Adolescent intermittent ethanol (AIE) sensitized fever in male Sprague Dawley rats exposed to poly I:C in adulthood.

Fever plays an indispensable role in host defense processes and is used as a rapid index of infection severity. Unfortunately, there are also substantial individual differences in fever reactions with biological sex, immunological history, and other demographic variables contributing to adverse outcomes of infection. The present series of studies were designed to test the hypothesis that a history of adolescent alcohol misuse may be a latent experiential variable that determines fever severity using polyinosinic:polycytidylic acid (poly I:C), a synthetic form of double-stranded RNA that mimics a viral challenge. Adult male and female Sprague Dawley rats were injected with 0 (saline) or 4 mg/kg poly I:C to first establish sex differences in fever sensitivity in Experiment 1 using implanted radiotelemetry devices for remote tracking. In Experiments 2 and 3, adolescent males and females were exposed to either water or ethanol (0 or 4 g/kg intragastrically, 3 days on, 2 days off, ∼P30-P50, 4 cycles/12 exposures total). After a period of abstinence, adult rats (∼P80-96) were then challenged with saline or poly I:C, and fever induction and maintenance were examined across a prolonged time course of 8 h using implanted probes. In Experiments 4 and 5, adult male and female subjects with a prior history of adolescent water or adolescent intermittent ethanol (AIE) were given saline or poly I:C, with tissue collected for protein and gene expression analysis at 5 h post-injection. Initial sex differences in fever sensitivity were minimal in response to the 4 mg/kg dose of poly I:C in ethanol-naïve rats. AIE exposed males injected with poly I:C showed a sensitized fever response as well as enhanced TLR3, IκBα, and IL-1ß expression in the nucleus of the solitary tract. Other brain regions related to thermoregulation and peripheral organs such as spleen, liver, and blood showed generalized immune responses to poly I:C, with no differences evident between AIE and water-exposed males. In contrast, AIE did not affect responsiveness to poly I:C in females. Thus, the present findings suggest that adolescent binge drinking may produce sex-specific and long-lasting effects on fever reactivity to viral infection, with preliminary evidence suggesting that these effects may be due to centrally-mediated changes in fever regulation rather than peripheral immunological mechanisms.

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.

A review on the reciprocal interactions between neuroinflammatory processes and substance use and misuse, with a focus on alcohol misuse.

Background: The last decade has witnessed a surge of findings implicating neuroinflammatory processes as pivotal players in substance use disorders. The directionality of effects began with the expectation that the neuroinflammation associated with prolonged substance misuse contributes to long-term neuropathological consequences. As the literature grew, however, it became evident that the interactions between neuroinflammatory processes and alcohol and drug intake were reciprocal and part of a pernicious cycle in which disease-relevant signaling pathways contributed to an escalation of drug intake, provoking further inflammation-signaling and thereby exacerbating the neuropathological effects of drug misuse.Objectives: The goal of this review and its associated special issue is to provide an overview of the emergent findings relevant to understanding these reciprocal interactions. The review highlights the importance of preclinical and clinical studies in testing and validation of immunotherapeutics as viable targets for curtailing substance use and misuse, with a focus on alcohol misuse.Methods: A narrative review of the literature on drug and neuroinflammation was conducted, as well as articles published in this Special Issue on Alcohol- and Drug-induced Neuroinflammation: Insights from Pre-clinical Models and Clinical Research.Results: We argue that (a) demographic variables and genetic background contribute unique sensitivity to drug-related neuroinflammation; (b) co-morbidities between substance use disorders and affect dysfunction may share common inflammation-related signatures that predict the efficacy of immunotherapeutic drugs; and (c) examination of polydrug interactions with neuroinflammation is a critical area where greater research emphasis is needed.Conclusions: This review provides an accessible and example-driven review of the relationship between drug misuse, neuroinflammatory processes, and their resultant neuropathological outcomes.

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.

TLR3 activation with poly I:C exacerbates escalated alcohol consumption in dependent male C57BL/6J mice.

Background: Activation of TLR3 receptors, which are sensitive to viral infection, has emerged as a possible mechanism that increases alcohol intake in rodents.Objectives: These studies examined whether a history of ethanol dependence exacerbated the increase in drinking driven by the TLR3 agonist poly I:C.Methods: Male C57BL/6J mice (>10 per group) were given access to ethanol (20% v/v) 2 hours a day following a history of home cage drinking or after having been rendered ethanol-dependent using a chronic intermittent ethanol (CIE) vapor model. After testing multiple doses, a 5 mg/kg repeated poly I:C challenge was used to probe the effects of repeated immune challenge, alone or in conjunction with repeated cycles of CIE, on voluntary drinking. An ethanol (12% v/v) operant self-administration model was used to test the effects of poly I:C on stress-induced reinstatement of ethanol seeking and consumption.Results: Poly I:C in naive animals resulted in transient, modest increases in ethanol intake in the home cage and in self-administration (p < 0.05). However, poly I:C challenge resulted in sensitized stress-induced ethanol consumption and evoked a strong and persistent escalation of drinking in mice with a history of dependence (p < 0.05 for both).Conclusion: Activation of viral immune defense may affect ethanol consumption in dependence and sensitivity to future stressors. As patients who suffer from alcohol use disorder are at a heightened risk for viral infection, this interaction could generate risk factors for exacerbating behaviors associated with Alcohol Use Disorders via an immune mechanism.

Prenatal and adolescent alcohol exposure programs immunity across the lifespan: CNS-mediated regulation.

For many individuals, first exposure to alcohol occurs either prenatally due to maternal drinking, or during adolescence, when alcohol consumption is most likely to be initiated. Prenatal Alcohol Exposure (PAE) and its associated Fetal Alcohol Spectrum Disorders (FASD) in humans is associated with earlier initiation of alcohol use and increased rates of Alcohol Use Disorders (AUD). Initiation of alcohol use and misuse in early adolescence correlates highly with later AUD diagnosis as well. Thus, PAE and adolescent binge drinking set the stage for long-term health consequences due to adverse effects of alcohol on subsequent immune function, effects that may persist across the lifespan. The overarching goal of this review, therefore, is to determine the extent to which early developmental exposure to alcohol produces long-lasting, and potentially life-long, changes in immunological function. Alcohol affects the whole body, yet most studies are narrowly focused on individual features of immune function, largely ignoring the systems-level interactions required for effective host defense. We therefore emphasize the crucial role of the Central Nervous System (CNS) in orchestrating host defense processes. We argue that alcohol-mediated disruption of host immunity can occur through both (a) direct action of ethanol on neuroimmune processes, that subsequently disrupt peripheral immune function (top down); and (b) indirect action of ethanol on peripheral immune organs/cells, which in turn elicit consequent changes in CNS neuroimmune function (bottom up). Recognizing that alcohol consumption across the entire body, we argue in favor of integrative, whole-organism approaches toward understanding alcohol effects on immune function, and highlight the need for more work specifically examining long-lasting effects of early developmental exposure to alcohol (prenatal and adolescent periods) on host immunity.

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.

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.

Gene expression profiling reveals a lingering effect of prenatal alcohol exposure on inflammatory-related genes during adolescence and adulthood.

Prenatal Alcohol Exposure (PAE) exerts devastating effects on the Central Nervous System (CNS), which vary as a function of both ethanol load and gestational age of exposure. A growing body of evidence suggests that alcohol exposure profoundly impacts a wide range of cytokines and other inflammation-related genes in the CNS. The olfactory system serves as a critical interface between infectious/inflammatory signals and other aspects of CNS function, and demonstrates long-lasting plasticity in response to alcohol exposure. We therefore utilized transcriptome profiling to identify gene expression patterns for immune-related gene families in the olfactory bulb of Long Evans rats. Pregnant dams received either an ad libitum liquid diet containing 35% daily calories from ethanol (ET), a pair-fed diet (PF) matched for caloric content, or free choice (FCL) access to the liquid diet and water from Gestational Day (GD) 11-20. Offspring were fostered to dams fed the FCL diet, weaned on P21, and then housed with same-sex littermates until mid-adolescence (P40) or young adulthood (P90). At the target ages of P40 or P90, offspring were euthanized via brief CO2 exposure and brains/blood were collected. Gene expression analysis was performed using a Rat Gene 1.0 ST Array (Affymetrix), and preliminary analyses focused on two moderately overlapping gene clusters, including all immune-related genes and those related to neuroinflammation. A total of 146 genes were significantly affected by prenatal Diet condition, whereas the factor of Age (P40 vs P90) revealed 998 genes significantly changed, and the interaction between Diet and Age yielded 162 significant genes. From this dataset, we applied a threshold of 1.3-fold change (30% increase or decrease in expression) for inclusion in later analyses. Findings indicated that in adolescents, few genes were altered by PAE, whereas adults displayed an increase of a wide range of gene upregulation as a result of PAE. Pathway analysis predicted an increase in Nf-κB activation in adolescence and a decrease in adulthood due to prenatal ethanol exposure, indicating age-specific and long-lasting alterations to immune signaling. These data may provide important insight into the relationship between immune-related signaling cascades and long-term changes in olfactory bulb function after PAE.

The role of oxytocin in alcohol and drug abuse.

The neuropeptide oxytocin (OXT) plays a key role in adaptive processes associated with reward, tolerance, memory and stress responses. Through interactions with brain reward and stress systems, OXT is known to play a role in several neuropsychiatric disorders, particularly those that involve altered social integration, such as alcohol and drug addiction (Heilig et al., 2016). As such, there is growing interest in the oxytocin system as a potential therapeutic target for the treatment of alcohol and substance use disorders. Accumulating preclinical evidence suggests that administration of OXT influences the development of tolerance, sensitization and withdrawal symptoms, and modulates numerous alcohol/drug-seeking and alcohol/drug-taking behaviors. Further, there is some evidence to suggest that OXT may help to reverse neuroadaptations that occur as a result of chronic alcohol or drug exposure. To date, there have been only a handful of clinical studies conducted in alcohol and drug dependent populations. This review summarizes the preclinical and clinical literature on the effects of OXT administration on alcohol- and drug-related behaviors. In addition, we discuss OXT interactions with the hypothalamic-pituitaryadrenal axis and multiple neurotransmitter systems within addiction circuitry.

Anhedonia and Increased Evoked Immune Response.

There is a growing body of evidence supporting the association between immune processes and psychopathology, including major depressive disorder (MDD). However, lack of diagnostic specificity has given rise to a search for specific symptom types, as opposed to more heterogeneous categorical diagnoses, linked to increased inflammation. One such symptom could be anhedonia, which is not only a key feature of MDD, but also a pervasive and persistent transdiagnostic symptom. To evaluate the specific role of anhedonia as well as categorical MDD diagnoses, we examined endotoxin-evoked immune responses in vitro in relation to current levels of anhedonia and history of recurrent MDD (rMDD) in a sample of adults recruited from the community. A total of 39 participants either had a history of rMDD (n = 20) or no lifetime history of any MDD episodes (n = 19). The average age of participants was 36.81 years and the majority were women (87.2%) and Caucasian (76.3%). We found that higher levels of current anhedonia, but not history of rMDD, were associated with increased lipopolysaccharide-stimulated levels of inflammatory markers even after we statistically controlled for the potential influence of participants' demographic (age, sex, ethnicity, income) and physiological (body temperature, BMI) characteristics, current symptoms of depression and anxiety, and the time of day of the sample collection. These findings highlight the relation of anhedonia specifically, rather than rMDD more generally, with inflammatory processes and identify endotoxin-stimulated cytokine production as a plausible biological marker of current anhedonia.

Rapid alterations in neuroimmune gene expression after acute ethanol: Timecourse, sex differences and sensitivity to cranial surgery.

Prior work has established that that an acute ethanol challenge mimicking high intensity alcohol consumption increased IL-6 and suppressed IL-1ß and TNFα mRNA in intoxication, with the opposite pattern seen in withdrawal. These experiments utilized Sprague-Dawley rats to further extend these results across time course (from 45 min to 6 h after ethanol), sex, and central versus peripheral expression. Furthermore, these data show that cannulation surgery may selectively modify the central neuroimmune response to ethanol. These findings highlight a unique plasticity of IL-6 that is specific to central structures and responsive to alterations by environmental factors.

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.

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

A cross-sectional comparison of ethanol-related cytokine expression in the hippocampus of young and aged Fischer 344 rats.

Our work in Sprague Dawley rats has shown rapid alterations in neuroimmune gene expression (RANGE) in the hippocampus and paraventricular nucleus of the hypothalamus (PVN). These manifest as increased interleukin (IL)-6 and IκBα, and suppressed IL-1ß and tumor necrosis factor alpha during acute ethanol intoxication. The present studies tested these effects across the lifespan (young adulthood at 2-3 months; senescence at 18 and 24 months), as well as across strain (Fischer 344) and sex. The hippocampus revealed age-dependent shifts in cytokine expression (IL-6, IL-1ß, and monocyte chemoattractant protein 1), but no changes were observed in the PVN at baseline or following ethanol. RANGE in adults was similar across sex and comparable with effects seen in Sprague Dawley rats. Plasma corticosterone levels increased with age, whereas the blood ethanol concentrations and loss of righting reflex were similar in all groups older than 2 months. These findings indicate that the RANGE effect is largely conserved across strain and is durable across age, even in the face of a shifting neuroimmune profile that emerges during immunosenescence.

Conditioned effects of ethanol on the immune system.

Several studies indicate that the immune system can be subjected to classical conditioning. Acute ethanol intoxication significantly modulates several pro-inflammatory cytokines (e.g. interleukins-1 and 6 [IL-1ß and IL-6, respectively] and tumor necrosis factor alpha [TNFα])) in several brain areas, including amygdala (AMG), paraventricular nucleus (PVN), and hippocampus (HPC). It is unknown, however, whether cues associated with ethanol can elicit conditioned alterations in cytokine expression. The present study analyzed, in male Sprague-Dawley rats, whether ethanol-induced changes in the central cytokine response may be amenable to conditioning. In Experiments 1 and 2, the rats were given one or two pairings between a distinctive odor (conditional stimulus, CS) and the post-absorptive effects of a high (3.0 or 4.0 g/kg, Experiments 1 and 2, respectively) ethanol dose. Neither of these experiments revealed conditioning of IL-6, IL-1ß, or TNFα, as measured via mRNA levels. Yet, re-exposure to the lemon-odor CS in Experiment 1 significantly increased C-Fos levels in the PVN. In Experiment 3, the rats were given four pairings between an odor CS and a moderate ethanol dose (2.0 g/kg), delivered intraperitoneally (i.p.) or intragastrically (i.g.). Re-exposure to the odor CS significantly increased IL-6 levels in HPC and AMG, an effect only evident in paired rats administered ethanol i.p. Overall, this study suggests that ethanol exposure can regulate the levels of IL-6 at HPC and AMG via classical conditioning mechanisms. These ethanol-induced, conditioned alterations in cytokine levels may ultimately affect the intake and motivational effects of ethanol. Impact statement This study examines, across three experiments, whether odor cues associated with ethanol exposure can condition changes in cytokine expression. The analysis of ethanol-induced conditioning of immune responses is a novel niche that can help understand the transition from social drinking to alcohol abuse and dependence. Ethanol-induced conditioning of the immune system could likely exacerbate neuroinflammation and drug-related toxicity, which in turn may facilitate further engagement in ethanol intake. The main new finding of the present study was that, after four pairings of ethanol's unconditioned effects and a distinctive odor, the latter CS increased IL-6 levels in HPC and AMG. This suggests that ethanol's effects upon IL-6 in HPC and AMG may come under conditioned control, particularly after repeated pairings between distinctive odor cues and ethanol's effects. This article advances our knowledge of conditioned increases in cytokine responses, which should help understand the mechanisms underlying alcohol use, abuse, and relapse.