Adolescent intermittent ethanol (AIE) produces lasting, sex-specific changes in rat body fat independent of changes in white blood cell composition.

Early initiation of alcohol use during adolescence, and adolescent binge drinking are risk factors for the development of alcohol use disorder later in life. Adolescence is a time of rapid sex-dependent neural, physiological, and behavioral changes as well as a period of heightened vulnerability to many effects of alcohol. The goal of the present studies was to determine age-related changes in blood (leukocyte populations) and body composition across adolescence and early adulthood, and to investigate whether adolescent intermittent ethanol (AIE) exposure would alter the trajectory of adolescent development on these broad physiological parameters. We observed significant ontogenetic changes in leukocyte populations that were mirrored by an age-related increase in cytokine expression among mixed populations of circulating leukocytes. Despite these developmental changes, AIE did not significantly alter overall leukocyte numbers or cytokine gene expression. However, AIE led to sex-specific changes in body fat mass and fat percentage, with AIE-exposed male rats showing significantly decreased fat levels and female rats showing significantly increased fat levels relative to controls. These changes suggest that while AIE may not alter overall leukocyte levels, more complex phenotypic changes in leukocyte populations could underlie previously reported differences in cytokine expression. Coupled with long-term shifts in adipocyte levels, this could have long-lasting effects on innate immunity and the capacity of individuals to respond to later immunological and physiological threats.

Cues associated with a single ethanol exposure elicit conditioned corticosterone responses in adolescent male but not female Sprague-Dawley rats.

It has been shown that ethanol-induced interleukin-6 (IL-6) in adult male Sprague-Dawley rats was sensitized by environmental stimuli paired with ethanol and was accompanied by a conditioned increase in corticosterone (CORT). Adolescent males showed ethanol-induced IL-6 conditioning more readily than adults. The present studies examined whether female adolescents display IL-6 conditioning and whether adolescents of either sex show CORT conditioning. Male and female (N = 212, n = 6-10) adolescent (postnatal day 33-40) rats were given ethanol (2 g/kg intraperitoneal injection; the unconditioned stimulus), either paired with a lavender-scented novel context (the conditioned stimulus) or explicitly unpaired from context. Rats were tested in the context without ethanol and brains/blood were collected. Adolescent females did not show signs of neuroimmune (Experiment 1) or CORT conditioning (Experiments 2-4). Paired males showed enhanced CORT to the scented context relative to unpaired counterparts when the interoceptive cue of a saline injection was used on test day (Experiment 2). Experiment 5 used a delayed conditioning procedure and showed that male paired adolescents showed significantly higher CORT in response to context, showing that classically conditioned CORT response was precipitated by environmental cues alone. These findings indicate that adolescent males may be predisposed to form conditioned associations between alcohol and environmental cues, contributing to adolescent vulnerability to long-lasting ethanol effects.

Acute Ethanol Challenge Differentially Regulates Expression of Growth Factors and miRNA Expression Profile of Whole Tissue of the Dorsal Hippocampus.

Acute ethanol exposure produces rapid alterations in neuroimmune gene expression that are both time- and cytokine-dependent. Interestingly, adolescent rats, who often consume binge-like quantities of alcohol, displayed reduced neuroimmune responses to acute ethanol challenge. However, it is not known whether growth factors, a related group of signaling factors, respond to ethanol similarly in adults and adolescents. Therefore, Experiment 1 aimed to assess the growth factor response to ethanol in both adolescents and adults. To test this, adolescent (P29-P34) and adult (P70-P80) Sprague Dawley rats of both sexes were injected with either ethanol (3.5 g/kg) or saline, and brains were harvested 3 h post-injection for assessment of growth factor, cytokine, or miRNA expression. As expected, acute ethanol challenge significantly increased IL-6 and IκBα expression in the hippocampus and amygdala, replicating our prior findings. Acute ethanol significantly decreased BDNF and increased FGF2 regardless of age condition. PDGF was unresponsive to ethanol, but showed heightened expression among adolescent males. Because recent work has focused on the PDE4 inhibitor ibudilast for treatment in alcohol use disorder, Experiment 2 tested whether ibudilast would alter ethanol-evoked gene expression changes in cytokines and growth factors in the CNS. Ibudilast (9.0 mg/kg s.c.) administration 1 h prior to ethanol had no effect on ethanol-induced changes in cytokine or growth factor changes in the hippocampus or amygdala. To further explore molecular alterations evoked by acute ethanol challenge in the adult rat hippocampus, Experiment 3 tested whether acute ethanol would change the miRNA expression profile of the dorsal hippocampus using RNASeq, which revealed a rapid suppression of 12 miRNA species 3 h after acute ethanol challenge. Of the miRNA affected by ethanol, the majority were related to inflammation or cell survival and proliferation factors, including FGF2, MAPK, NFκB, and VEGF. Overall, these findings suggest that ethanol-induced, rapid alterations in neuroimmune gene expression were (i) muted among adolescents; (ii) independent of PDE4 signaling; and (iii) accompanied by changes in several growth factors (increased FGF2, decreased BDNF). In addition, ethanol decreased expression of multiple miRNA species, suggesting a dynamic molecular profile of changes in the hippocampus within a few short hours after acute ethanol challenge. Together, these findings may provide important insight into the molecular consequences of heavy drinking in humans.

Adolescent intermittent ethanol exposure produces Sex-Specific changes in BBB Permeability: A potential role for VEGFA.

Binge drinking that typically begins during adolescence can have long-lasting neurobehavioral consequences, including alterations in the central and peripheral immune systems. Central and peripheral inflammation disrupts blood-brain barrier (BBB) integrity and exacerbates pathology in diseases commonly associated with disturbed BBB function. Thus, the goal of the present studies was to determine long-lasting effects of adolescent intermittent ethanol (AIE) on BBB integrity. For AIE, male and female Sprague Dawley rats were repeatedly exposed to ethanol (4 g/kg, intragastrically) or water during adolescence between postnatal day (P) 30 and P50. In adulthood (∼P75), rats were challenged with fluorescein isothiocyanate (FITC)-tagged Dextran of varying molecular weights (4, 20, & 70 kDa) for assessment of BBB permeability using gross tissue fluorometry (Experiment 1). Experiment 2 extended these effects using immunofluorescence, adding an adult ethanol-exposed group to test for a specific developmental vulnerability. Finally, as a first test of hypothesized mechanism, Experiment 3 examined the effect of AIE on Vascular Endothelial Growth Factor A (VEGFA) and its co-localization with pericytes (identified through expression of platelet derived growth factor receptor beta (PDGFRß), a key regulatory cell embedded within the BBB. Male, but not female, rats with a history of AIE showed significantly increased dextran permeability in the nucleus accumbens (NAc), cingulate prefrontal cortex (cPFC), and amygdala (AMG). Similar increases in dextran were observed in the hippocampus (HPC) and ventral tegmental area (VTA) of male rats with a history of AIE or equivalent ethanol exposure during adulthood. No changes in BBB permeability were evident in females. When VEGFa expression was examined, male rats exposed to AIE were challenged with 3.5 g/kg ethanol (i.p.) or vehicle acutely in adulthood to assess long-lasting versus acute actions of ethanol. Adult rats with a history of AIE showed significantly fewer total cells expressing VEGFa in the AMG and dHPC following the acute ethanol challenge in adulthood. They also showed a significant reduction in the number of PDGFRß positive cells that also expressed VEGFa signal. The anatomical distribution of these effects corresponded with increased BBB permeability after AIE (i.e., differential effects in the PVN, AMG, and dHPC). These studies demonstrated sex-specific effects of AIE, with males, but not females, demonstrating long-term increases in BBB permeability that correlated with changes in VEGFa and PDGFRß protein, two factors known to influence BBB permeability.

Circulating corticosterone levels mediate the relationship between acute ethanol intoxication and markers of NF-κB activation in male rats.

Binge drinking is a harmful pattern of alcohol use that is associated with a number of serious health problems. Of particular interest are the rapid alterations in neuroimmune gene expression and the concurrent activation of the hypothalamic-pituitary-adrenal (HPA) axis activation associated with high intensity drinking. Using a rat model of acute binge-like ethanol exposure, the present studies were designed to assess the role of corticosterone (CORT) in ethanol-induced neuroimmune gene expression changes, particularly those associated with the NFκB signaling pathway, including rapid induction of IL-6 and IκBα, and suppression of IL-1ß and TNFα gene expression evident after administration of moderate to high doses of ethanol (1.5-3.5 g/kg ip) during intoxication (3 h post-injection). Experiment 1 tested whether inhibition of CORT synthesis with metyrapone and aminoglutethimide (100 mg/kg each, sc) would block ethanol-induced changes in neuroimmune gene expression. Results indicated that rapid alterations in IκBα, IL-1ß, and TNFα expression were completely blocked by pretreatment with the glucocorticoid synthesis inhibitors, an effect that was reinstated by co-administration of exogenous CORT (3.75 mg/kg) in Experiment 2. Experiment 3 assessed whether these rapid alterations in neuroimmune gene expression would be evident when rats were challenged with a subthreshold dose of ethanol (1.5 g/kg) in combination with 2.5 mg/kg CORT, which showed limited evidence for additive effects of low-dose CORT combined with a moderate dose of ethanol. Acute inhibition of mineralocorticoid (spironolactone) or glucocorticoid (mifepristone) receptors, alone (Experiment 4) or combined (Experiment 5) had no effect on ethanol-induced changes in neuroimmune gene expression, presumably due to poor CNS penetrance of these drugs. Finally, Experiments 6 and 7 showed that dexamethasone (subcutaneous; a GR agonist) recapitulated effects of ethanol. Overall, we conclude that ethanol-induced CORT synthesis and release is responsible for suppression of IL-1ß, TNFα, and induction of IκBα in the hippocampus through GR signaling. Interventions designed to curb these changes may reduce drinking, and subdue detrimental neuroimmune activation induced by ethanol.

Male, but not female, Sprague Dawley rats display enhanced fear learning following acute ethanol withdrawal (hangover).

The present studies investigated the effects of withdrawal from a single binge-like dose of ethanol (hangover) on fear conditioning in male and female Sprague Dawley rats. In Experiment 1, males and females were given 0 or 3.5 g/kg ethanol intraperitoneally (i.p.) and then conditioned to contextual fear 24 h post injection. Withdrawal from acute ethanol enhanced expression of the conditioned freezing response in males, but not in females. Experiment 2 demonstrated that in males, withdrawal from acute ethanol administered 24 h prior to conditioning enhanced contextual fear conditioning, but not auditory-cued fear conditioning. In Experiment 3, male and female rats were given 3.5 g/kg ethanol, and blood ethanol concentrations (BECs) were assessed at various time points for determination of ethanol clearance. Female rats cleared ethanol at a higher rate than males, with 10 h required for females and 14 for males to eliminate ethanol from their systems. Because females cleared ethanol faster than males, in Experiment 4, females were conditioned 18 h after ethanol administration to keep the interval between ethanol clearance and fear conditioning similar to that of males. Withdrawal from acute ethanol given 18 h prior to conditioning did not affect both contextual and auditory-cued fear conditioning in females. In summary, these results highlight sex differences in the impact of withdrawal from acute ethanol (hangover) on fear learning; suggesting that males are more sensitive to hangover-associated enhancement of negative affect than females.

Adolescent intermittent ethanol (AIE) produces sex specific alterations in adult neuroimmune gene expression and ethanol sensitivity that are independent of ethanol metabolism.

The goal of the present studies was to determine long-lasting effects of adolescent intermittent ethanol (AIE), a rodent model of binge patterns of ethanol consumption, on (i) behavioral sensitivity to ethanol challenge in adulthood using the Loss of Righting Reflex (LORR) test; (ii) ethanol pharmacokinetics and ethanol-metabolizing enzyme expression when re-challenged with ethanol as adults; and (iii) induction of neuroimmune gene expression during an adult binge-like ethanol challenge. To evaluate the impact of AIE on ethanol sensitivity in adulthood, adult rats received a sedative ethanol dose of 3.5 g/kg and were tested for the LORR. Sexually dimorphic effects were observed, with AIE males showing more rapid recovery than vehicle exposed controls, an effect that was completely absent in females. Rats exposed to the same AIE procedure were challenged with 0.75, 1.5, or 3.0 g/kg i.p. ethanol in adulthood. Female rats with a history of AIE displayed a small increase in ethanol clearance rate when challenged with 0.75 g/kg, however no other significant differences in ethanol pharmacokinetics were noted. To assess persistent AIE-associated changes in neuroimmune gene expression, rats were challenged with 0 or 2.5 g/kg ethanol. Both male and female adult rats with a history of AIE displayed sensitized hippocampal IL-6 and IκBα gene expression in response to ethanol challenge. Changes in cytokine gene expression as well as ethanol sensitivity assessed by LORR were not shown to be the result of changes in ethanol pharmacokinetics and point to AIE altering other mechanisms capable of significantly altering the neuroimmune and behavioral response to ethanol.

Conditioning the neuroimmune response to ethanol using taste and environmental cues in adolescent and adult rats.

IMPACT STATEMENT: A combined odor and taste cue was paired with a binge-like ethanol exposure (4 g/kg intraperitoneal) using a single-trial learning paradigm. Re-exposure to the CS alone was sufficient to evoke a conditioned Interleukin (IL)-6 elevation in the amygdala in adolescents, an effect that was not observed in young adults. This demonstrates a particular sensitivity of adolescents to alcohol-associated cues and neuroimmune learning, whereas prior work indicated that adults require multiple pairings of ethanol to the CS in order to achieve a conditioned amygdala IL-6 response. While the role of immune conditioning has been studied in other drugs of abuse, these findings highlight a previously unknown aspect of alcohol-related learning. Given the emergent importance of the neuroimmune system in alcohol abuse, these findings may be important for understanding cue-induced reinstatement of alcohol intake among problem drinkers.

The influence of central interleukin-6 on behavioral changes associated with acute alcohol intoxication in adult male rats.

Recent studies have demonstrated brain cytokine fluctuations associated with acute ethanol intoxication (increased IL-6) and withdrawal (increased IL-1ß and TNFα). The purpose of the present studies was to examine the potential functional role of increased central interleukin-6 (IL-6). We utilized two tests of ethanol sensitivity to establish a potential role for IL-6 after high (3.5-4.0 g/kg, intraperitoneally [i.p.]) or moderate (2.0 g/kg, i.p.) doses of ethanol: loss of righting reflex (LORR) and conditioned taste aversion (CTA), respectively. Briefly, guide cannulae were implanted into the third ventricle of adult male Sprague-Dawley rats. In the first experiments, rats were infused with 25, 50, 100, or 200 ng of IL-6; or 0.3, 3.0, or 9.0 µg of the JAK/STAT inhibitor AG490 30 min prior to a high-dose ethanol challenge. Although sleep time was not affected by exogenous IL-6, infusion of AG490 increased latency to lose the righting reflex relative to vehicle-infused rats. Next, we assessed whether IL-6 was sufficient to produce a CTA. Moderately water-deprived rats received intracerebroventricular (i.c.v.) infusions of 25, 50, or 100 ng IL-6 immediately after 60-min access to 5% sucrose solution. Forty-eight hours later, rats were returned to the context and given 60-min access to sucrose solution. IL-6 infusion had no significant effect on sucrose intake when all rats were considered together. However, a median split revealed that low sucrose-consuming rats significantly increased their drinking on test day, an effect that was not seen in rats that received 50 or 100 ng of IL-6. In the last study, AG490 had no effect on ethanol-induced CTA (2 g/kg). Overall, these studies suggest that IL-6 had only a minor influence on ethanol-induced behavioral changes, yet phenotypic differences in sensitivity to IL-6 were apparent. These studies are among the first to examine a potential functional role for IL-6 in ethanol-related behaviors, and may have important implications for understanding the relationship between acute ethanol intoxication and its associated behavioral alterations.