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. Author manuscript; available in PMC: 2020 Sep 1.
Published in final edited form as: Alcohol. 2018 Nov 22;79:37–45. doi: 10.1016/j.alcohol.2018.11.004

The influence of central Interleukin-6 on Behavioral Changes Associated with Acute Alcohol Intoxication in adult male rats

Thaddeus M Barney 1, Andrew S Vore 1, Anny Gano 1, Jamie E Mondello 1, Terrence Deak 1,*
PMCID: PMC6531375  NIHMSID: NIHMS1514239  PMID: 30472309

Abstract

Recent studies have demonstrated brain cytokine fluctuations associated with acute ethanol intoxication (increased IL-6) and withdrawal (increased IL-1β 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 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 cannula 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 i.c.v. infusions of 25, 50, or 100 ng IL-6 immediately after 60 min access to 5% sucrose solution. 48 hr 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.

Keywords: intracerebroventricular, cytokine, conditioned taste aversion, loss of righting reflex, inflammation, neuroimmune, interleukin-6

Introduction

Alcohol consumption poses a significant public health concern and economic burden to society. In the United States alone, the Center for Disease Control estimated that 88,000 deaths and 32,400 cases of liver disease per year were due to excessive alcohol use (CDC, 2013). Sacks (2010) estimated that alcohol use represents a $200 billion burden on the country annually. Increased incidence of liver disease, brain damage, and other chronic disease states associated with alcohol consumption are often tied to chronic patterns of alcohol use and dependence. However, negative consequences are also prevalent after short-term, binge-like consumption of alcohol. The National Institute on Alcohol Abuse and Alcoholism (NIAAA) defines binge drinking as consumption of alcoholic beverages that results in blood ethanol concentrations (BECs) of greater than 80 mg/dL, approximately 4–5 standard drinks in a two-hr period. These levels of acute intoxication have been linked to myriad cognitive impairments and negative health consequences. Among these are impairments in motor coordination (Chuck, McLaughlin, Arizzi-LaFrance, Salamone, & Correa, 2006), motivated lever pressing (Chuck et al., 2006), spatial learning (Gawal, 2016; West et al., 2018) and altered fear conditioning (Broadwater & Spear, 2014; Gould, 2003; Tipps, Raybuck, Buck, & Lattal, 2015).

Recently, studies of binge-like ethanol effects have highlighted the importance of the immune system (both peripherally and centrally) as an important mechanism contributing to long-lasting effects of binge ethanol (Vore, Doremus-Fitzwater, Gano, & Deak, 2017). Most commonly known for their role in orchestration of peripheral immune responses, cytokines such as the interleukins (IL) in the CNS are potently modulated by acute ethanol and may contribute to the consequences of binge-like ethanol exposure. For example, it has been shown that blocking IL-1 signaling specifically in the basolateral amygdala reduced voluntary ethanol consumption (Marshall et al., 2016). In particular, research has outlined a role for IL-1 in the sedative effects of ethanol. For example, knockout (KO) of the type 1 IL-1 receptor (IL-1R) reduced the loss of righting reflex (LORR) time. KO of IL-1 receptor antagonist (an endogenous ligand that binds to IL-1R type 1 without activating signal transduction; Granowitz, E. V.; Vannier, E.; Poutsiaka, D. D.; Dinarello, 1992) produced opposite effects, indicating that interleukin-1 (IL–1) signaling during intoxication affected the LORR of mice (Blednov, Benavidez, Black, Mayfield, & Harris, 2015). These results demonstrate a role for IL-1 signaling in modulating sensitivity to the sedative properties of ethanol. Studies have shown that IL-1β mRNA levels were reduced during intoxication and increased predominantly during withdrawal from ethanol, suggesting the role of cytokines in ethanol-mediated behavior is complex and phase-specific. Consistent with this, cytokines such as IL-6 were more consistently elevated during intoxication as opposed to withdrawal (Doremus-Fitzwater et al., 2014; Doremus-Fitzwater, Gano, Paniccia, & Deak, 2015; Gano, Doremus-Fitzwater, & Deak, 2017). Some studies have illuminated possible IL-6/ethanol interactions during withdrawal from ethanol such as a neuroadaptive effect of IL-6 overexpression as measured by electroencephalography (EEG) (Gruol, Huitron-Resendiz, & Roberts, 2018). Yet during intoxication the effects of IL-6 on ethanol-induced behaviors such as LORR and conditioned taste aversion (CTA) have not been examined. Therefore, we investigated the potential influence of central IL-6 in behavioral changes associated with acute ethanol intoxication (LORR and CTA).

In the central nervous system, IL-6 signaling modulates a variety of stress-related and sickness-like behaviors. For example, IL-6 immunoneutralization via intracerebroventricular (i.c.v.) infusion exacerbated impairments in reversal learning produced by chronic cold stress exposure (Donegan, Girotti, Weinberg, & Morilak, 2014), and IL-6 infused i.c.v. rescued deficits caused by trimethyltin injection in IL-6 KO mice in passive avoidance and Y-maze tasks (Kim et al., 2013). Additionally, in rats given lipopolysaccharide (LPS) blocking IL-6 signaling via i.c.v. infusion of the Janus kinase (JAK) inhibitor AG490 produced higher fevers but less adipsia and locomotor impairment in rats (Damm, Harden, Gerstberger, Roth, & Rummel, 2013). Studies have also outlined a role for IL-6 in fear conditioning, indicating that by blocking IL-6 signaling in aged mice given (LPS), LPS-induced fear conditioning deficits were reversed (Burton & Johnson, 2012). Furthermore, IL-6 infusion into the basolateral amygdala (BLA) 4 hr prior to fear conditioning (acquisition) impaired fear learning. This deficit was reversed by co-administration of the JAK/signal transducer and activator of transcription (STAT) inhibitor JSI-124 (Hao et al., 2014). Thus, IL-6 participates in a variety of centrally-mediated alterations in behavior and has the potential to play an important role in ethanol-related behavioral changes when given either icv or within discrete CNS sites.

Many of these effects are likely to involve IL-6 action at its cognate receptor and downstream activation of JAK/STAT signaling (Brasier, 2010; Lacroix et al., 2015). In the canonical signaling pathway, IL-6 interacts with a high affinity, membrane-bound glycoprotein 130 (gp130) coupled IL-6 receptor (IL-6R). When bound, the IL-6/IL-6R /gp130 trimer homodimerizes and binds JAK2. JAK2 is autophosphorylated, which causes phosphorylation of a tyrosine residue on gp130, revealing a STAT3 binding site. Once STAT3 binds, it is phosphorylated at the Y705 tyrosine site and the S727 serine site (Levy & Lee, 2002). It then forms a homo- or hetero-dimer, and translocates to the nucleus, where it can regulate the transcription of a wide array of genes. In addition, IL-6 can signal through a secondary pathway referred to as trans-signaling. The primary mechanistic difference between the canonical and trans-signaling pathways is that in the trans pathway, the IL-6R is soluble and not bound to the membrane, and only binds to the gp130 once IL-6 has become associated with the IL-6R. IL-6R levels bound to neurons is relatively low (Burton & Johnson, 2012; Lacroix et al., 2015), and it is therefore important to recognize that much of the IL-6 signaling in the brain may be through the trans-signaling pathway (Burton & Johnson, 2012). Although these two pathways may activate different signal transducers, they both rely on JAK/STAT signaling (Brasier, 2010; Lacroix, 2018), making JAK/STAT signaling an effective functional target for inhibiting both canonical and trans-signaling pathways for IL-6.

With this in mind, the present series of studies was designed to test the potential functional role that IL-6 may play in the coordination of ethanol-induced behaviors typically associated with acute intoxication (LORR, CTA) (Figure 1). Specifically, the effects of i.c.v IL-6 administration on LORR (Exp 1–2), and CTA (Exp 4) were assessed. Since no specific antagonists to the IL-6 receptor are commercially available, the broad spectrum JAK/STAT inhibitor AG490 was used as a plausible for test to investigate the potential effect of IL-6 signaling on ethanol-induced LORR (Exp 3) or CTA (Exp 5). We utilized male rats since our prior work has shown that both males and females evince robust ethanol-induced IL-6 responses (Gano et al., 2017), though we recognize the importance of testing mechanisms in both sexes. These studies are among the first to test the potential functional role of IL-6 in acute ethanol-related changes in behavior.

Figure 1:

Figure 1:

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Schematic representation of the design of the studies. Cannulation coordinates are shown (top) along with an experimental timeline for each study (below). Experimental timelines are shaded dark grey if testing high dose ethanol, and CTA study timelines are shaded light grey.

Materials and Methods

General Methods

Subjects.

Adult, male Sprague-Dawley rats were purchased from Envigo (200–250 grams) and acclimated to the colony for a minimum of 1 week. Rats were pair-housed in standard Plexiglas® bins and given free access to water and food. Each cage was provided with enrichment in the form of a wooden chew-block. The colony rooms were on a 12:12 hr light:dark cycle (lights on at 0700) and kept at approximately 22°C. In all experiments, rats were maintained and treated in accordance with Public Health Service policy, and with protocols approved by the Institutional Animal Care and Use Committee at Binghamton University.

Intracerebroventricular (i.c.v.) Surgery.

Rats were maintained under anesthesia using isoflurane (2–5% in oxygen) throughout the cannulation procedure. The rat’s head was shaved and a midline incision was made across the top of the skull. After cleaning the periosteum, a 1 mm hole was drilled and a 22 gauge stainless steel guide cannula (Plastics One, Roanoke, VA) was lowered into the third ventricle targeting the following coordinates (Paxinos & Watson, 2007) AP: −1.0-mm, ML: ± 0.0-mm, DV: −6.0-mm (see Figure 1 for a schematic). The guide cannula was fixed in place with dental cement (Henry Schein, Melville, NY) and three screws. Patency was maintained with a 22 gauge stylette (Plastics One, Roanoke, VA). Rats were injected subcutaneously with 0.03 mg/kg of buprenorphine prior to, 12 hr, and 24 hr post-surgery to mitigate pain.

Verification of cannula placement.

After completion of experimental protocols (described below), we utilized two procedures to verify placement of cannula within the 3rd ventricle. A positive behavioral response to angiotensin challenge, defined as a rat consuming at least 8 ml of water in 30 min immediately following infusion of 2.5 μg angiotensin, qualified the animal for inclusion in the final analysis (Arakawa, 2009; Richey, Doremus-Fitzwater, Buck, & Deak, 2012). Rats that failed to display a dipsogenic response to angiotensin were rapidly decapitated, the brain removed and frozen in chilled methyl-butane and stored at −80 °C. Brains were then sliced at 20 microns using a Leica Cryostat at −20 °C and stained with cresyl violet (2%) for cannula verification via microscopy. For experiments 3–5, secondary verification was performed with Evans blue infusion. Rats were infused i.c.v using the same procedure described above with 2.5 μl of 5% Evans blue dye, brains were harvested and flash-frozen. Cannula were considered correctly placed if the lateral ventricles were stained blue.

Drugs.

On each day of injection, a fresh stock of ethanol was diluted from 95% to 20% (v/v) in pyrogen free physiological saline (Sigma Aldrich, S8776). Ethanol was injected intraperitoneally (i.p.). Recombinant rat IL-6 (rrIL-6; R&D Systems, 506-RL/CF) carrier-free protein was diluted initially in pyrogen-free physiological saline and stored at −20 °C until the day of testing, when a stock was then diluted to achieve the doses described in individual experiments below. Dose response functions for IL-6 were chosen based on effective doses and using a log-base 2 dose response curve. For instance, 100 ng of IL-6 produced HPA activation and increased in body temperature, but not social behavior changes (Lenczowski et al., 1999). Doses in a range of 10 ng to 400 ng have been shown to affect neuronal activity such as splenic nerve discharge (Helwig, Craig, Fels, Blecha, & Kenney, 2008), whereas a dose as low as 6 ng recovered memory deficits in a passive avoidance task induced by trimethyltin injection in IL-6 KO mice (Kim et al., 2013). The JAK/STAT inhibitor AG490 (Tocris, 0414) was diluted initially to a concentration of 30 mg/ml in dimethyl sulfoxide (DMSO), and further diluted with artificial cerebrospinal fluid (aCSF) to working concentrations on the day of testing. Final doses of AG490 (0.3, 3.0, 9.0 µg) were chosen because they represented a broad range of the effective doses in published studies; approximately 0.3 µg was sufficient to block leptin-induced reductions in food consumption when AG490 was infused into the VTA before a leptin microinfusion (Morton, Blevins, Kim, Matsen, & Figlewicz, 2009), whereas 1 or 5 µg infusions into the spinal cord increased the mechanical pain threshold in rats previously exposed to chronic constriction injury (Wang et al., 2014). A dose of 3 µg reduced the STAT phosphorylation induced by LPS injection 4 hr after infusion (Damm et al., 2013). The highest dose (9 µg) was chosen to ensure we captured the high end of the dose response curve. Artificial cerebrospinal fluid (aCSF) was prepared (127.7 mM NaCl, 4.02 mM KCl, 0.65mM NaH2PO4 (monobasic), 2.04 mM NaH2PO4 (dibasic), 1.0 mM Dextrose, 1.29 mM CaCl2(2*H2O), 0.93 mM MgCl2(6*H2O); pH = 7.4 ± 0.1) and used within a week. Angiotensin (Sigma Aldrich, A9525) was diluted to 1 μg/μl with sterile physiological saline and stored at −20 °C until testing. Angiotensin was used solely for the verification of cannula placement in the third ventricle, since ICV angiotensin elicits a rapid dipsogenic response. rrIL-6 protein, AG490, and angiotensin were each administered i.c.v in a volume of 2.5–2.65 μl over the course of one minute using a 10 μl Hamilton syringe connected to PE10 tubing (Plastics One, Roanoke, VA) and a microinjector that extended 1.0 mm beyond the tip of the cannula. The Hamilton syringe was operated by a syringe pump. Following administration, the microinjector remained undisturbed in the cannula for one minute to allow for diffusion of the drug.

Loss of righting reflex (LORR) test.

Immediately after injection of ethanol, rats were placed in a fresh cage with bedding spread to form a trough. Experimenters flipped rats onto their backs and placed them in these troughs every 5 sec until the rat did not immediately right itself. Upon observing that the rat failed to regain the righting reflex for one whole minute, this time was recorded and the time between injection (T0) and loss of righting reflex (LORR) defined the latency to lose the righting reflex. If at any point within that minute the rodent regained righting reflex, this time was not used and the procedure was repeated. After observing LORR, rats were left undisturbed and an observer monitored the rats’ behavior. When a rat was observed righting itself, it was returned to its back, and if it could right itself a second time within one minute of being returned to its back, the rat was considered awake. The time between the loss and the regaining of the righting reflex constitutes sleep time.

Blood collection and Blood Ethanol Concentrations (BEC) determinations.

To measure BECs at awakening, a tail blood sample was collected in 0.65 ml microfuge tubes upon awakening. To do this, rats were briefly placed into a Plexiglas restraint tube, the tip of the tail was transected, and the tail was gently massaged to collect blood into the tube. Blood was centrifuged within 20 min after collection at 4 °C for 15 min (3220 g) and serum was pipetted from the fractionated blood into a new tube and frozen at −20 °C until time of assay. BECs were measured using an Analox AM-1 alcohol analyzer (Analox Instruments, Lunenburg, MA).

Conditioned Taste Aversion.

To motivate drinking, rats were fluid restricted to 50% of their ad libitum daily consumption of water for 24 hr prior to testing to improve the proportion of rats that sample the sucrose on day 1 (Anderson et al., 2010; Vetter-O’Hagen et al., 2009). On the day of testing, rats were transferred from their home colony into a procedural room and placed into a clean, novel cage, then allowed 15 min to acclimate. Rats then received ad libitum access for one hr to a 100 mL graduated bottle of sucrose solution (5% in tap water). Amount of solution consumed was measured as difference in volume from start to end of testing in 100 ml graduated drinking bottles. Rats that consumed fewer than 2 ml of sucrose were excluded from analysis on the grounds that they did not have sufficient exposure to the sucrose to be able to form a CTA. Drugs (IL-6 or AG490+EtOH) were administered immediately after the first exposure. After one day of rest, rats were again water deprived to 50% for 24 hr. Rats were then returned to the testing environment and allowed access to sucrose solution for 60 min.

Statistics.

In studies that included unoperated controls, these groups were compared initially with a t-test to the vehicle control group to ascertain the potential influence of cranial surgery on the dependent measures of interest. If no differences were observed, then the unoperated control groups were not included in the ANOVA. Data were analyzed using omnibus ANOVAs with follow-up post hoc tests (Fisher LSD) run only if the results of the ANOVA indicated a significant main effect. In cases where unoperated controls were statistically different from vehicle injected controls, all groups were included in the omnibus ANOVA. Criterion for rejection of the null hypothesis was always α = 0.05.

Specific Experimental Methods

Experiment 1: dose response to i.c.v IL-6 in LORR.

A range of IL-6 doses (25, 50, 100 ng) were tested on LORR as a way to assess the potential effects of IL-6 on the sedative properties of ethanol. Rats (n=13 per group) were randomly assigned to one of four experimental groups that underwent i.c.v surgery, whereas a fifth group was not cannulated (n=8, N=60) and served as unoperated controls. On test day, cannulated rats were infused with saline, 25 ng, 50 ng or 100 ng of rrIL-6 and then returned to the home cage for 30 min. Rats were then injected i.p. with ethanol (4 g/kg) and the latency to lose the righting reflex was measured. Latency to regain the righting reflex was recorded and blood samples were taken immediately upon awakening.

Experiment 2: test of high-dose IL-6 effects in LORR.

A dose response curve with a higher dose of IL-6 and a larger sample was tested to replicate and extend the findings from the first experiment. Three experimental groups underwent surgery while a fourth group was not cannulated (n=18 per group, N=72). On test day, cannulated rats were infused with saline, 100 ng, or 200 ng of IL-6 and then returned to the home cage for 30 min. Rats were then injected i.p. with ethanol (4 g/kg) and the latency to lose the righting reflex was measured. Latency to regain the righting reflex was recorded and tail bloods were taken upon awakening.

Experiment 3: dose response to AG490 in LORR.

In order to assess the effects of JAK/STAT signaling on ethanol-induced sedation, rats were infused with AG490 and their LORR response observed. Four experimental groups underwent surgery (n=12) while a fifth group was not cannulated (n=8; N=56). On test day, cannulated rats were infused with vehicle (40% DMSO in aCSF), 0.3, 3, or 9 µg of AG490 in DMSO/aCSF and then returned to the home cage for 30 min. Rats were then injected i.p. with ethanol (3.5 g/kg) and the latency to lose the righting reflex was measured. A lower dose of ethanol was used (compared to Exp 1–2) in order to reduce the long sleep time in the LORR test observed in the first two experiments. Latency to regain the righting reflex was recorded and blood samples were taken immediately upon awakening.

Experiment 4: CTA dose response to IL-6.

Illness-inducing agents such as lithium chloride and lipopolysaccharide induce a robust CTA (Cross-Mellor, Kavaliers, & Ossenkopp, 2004). The general sickness response to some cytokines has been well documented (Harden, Plessis, Poole, & Laburn, 2008). In addition, evidence suggests that IL-1 can be used effectively as an unconditioned stimulus (US) in CTA training (Janz et al., 1991). However, using IL-6 as the US has not been tested. Therefore, rats were infused with a range of doses of IL-6 after an initial exposure to sucrose solution to test the hypothesis that IL-6 might act as an unconditioned stimulus in establishment of a CTA. Four experimental groups underwent surgery (n = 15, N=60). Rats were infused with saline, 25, 50, or 100 ng IL-6 after one hr of exposure to sucrose solution. Conditioned taste aversion was assessed at approximately 48 hr after initial exposure to sucrose.

Experiment 5: dose response to AG490 in CTA.

We hypothesized that IL-6 signaling through the JAK/STAT pathway in the brain may in part be responsible for the development of a CTA following moderate ethanol exposure. This is due to the role of the JAK/STAT pathway in both the canonical and trans signaling pathways. To test this, rats were infused with AG490 following exposure to sucrose and then injected with ethanol. Four experimental groups underwent surgery (n=12–13; N=59) while a fifth group was not cannulated (n=8). On test day, cannulated rats were infused with vehicle (40% DMSO in aCSF), 0.3, 3, or 9 µg of AG490 in DMSO/aCSF after one hr access to sucrose solution. Immediately after the infusion, rats were injected with 2 g/kg of ethanol and returned to the home cage. CTA was assessed at approximately 48 hr after initial exposure to sucrose.

Results

Experiment 1: dose response to i.c.v IL-6 in LORR.

Data for Experiment 1 were analyzed with a one-way between subjects ANOVA with 4 groups. No differences between drug conditions were observed for latency to lose the righting reflex (F (3, 27) = 0.55, p = 0.65) or sleep time (F (3, 27) = 0.27, p = 0.85) (Figure 2). When BECs were measured at awakening, ethanol injection produced BECs of ~300–350 mg/dl that also did not differ significantly across drug conditions (F (3, 27) = 0.34, p = 0.79).

Figure 2:

Figure 2:

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Experiments 1 (A, B, C) and 2 (D, E, F) had similar designs but with different doses of IL-6 tested. Rats were given 4 g/kg i.p. ethanol after an infusion of IL-6 or vehicle, and latency to LORR (A, D) and sleep time were measured (B, E). Blood was taken from the tail upon awakening and used to assess BEC (C, F). One-way ANOVAs comparing drug conditions did not reveal a significant impact of IL-6 at any dose on any measure.

Experiment 2: test of high-dose IL-6 effects in LORR.

A one-way ANOVA revealed no effect of IL-6 treatment on latency to sleep (F (2, 36) = 0.41, p = 0.67), sleep time (F (2, 36) = 2.72, p = 0.08), or BECs (F (2, 36) = 0.63, p = 0.54) even at the highest dose tested (Figure 2). Similar BECs upon awakening were observed as in the previous study.

Experiment 3: dose response to AG490 in LORR.

A one-way ANOVA, after a 1/y transformation to account for non-homogeneity of variance, showed a significant effect of AG490 on latency to lose the righting reflex (F (3, 29) = 6.04, p < 0.05). Follow-up Fisher’s LSD tests determined that vehicle infused rats had shorter latencies to lose the righting reflex as compared to the 0.3 µg (t (16) = 3.97, p < 0.05), 3.0 µg (t (16) = 3.41, p < 0.05), and 9 µg (t (15) = 2.16, p < 0.05) groups. Note that data are shown in standard units (not transformed) to be comparable to other figures (Figure 3A). However, sleep time (F (3, 29) = 0.55, p = 0.65) and BECs (F (3, 29) = 1.0, p = 0.39) were unaffected by AG490 (Figure 3B, 3C).

Figure 3:

Figure 3:

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In experiment 3, rats were injected with 3.5 g/kg i.p. ethanol after infusion of AG490 or vehicle, and their latency to LORR (A), sleep time (B), and BECs (via tailblood sample) upon awakening (C) were assessed. A one-way ANOVA with post-hoc Fisher’s LSD determined a significant prolonged latency to LORR in all groups that received AG490 as compared to saline-infused rats (indicated by #).

Experiment 4: CTA dose response to IL-6.

The effects of day (exposure vs. test day) and drug exposure were determined using a 2×4 repeated measures ANOVA. Our results indicated that there was no effect of drug on consumption of sucrose when all rats were included in the analysis (F (3, 43) = 0.27, p = 0.85) (Figure 4A). However, due to the variability in CTAs observed, we then performed a median split based on the first day of sucrose consumption to separate low versus high sucrose consumers. When these groups were analyzed separately, a significant difference between sucrose consumption on the initial exposure day and the test day emerged among the low consumers (F (3, 20) = 7.43, p < 0.05) (Figure 4B). Post-hoc Fisher’s LSD determined that this effect was driven by an increase in drinking from exposure to test day in the vehicle (t = 2.14, p < 0.05) and 25 ng IL-6 (t = 2.67, p <0.05) groups. This pattern was not evident for the groups that received 50 or 100 ng of IL-6, indicating that IL-6 prevented the increase in drinking from exposure to test day, specifically in low-consuming rats. In rats with a high consuming phenotype, there was no significant escalation of drinking from the first to the second day of testing, and IL-6 administration did not affect drinking on the test day (Figure 4C).

Figure 4:

Figure 4:

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Rats received either IL-6 (experiment 4) or AG490 and 2 g/kg ethanol (experiment 5) before an initial exposure to 5% sucrose in a novel context. After 48 hr, rats were returned to the context and exposed to sucrose. Intake of sucrose was measured on both days. Data were examined using a two-way repeated measures ANOVA. Experiment 4: analyzed together, the ANOVA did not reveal any effect of treatment or test day (A). After a median split, the ANOVA with follow up Fisher’s LSD test in the low consumers revealed a main effect of day, such that rats escalated their sucrose intake on the second day. This pattern was only true for the groups that received either vehicle or the lowest dose of IL-6 (B). This effect was not apparent in the high consumers (C). Experiment 5: All data was analyzed together and showed that there was a main effect of day when rats were exposed to ethanol, indicating a successful induction of CTA (D). This CTA was not impacted by AG490 at any dose given. This pattern was the same for both low (E) and high (F) consumers assessed alone.

Experiment 5: dose response to AG490 in CTA.

For this study, a 2×4 repeated measures ANOVA was conducted after determining statistical similarity between the unoperated control and vehicle groups. As expected, ethanol administration immediately after initial exposure to sucrose produced a robust CTA as evidenced by a main effect of test day (F (1, 44) = 248.1, p < 0.05). AG490 had no effect on consumption of sucrose on test day (F (4, 44) = 0.07244, p = 0.99) (Figure 4D). A median split did not reveal any significant differences between low and high consumers of sucrose (Figure 4E, 4F).

Discussion

Acute ethanol exposure induces changes in cytokine expression that vary as a function of intoxication and withdrawal (Doremus-Fitzwater et al., 2015; Gano et al., 2017; Vore et al., 2017). Of particular interest, IL-6 is a cytokine with both pro- and anti-inflammatory effects that displays increased gene expression during intoxication (Doremus-Fitzwater et al., 2015; Gano et al., 2017; Vore et al., 2017). The focus of our studies was to determine the functional impact this increase might produce in two standard tests of ethanol sensitivity: the LORR procedure and CTA. It should be noted that several doses of ethanol (2–4 g/kg) were tested, allowing us to probe behavioral effects of ethanol that are apparent across a range of moderate-to-high doses.

Our results indicated that JAK/STAT signaling (and therefore potentially IL-6 signaling) in the brain had a moderate influence on sensitivity to the sedative effects of ethanol, evidenced by an increase in the latency to LORR when AG490 was administered. The exact mechanisms by which ethanol produces the LORR are unclear, yet the potential involvement of cytokine signaling has been shown in the literature. A study in which injections of a phosphodiesterase E4 (PDE4) inhibitor known to affect cytokine production (Crilly et al., 2011; Schafer et al., 2014) induced extended LORR sleep times in addition to affecting other sedation and locomotion measures (Blednov et al., 2017). Similarly, the putative microglial inhibitor minocycline (Romero-Sandoval, Horvath, & DeLeo, 2008) was injected i.p. three times before exposure to ethanol and significantly reduced sleep time, indicating a potential role for cytokine signaling mediated by microglia in prolonging the sedative effects of ethanol (Wu et al., 2011). In summary, the literature suggests a potential role for cytokine signaling in modulating the LORR response, but exactly how cytokines exert their effects on the described signaling mechanisms remains unclear. More work determining the relationship between cytokine, neurotransmitter, and calcium signaling is needed to understand the intricacies of ethanol-induced LORR.

Although blocking endogenous JAK signaling with AG490 had no effect on ethanol-induced CTA, the effect of exogenous IL-6 (50 or 100 ng) on sucrose consumption depended on phenotypic differences within subjects. Infusion of IL-6 prevented the escalation of sucrose intake from exposure to test day exclusively in rats that drank low amounts on the initial exposure day. This finding may indicate the presence of two distinct phenotypes of rat in the sample indicative of individual differences in IL-6 sensitivity. The individual variability in this case may be driven by relative neophobia in the low-consuming rats, or by a lower baseline preference for sucrose. Studies that have investigated differences between low and high consumers of sucrose have observed that high consumers self-administer more amphetamine (DeSousa, Bush, & Vaccarino, 2000). Additionally, they found that low consumers displayed a more anxious phenotype as measured in the elevated plus maze and the acoustic startle response test (Desousa, Wunderlich, De Cabo, & Vaccarino, 1998). This finding in particular informs the interpretation that the low consumers in the present study may represent an anxious or neophobic phenotype that is uniquely sensitive to any aversive effects of IL-6 being produced during intoxication. In previous studies, our lab has demonstrated that cytokine expression is variable within dyads of pair-housed rats. Specifically, basal IL-1β levels in the hypothalamus were shown to be significantly higher in rats that were classified as submissive in their dyad (Barnum, Blandino, & Deak, 2008). With that said, the present studies utilized rats that were single-housed post- surgery, suggesting that existing dominance hierarchies probably cannot account for the individual differences observed here. Nevertheless, future studies should explore individual differences in basal and evoked IL-6 in ethanol-related behaviors.

The selective effect on sucrose consumption (only low-consumers were affected) indicates that IL-6 signaling is not sufficient to change sucrose drinking behavior in all rats. IL-6 produced endogenously is often accompanied by cascading changes in other cytokines as well, and as such it may be that we did not replicate the cytokine environment sufficiently to reproduce the CTA produced by ethanol. This idea is supported by co-administration studies, which have shown robust effects. For example, when IL-6 and IL-1β were infused together, but not alone, in the locus coeruleus, subjects displayed reduced locomotion 4 hr later, and enhanced depressive behaviors such as the forced swim test (Kurosawa, Shimizu, & Seki, 2016). Another study showed that non-pyrogenic doses of either IL-6 or IL-1β infused i.c.v. had no effect on fever or food consumption, but when these low doses were applied concurrently, the synergistic effect of the two cytokines produced both anorexia and fever (Harden et al., 2008). Our results indicate that IL-6 is unlikely to be a necessary mediator of ethanol-induced CTA, however, it may be that IL-6 is working in conjunction with other cytokines to produce the CTA. In light of these studies, combining administration of IL-6 with TNF-α or IL-1β could shed light on the role of the synergistic effects of the cytokine response to ethanol.

One limitation of the studies performed with AG490 is that it has effects on signaling that are not restricted to IL-6. AG490 acts by competing for ATP binding on JAK2, thereby inhibiting its activation (Rashid, Bibi, Parveen, & Shafique, 2015). As such, it is not entirely specific to the IL-6 pathway, as other molecules, such as the IL-10 family cytokines use JAK2 signaling (Truong, Hong, Hoang, Lee, & Hong, 2017). Furthermore, AG490 also inhibits, to a lesser extent, JAK1 (Xuan, Guo, Han, Zhu, & Bolli, 2001) and JAK3 (Rashid et al., 2015). Finally, AG490 has been shown to decrease levels of gp130 (Seo et al., 2009), which is also responsible for transducing signals from other ligands like ciliary neurotrophic factor (Cron, Allen, & Febbraio, 2016). Therefore, it is possible that the effects produced by AG490 (or the lack thereof) could be due to off-target effects of the drug. Although this is a limitation, it is a minor one given previous studies that demonstrated its ability to effectively block IL-6 signaling effects in the brain on STAT3 phosphorylation, and also on behavioral processes known to be affected by IL-6 (Damm et al., 2013; Kim et al., 2013). Another possible explanation for the lack of AG490 effects in the CTA compared to the LORR study is that there was no 30 minute incubation time between the AG490 administration and the ethanol. It is therefore possible that ethanol produced a CTA before AG490 could effectively block JAK/STAT signaling. Ethanol was given as quickly as possible following the sucrose drinking to ensure an effective CTA development, although in future studies a lag of 30 minutes may be used. Additionally, IL-6 may act on other JAKs to produce effects that AG490 failed to block. Overall, although AG490 may operate through several mechanisms to modulate cytokine signaling pathways, its effectiveness at blocking STAT3 phosphorylation following increases in IL-6 provides reasonable justification in the case of null results that JAK2/STAT3 activation is not involved in the behavior in question. In particular, we can tentatively conclude that CTA formation to ethanol is not dependent on JAK2/STAT3 signaling. On the other hand, this pathway may modulate the effect of ethanol on the sedative properties of ethanol. This could be initiated by IL-6 binding, or by some other activator of JAK/STAT as discussed above.

A drawback of all experiments that employ i.c.v surgery and drug delivery is the inherent damage and ensuing inflammation that occur in the brain. The ability to deliver these drugs in the ventricle where they presumably diffuse to a relatively wide portion of the brain outweighs this drawback, but the potential confounds of ongoing inflammation or altered basal cytokines must be considered. Indeed, the potential influence of cranial surgery motivated us to incorporate unoperated controls in most studies. The similarity of unoperated controls to vehicle infused rats in these experiments lends confidence to the assertion that the baseline behaviors that we measured were not altered by implantation of indwelling cannula. Of particular concern to the studies which include infusion of IL-6 are the data which suggest that the diffusion of IL-6 from an i.c.v. infusion extends only marginally beyond the periventricular region (Helwig et al., 2008). However, i.c.v. infusion of IL-6 or drugs targeting the IL-6R have demonstrated unequivocal effects, suggesting the i.c.v. route as a reasonable first step toward assessing a central role of IL-6 in ethanol-mediated behaviors. Nevertheless, it is possible that IL-6 is playing roles via brain regions that were not reached by the infusions, and future studies should test for site-specific effects of IL-6. For example, the hippocampus could be targeted because electrophysiological studies have demonstrated that overexpression of IL-6 in the astrocytes of the hippocampus can protect long-term potentiation to some extent from the disrupting effect of ethanol (Hernandez et al., 2016). Use of global KO, inducible KO, or targeted infusion models offer advantages and viable approaches for future studies.

Ethanol elicits broad changes in neuroimmune function characterized by specific cytokines demonstrating peak expression predominantly during intoxication or withdrawal, yet the functional ramifications of these changes remain undetermined (Doremus-Fitzwater et al., 2015; Gano et al., 2017; Richey et al., 2012). Cytokines can influence systems such as the HPA axis, changing the response of the organism to stress (Arakawa, Blandino, & Deak, 2009; Barnum et al., 2008; Blandino, Barnum, & Deak, 2006), and can also modulate the ability to respond to immune threats (Burrack et al., 2018; Iwasaki & Medzhitov, 2015). Cytokines in particular have been established as important clinical markers of various neuropsychiatric conditions involving affective dysregulation, including anxiety and depression (Kudinova et al., 2016; Oliveira Miranda et al., 2014; Pascual, Baliño, Aragón, & Guerri, 2015). The above studies represent the first strides toward understanding a potential functional role for IL-6 in the context of acute ethanol intoxication and withdrawal. As discussed, IL-6 can have effects on behavior when introduced into specific brain areas associated with behavior (e.g. the amygdala and fear conditioning; Hao et a., 2014), and thus future studies in the field employing IL-6 infusion should apply doses in regions of interest that are related to the behavior under study. The results of our studies using i.c.v. infusion in an attempt to affect the whole brain highlight the importance of individual variability as a moderator of the cytokine and ethanol interaction. Further, they indicate that IL-6 signaling may play a role in both ethanol sensitivity and CTA responses.

Table1:

Summary of subject exclusions and final N

Experiment Starting Subjects (N) Excluded Subjects Final Subjects (N)
1 60 22 38 (n=7-9)
2 72 20 52 (n=12-14)
3 56 16 40 (n=7-9)
4 60 13 47 (n=9-14)
5 59 10 49 (n=7-12)
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Research Highlights:

  • ICV IL-6 had no effect on loss of righting reflex (LORR)

  • ICV IL-6 produced conditioned taste aversion (CTA) only in low sucrose consumers

  • Individual differences may determine sensitivity to ICV IL-6

  • ICV AG490, a JAK/STAT inhibitor, modestly increased LORR

  • AG490 had no effect on ethanol-induced CTA

Acknowledgements:

Research reported in this publication was supported by the National Institute of Health Grants P50AA017823 and T32AA025606, and the Center for Development and Behavioral Neuroscience at Binghamton University. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the above stated funding agencies. The authors have no conflicts of interest to declare.

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