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.

Cues associated with repeated ethanol exposure facilitate the corticosterone response to ethanol and immunological challenges in adult male Sprague Dawley rats: implications for neuroimmune regulation.

Background: We previously found a conditioned increase in central neuroinflammatory markers (Interleukin 6; IL-6) following exposure to alcohol-associated cues. Recent studies suggest (unconditioned) induction of IL-6 is entirely dependent on ethanol-induced corticosterone.Objectives: The goals of these present studies were to test whether alcohol-paired cues facilitated the hypothalamic-pituitary-adrenal (HPA) axis response to either a subthreshold priming alcohol dose or an immune or psychological stress challengeMethods: In Experiment 1 (N = 64), adult male Sprague Dawley rats were trained (paired or unpaired, four pairings total) with either vehicle or 2 g/kg alcohol [intragastric (i.g.) or intraperitoneal (i.p.)] injections. In Experiments 2 (N = 28) and 3 (N = 30), male rats were similarly trained but with 4 g/kg alcohol i.g. intubations. On test day, all rats were either administered a 0.5 g/kg alcohol dose (i.p. or i.g. Experiment 1), a 100 µg/kg i.p. lipopolysaccharide (LPS) challenge (Experiment 2), or a restraint challenge (Experiment 3), and exposed to alcohol-associated cues. Blood plasma was collected for analysis.Results: Alcohol-associated cues facilitated the plasma corticosterone response to a subthreshold dose of alcohol (F1,28 = 4.85, p < .05) and an immune challenge (F8,80 = 6.23, p < .001), but not a restraint challenge (F2,27 = 0.18, p > .05).Conclusion: These findings reveal that the impact of the cues associated with alcohol intoxication on the HPA axis may be context-specific. This work illustrates how HPA axis learning processes form in the early stages of alcohol use and has important implications for how the HPA and neuroimmune conditioning may develop in alcohol use disorder in humans and facilitate the response to a later immune challenge.

Sex-specific effects of ethanol consumption in older Fischer 344 rats on microglial dynamics and Aß(1-42) accumulation.

Chronic alcohol consumption, Alzheimer's disease (AD), and vascular dementia are all associated with cognitive decline later in life, raising questions about whether their underlying neuropathology may share some common features. Indeed, recent evidence suggests that ethanol exposure during adolescence or intermittent drinking in young adulthood increased neuropathological markers of AD, including both tau phosphorylation and beta-amyloid (Aß) accumulation. The goal of the present study was to determine whether alcohol consumption later in life, a time when microglia and other neuroimmune processes tend to become overactive, would influence microglial clearance of Aß(1-42), focusing specifically on microglia in close proximity to the neurovasculature. To do this, male and female Fischer 344 rats were exposed to a combination of voluntary and involuntary ethanol consumption from ∼10 months of age through ∼14 months of age. Immunofluorescence revealed profound sex differences in microglial co-localization, with Aß(1-42) showing that aged female rats with a history of ethanol consumption had a higher number of iba1+ cells and marginally reduced expression of Aß(1-42), suggesting greater phagocytic activity of Aß(1-42) among females after chronic ethanol consumption later in life. Interestingly, these effects were most prominent in Iba1+ cells near neurovasculature that was stained with tomato lectin. In contrast, no significant effects of ethanol consumption were observed on any markers in males. These findings are among the first reports of a sex-specific increase in microglia-mediated phagocytosis of Aß(1-42) by perivascular microglia in aged, ethanol-consuming rats, and may have important implications for understanding mechanisms of cognitive decline associated with chronic drinking.

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.

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.

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.

Neuroendocrine and neuroimmune responses in male and female rats: evidence for functional immaturity of the neuroimmune system during early adolescence.

Adolescence is a developmental period characterized by rapid behavioral and physiological changes, including enhanced vulnerability to stress. Recent studies using rodent models of adolescence have demonstrated age differences in neuroendocrine responses and blunted neuroimmune responding to pharmacological challenges. The present study was designed to test whether this neuroimmune insensitivity would generalize to a non-pharmacological stress challenge. Male and female adolescent (P29-33) and adult (P70-80) Sprague Dawley rats were exposed to intermittent footshock for one-, two-, or two-hours + recovery. Plasma corticosterone and progesterone levels as well as gene expression of several cytokines and c-Fos gene expression in the paraventricular nucleus of the hypothalamus (PVN), the medial amygdala (MeA), and the ventral hippocampus (vHPC) were analyzed. The results of the present study demonstrated differences in response to footshock, with these differences dependent on age, sex, and brain region of interest. Adult males and females demonstrated time-dependent increases in IL-1ß and IL-1R2 in the PVN, with these changes not evident in adolescent males and substantially blunted in adolescent females. TNFα expression was decreased in all regions of interest, with adults demonstrating more suppression relative to adolescents and age differences more apparent in males than in females. IL-6 expression was affected by footshock predominantly in the vHPC of adolescent and adult males and females, with females demonstrating prolonged elevation of IL-6 gene expression. In summary, central cytokine responses to acute stressor exposure are blunted in adolescent rats, with the most pronounced immaturity evident for the brain IL-1 signaling system.

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.

Assessment of neuroinflammation in the aging hippocampus using large-molecule microdialysis: Sex differences and role of purinergic receptors.

Aging is associated with an enhanced neuroinflammatory response to acute immune challenge, often termed "inflammaging." However, there are conflicting reports about whether baseline levels of inflammatory markers are elevated under ambient conditions in the aging brain, or whether such changes are observed predominantly in response to acute challenge. The present studies utilized two distinct approaches to assess inflammatory markers in young and aging Fischer 344 rats. Experiment 1 examined total tissue content of inflammatory markers from hippocampus of adult (3 month), middle-aged (12 month), and aging (18 month) male Fischer (F) 344 rats using multiplex analysis (23-plex). Though trends emerged for several cytokines, no significant differences in basal tissue content were observed across the 3 ages examined. Experiment 2 measured extracellular concentrations of inflammatory factors in the hippocampus from adult (3 month) and aging (18 month) males and females using large-molecule in vivo microdialysis. Although few significant aging-related changes were observed, robust sex differences were observed in extracellular concentrations of CCL3, CCL20, and IL-1α. Experiment 2 also evaluated the involvement of the P2X7 purinergic receptor in neuroinflammation using reverse dialysis of the selective agonist BzATP. BzATP produced an increase in IL-1α and IL-1ß release and rapidly suppressed the release of CXCL1, CCL2, CCL3, CCL20, and IL-6. Other noteworthy sex by aging trends were observed in CCL3, IL-1ß, and IL-6. Together, these findings provide important new insight into late-aging and sex differences in neuroinflammation, and their regulation by the P2X7 receptor.

Lingering Effects of Prenatal Alcohol Exposure on Basal and Ethanol-Evoked Expression of Inflammatory-Related Genes in the CNS of Adolescent and Adult Rats.

Emerging data suggest that alcohol's effects on central inflammatory factors are not uniform across the lifespan. In particular, prenatal alcohol exposure (PAE) significantly alters steady-state levels of neuroimmune factors, as well as subsequent reactivity to later immune challenge. Thus, the current experiment investigated developmental sensitivities to, and long-lasting consequences of, PAE on ethanol-evoked cytokine expression in male and female adolescent and adult rats. Pregnant dams received either an ad libitum ethanol liquid diet (2.2% GD 6-8; 4.5% GD 9-10; 6.7% GD11-20; 35% daily calories from ethanol) or free-choice access to a control liquid diet and water. At birth, offspring were fostered to dams given free-choice access to the control liquid diet. Pups then matured until mid-adolescence [postnatal day (PD) 35] or adulthood (PD90), at which time they were challenged with either a binge-like dose of ethanol (4 g/kg; intragastrically) or tap water. During intoxication (3 h post-ethanol challenge), brains and blood were collected for assessment of neuroimmune gene expression (reverse transcription-polymerase chain reaction; RT-PCR) in the hippocampus, amygdala, and PVN, as well as for blood ethanol concentrations (BEC) and plasma corticosterone levels. Results revealed that rats challenged with ethanol at either PD35 or PD90 generally exhibited a characteristic cytokine signature of acute intoxication that we have previously reported: increased Il-6 and IkBα expression, with decreased Il-1ß and Tnfα gene expression. With a few exceptions, this pattern of gene changes was observed in all three structures examined, at both ages of postnatal ethanol challenge, and in both sexes. While few significant effects of PAE were observed for ethanol-induced alterations in cytokine expression, there was a consistent (but nonsignificant) trend for PAE to potentiate the expression of Il-6 and IkBα in all groups except adult females. Although these data suggest that later-life ethanol challenge was a far greater driver of inflammatory signaling than PAE, the current results demonstrate PAE resulted in subtle long-term alterations in the expression of many key neuroinflammatory factors associated with NF-κB signaling. Such long-lasting impacts of PAE that may engender vulnerability to later environmental events triggering neuroinflammatory processes, such as chronic ethanol exposure or stress, could contribute to heightened vulnerability for PAE-related alterations and deficits.

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.

Assessment of Extracellular Cytokines in the Hippocampus of the Awake Behaving Rat Using Large-Molecule Microdialysis Combined with Multiplex Arrays After Acute and Chronic Ethanol Exposure.

BACKGROUND: Studies have demonstrated persistent changes in central nervous system (CNS) cytokine gene expression following ethanol (EtOH) exposure. However, the low endogenous expression and short half-lives of cytokines in the CNS have made cytokine protein detection challenging. The goal of these studies was to establish parameters for use of large-molecule microdialysis and sensitive multiplexing technology for the simultaneous detection of brain cytokines, corticosterone (CORT), and EtOH concentrations in the awake behaving rat. METHODS: Adult (P75+) male Sprague Dawley rats that were either naïve to EtOH (Experiment 1) or had a history of adolescent chronic intermittent EtOH (CIE; Experiment 2) were given an acute EtOH challenge during microdialysis. Experiment 1 examined brain EtOH concentrations, CORT and a panel of neuroimmune analytes, including cytokines associated with innate and adaptive immunity. The natural time course of changes in these cytokines was compared to the effects of an acute 1.5 or 3.0 g/kg intraperitoneal (i.p.) EtOH challenge. In Experiment 2, rats with a history of adolescent CIE or controls exposed to vehicle were challenged with 3.0 g/kg i.p. EtOH during microdialysis in adulthood, and a panel of cytokines was examined in parallel with brain EtOH concentrations and CORT. RESULTS: The microdialysis procedure itself induced a cytokine-specific response that replicated across studies, specifically a sequential elevation of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and IL-10. Surprisingly, acute EtOH did not significantly alter this course of cytokine fluctuations in the hippocampus. However, a history of adolescent CIE showed drastic effects on multiple neuroimmune analytes when rechallenged with EtOH as adults. Rats with a history of adolescent EtOH displayed a severely blunted neuroimmune response in adulthood, evinced by suppressed IL-1ß, IL-10, and TNF-α. CONCLUSIONS: Together, these findings provide a methodological framework for assessment of cytokine release patterns, their modulation by EtOH, and the long-lasting changes to neuroimmune reactivity evoked by a history of adolescent CIE.

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.

Late aging alters behavioral sensitivity to ethanol in a sex-specific manner in Fischer 344 rats.

Responsiveness to ethanol (EtOH) differs as a function of age. Adolescent rodents are less sensitive than adults to the sedative effects of EtOH, whereas they show enhanced sensitivity to EtOH-induced social facilitation. Late aging is associated with a natural decline in social behavior and aging-related peculiarities in sensitivity to EtOH have been largely unexplored. Whether there are sex differences in the behavioral response to EtOH during late aging remains unknown. Thus, behavioral responses to EtOH in male and female Fischer (F) 344 rats aged 4-5 months (adult) and 19-20 months (aging) were examined. First, the effects of saline and EtOH (0.5 and 0.75 g/kg) on social interaction were assessed. Social investigation and contact behavior were lower in aging animals and higher in females. Interestingly, in aged females, social contact behavior was increased following a 0.5 g/kg EtOH dose, whereas the same dose suppressed social contact in aged males. Behavioral sensitivity to the sedative effects of 3.0 and 3.5 g/kg EtOH was assessed with the loss of righting reflex (LORR) test. Although latency to LORR did not differ as a function of age or sex, aged rats showed significantly greater LORR duration and significantly lower blood ethanol concentrations (BECs) at regaining of the righting reflex relative to adults. In addition, females had a lower LORR duration, regardless of age; no sex differences were evident in BECs at awakening. In a second experiment, blood ethanol concentrations (BECs) over time were assessed following 0.75, 1.5, and 3.0 g/kg EtOH in 3-, 12-, and 18-month-old male and female F344 rats. Aged rats had higher peak BECs following 3.0 g/kg EtOH, whereas few age or sex differences were apparent at lower doses. Taken together, these data indicate that late aging is associated with altered sensitivity to the social facilitating effects and sedative effects of EtOH.

Differential effects of acute versus chronic stress on ethanol sensitivity: Evidence for interactions on both behavioral and neuroimmune outcomes.

Acute alcohol intoxication induces significant alterations in brain cytokines. Since stress challenges also profoundly impact central cytokine expression, these experiments examined the influence of acute and chronic stress on ethanol-induced brain cytokine responses. In Experiment 1, adult male rats were exposed to acute footshock. After a post-stress recovery interval of 0, 2, 4, or 24 h, rats were administered ethanol (4 g/kg; intragastric), with trunk blood and brains collected 3 h later. In non-stressed controls, acute ethanol increased expression of Il-6 and IκBα in the hippocampus. In contrast, rats exposed to footshock 24 h prior to ethanol demonstrated potentiation of hippocampal Il-6 and IκBα expression relative to ethanol-exposed non-stressed controls. Experiment 2 subsequently examined the effects of chronic stress on ethanol-related cytokine expression. Following a novel chronic escalating stress procedure, rats were intubated with ethanol. As expected, acute ethanol increased Il-6 expression in all structures examined, yet the Il-6 response was attenuated exclusively in the hippocampus in chronically stressed rats. Later experiments determined that neither acute nor chronic stress affected ethanol pharmacokinetics. When ethanol hypnosis was examined, however, rats exposed to chronic stress awoke at significantly lower blood ethanol levels compared to acutely stressed rats, despite similar durations of ethanol-induced sedation. These data indicate that chronic stress may increase sensitivity to ethanol hypnosis. Together, these experiments demonstrate an intriguing interaction between recent stress history and ethanol-induced increases in hippocampal Il-6, and may provide insight into novel pharmacotherapeutic targets for prevention and treatment of alcohol-related health outcomes based on stress susceptibility.

Adolescent Ethanol Exposure Leads to Stimulus-Specific Changes in Cytokine Reactivity and Hypothalamic-Pituitary-Adrenal Axis Sensitivity in Adulthood.

Adolescent alcohol use comprises a significant public health concern and is often characterized by binge-like consumption patterns. While ethanol exposure in adulthood has been shown to alter the stress response, including the Hypothalamic-Pituitary-Adrenal (HPA) axis, few studies have examined whether binge-like ethanol exposure during adolescence results in enduring changes in HPA axis sensitivity in adulthood. In the present studies, adolescent Sprague-Dawley rats were given intragastric (i.g.) intubations of ethanol (4 g/kg) or vehicle once per day for three consecutive days, beginning on postnatal day (P) 30 (±1). This exposure was followed by a 2-day period of rest/withdrawal. Rats received a total of either two (Experiments 1, 2 and 3) or four (Experiment 4) cycles of ethanol exposure and were subsequently allowed to age normally until adulthood. In Experiment 1, adult, (P71-75), ethanol- or vehicle-exposed rats received a 60 min restraint stress challenge. In Experiment 2, rats received a 50 µg/kg injection of lipopolysaccharide (LPS). In Experiment 3, rats received a challenge of 2.5 g/kg ethanol (intraperitoneally; i.p.). In Experiment 4, male and female ethanol- or vehicle- exposed rats received a 50 µg/kg injection of LPS. In all experiments, blood samples were collected for later assessment of corticosterone (CORT), blood ethanol concentrations (BECs), and the cellular fraction of blood was analyzed for cytokine gene expression. As expected, all three challenges led to a time-dependent surge in CORT. Gene expression analyses of cytokines (Interleukin [IL]-6, IL-1ß, and Tumor necrosis factor alpha [TNFα]) from the cellular fraction of blood revealed unique, time-dependent patterns of cytokine expression depending upon the nature of the adult challenge incurred (restraint, LPS, or EtOH). Importantly, adolescent ethanol exposure led to attenuated restraint and LPS-induced cytokine expression in males, whereas female rats displayed an absence of cytokine alterations, and a tendency toward heightened HPA axis reactivity. These findings suggest that adolescent ethanol exposure may cause lasting alterations in cytokine regulation and HPA axis sensitivity that (a) persist into adulthood; (b) may vary depending on the nature of the challenge incurred during adulthood; and that