AIS groups. Rats in the impoverished group demonstrated increased alcohol consumption following a period of deprivation, the “alcohol deprivation effect”, at the first day post-abstinence (PAD1), but intake returned to baseline by PAD2 (I). n = 7–8/group ** p < 0.01.

Figure 4

FluoroJade B (FJB) staining and quantification in the IP group (PND 44) and the IP+2BC group (PND 111). Representative images of FJB staining in the entorhinal cortex (A–D). A few FJB+ cells were detected in the peri-/entorhinal cortex of a few rats (arrow in (B)) as quantified in (E), but the difference between groups was not significant. n = 7–8/group.

Figure 5

Microglia in the hippocampus (A–D) and entorhinal cortex (E–H). Iba1 immunoreactivity was higher in the IP groups compared to the IP+2BC groups for both regions (I,J). In the hippocampus, microglia soma size was decreased in the IP+2BC group compared to IP, for AIE rats (K). Soma size in the entorhinal cortex did not differ between groups (L). n = 5–8/group, * p < 0.05, **** p < 0.0001.

Figure 6

Vimentin immunoreactivity. Approximate location of images taken in the forceps minor of the corpus callosum (CC_fm, (A)). Image adapted from [76]. Representative images of vimentin immunoreactivity in the CC_fm (B–E) and the hippocampus (F–I). In the CC_fm, AIE rats had a 33 ± 0.02% increase in astrocyte reactivity following i.p. injection that resolved even after 2BC drinking (J). When quantifying vimentin+ cells in the hippocampus, there were fewer cells counted in AIE rats regardless of treatment condition, but this effect was not statistically significant (K). Fluorescent double-labelled images verifying that vimentin+ immunoreactivity (red) co-localized with astrocyte marker, GFAP (green) (L) but not with microglia marker Iba1 (green) (M) in the hippocampus. n = 7–8/group, * p < 0.05.