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Neuropharmacology 103 (2016) 27e43

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Neuropharmacology

journal homepage: www.elsevier .com/locate/neuropharm

Transgenic mice with increased astrocyte expression of IL-6 showaltered effects of acute ethanol on synaptic function

Ruben V. Hernandez, Alana C. Puro, Jessica C. Manos, Salvador Huitron-Resendiz,Kenneth C. Reyes, Kevin Liu, Khanh Vo, Amanda J. Roberts, Donna L. Gruol*

Molecular and Cellular Neuroscience Department, The Scripps Research Institute, La Jolla, CA 92037, USA

a r t i c l e i n f o

Article history:Received 15 September 2015Received in revised form21 November 2015Accepted 14 December 2015Available online 17 December 2015

Keywords:Synaptic plasticityEEGGamma frequencyGliaEthanol withdrawal hyperexcitabilityNeuroimmuneSTAT3Long-term potentiation

* Corresponding author. MCND, SP30-1522, The ScriNorth Torrey Pines Road, La Jolla, CA 92037, USA.

E-mail address: gruol@scripps.edu (D.L. Gruol).

http://dx.doi.org/10.1016/j.neuropharm.2015.12.0150028-3908/© 2015 Elsevier Ltd. All rights reserved.

a b s t r a c t

A growing body of evidence has revealed that resident cells of the central nervous system (CNS), andparticularly the glial cells, comprise a neuroimmune system that serves a number of functions in thenormal CNS and during adverse conditions. Cells of the neuroimmune system regulate CNS functionsthrough the production of signaling factors, referred to as neuroimmune factors. Recent studies showthat ethanol can activate cells of the neuroimmune system, resulting in the elevated production ofneuroimmune factors, including the cytokine interleukin-6 (IL-6). Here we analyzed the consequences ofthis CNS action of ethanol using transgenic mice that express elevated levels of IL-6 through increasedastrocyte expression (IL-6-tg) to model the increased IL-6 expression that occurs with ethanol use. Re-sults show that increased IL-6 expression induces neuroadaptive changes that alter the effects of ethanol.In hippocampal slices from non-transgenic (non-tg) littermate control mice, synaptically evoked den-dritic field excitatory postsynaptic potential (fEPSP) and somatic population spike (PS) at the Schaffercollateral to CA1 pyramidal neuron synapse were reduced by acute ethanol (20 or 60 mM). In contrast,acute ethanol enhanced the fEPSP and PS in hippocampal slices from IL-6 tg mice. Long-term synapticplasticity of the fEPSP (i.e., LTP) showed the expected dose-dependent reduction by acute ethanol in non-tg hippocampal slices, whereas LTP in the IL-6 tg hippocampal slices was resistant to this depressiveeffect of acute ethanol. Consistent with altered effects of acute ethanol on synaptic function in the IL-6 tgmice, EEG recordings showed a higher level of CNS activity in the IL-6 tg mice than in the non-tg miceduring the period of withdrawal from an acute high dose of ethanol. These results suggest a potential rolefor neuroadaptive effects of ethanol-induced astrocyte production of IL-6 as a mediator or modulator ofthe actions of ethanol on the CNS, including persistent changes in CNS function that contribute tocognitive dysfunction and the development of alcohol dependence.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Neuroimmune factors play critical roles in homeostatic regula-tion of CNS function, neuronal development, defense against insultand infection, and repair mechanisms. Abnormal production ofneuroimmune factors is considered an important contributingfactor to many neuropsychiatric and neurologic condition, such asmajor depression (Sukoff Rizzo et al., 2012; Young et al., 2014),dementia (Trapero and Cauli, 2014), Alzheimer's disease (Lutermanet al., 2000), epilepsy (Li et al., 2011), schizophrenia (Schwieler

pps Research Institute, 10550

et al., 2015), autism (Wei et al., 2013), sleep disturbances (Zhuet al., 2012), infection (Jurgens et al., 2012), and trauma (Lloydet al., 2008). Importantly, recently studies have identified thatexcessive alcohol (ethanol) exposure, which is known to producecognitive impairment, induces elevated glial production of IL-6 andother neuroimmune factors (Alfonso-Loeches et al., 2010;Doremus-Fitzwater et al., 2014). Excessive alcohol use is often co-morbid with neuropsychiatric and neurologic conditions, and isthought to impact negatively on these conditions (e.g., depression(Briere et al., 2014), epilepsy (Shield et al., 2013), trauma(Kachadourian et al., 2014), HIV infection (Silverstein et al., 2014)).Thus, insight into actions of IL-6 and interactions between IL-6 andethanol are an important step toward an understanding CNSmechanism involved in the effects of ethanol on the brain andpotential interactions with mechanisms underlying

R.V. Hernandez et al. / Neuropharmacology 103 (2016) 27e4328

neuropsychiatric and neurologic conditions co-morbid withalcohol use disorders.

A number of in vivo and in vitro studies have established thatboth acute and chronic ethanol alter CNS expression of IL-6 andother neuroimmune factors, primarily due to an action of ethanolon astrocytes and microglia. These neuroimmune factors includethe proinflammatory cytokines IL-6, IL-1b and TNF-a and the che-mokine CCL2 (Alfonso-Loeches et al., 2010; Blanco et al., 2005; Heand Crews, 2008; Kane et al., 2013, 2014; Lippai et al., 2013; Qinand Crews, 2012; Tiwari et al., 2009; Valles et al., 2004;Vongvatcharanon et al., 2010; Ward et al., 2009; Zhang et al.,2014; Zhao et al., 2013). Ethanol-induced CNS expression of theseneuroimmune factors appears to depend on brain region, cell type,and/or dose, duration and method of ethanol exposure. In recentstudies acute ethanol (4 g/kg, i.p.) was shown to produce a prom-inent and prolonged increase (3e9 h) in the level of IL-6 mRNA inthe hippocampus of rats, whereas the level of TNF-a mRNA wasreduced, and the level of IL-1 b mRNA was unaffected (Doremus-Fitzwater et al., 2014). Chronic ethanol (6 g/kg per day for 10days, by gavage) increased IL-6 mRNA levels in the CNS of adultmice, but only in cerebellum, whereas increased levels of CCL2mRNA were observed in hippocampus, cerebellum and cortex, andTNF-a mRNA levels were not altered in any of these three CNS re-gions (Kane et al., 2014). Chronic ethanol consumption (13 g/kg perday for 5 months) induced a toll-like receptor 4 (TLR4) response inthe cortex of mice that was associated with astrocyte activation andproduction of IL-6 mRNA, and IL-1 and TNF-a protein, an effect thatwas not observed in TLR4 knockout mice (Alfonso-Loeches et al.,2010). Increased levels of IL-6, IL-1b and TNF-a protein in the CNSof mice were also observed following three weeks of ethanol con-sumption using a two-bottle choice drinking paradigm (Zhanget al., 2014). Both acute (100 mM, 24 h) and chronic (50 mM, 7day) ethanol increased IL-6 production in primary cultures ofcortical astrocytes, whereas there was no effect of ethanol on TNF-aproduction (Sarc et al., 2011). Ethanol (50e100 mM, 24 h) alsoincreased secretion of cytokines in primary cultures of cerebralmicroglia, including IL-6, TNF-a, MIP-1-a, and MIP-2 (Boyadjievaand Sarkar, 2010).

While these and other studies have established that both acuteand chronic ethanol alter CNS expression of neuroimmune factors,little is known about the consequences of this action of ethanol andunderlying mechanisms. Fundamental to this issue is an under-standing of how neuroimmune factors affect basic neuronal func-tions such synaptic transmission and cell excitability, which aretarget sites of ethanol action, and if or how the actions of neuro-immune factors and ethanol interact.

In the hippocampus, ethanol has been shown to alter hippo-campal function through modifications of cellular mechanismsmediating synaptic transmission and plasticity (Peris et al., 1997;Weiner and Valenzuela, 2006; White and Swartzwelder, 2004;Zorumski et al., 2014). IL-6 and other proinflammatory cytokines(e.g. IL-1b and TNF-a) also alter hippocampal synaptic transmissionand plasticity (Beattie et al., 2002; Bellinger et al., 1993; Coogan andO'Connor, 1997; Ikegaya et al., 2003; Katsuki et al., 1990; Li et al.,1997; Nelson et al., 2012; O'Connor and Coogan, 1999; Pribiagand Stellwagen, 2013; Steffensen et al., 1994; Stellwagen andMalenka, 2006; Tancredi et al., 2000, 1992; Wheeler et al., 2009;Yang et al., 2005; Zhang et al., 2010). This commonality of ethanoland cytokine action raises the possibility that increased levels ofproinflammatory cytokines produced by ethanol could result,either directly or indirectly, in transient or long-term neuro-adaptive changes that then alter the effects of ethanol. Our studiesfocus on this possibility.

In the current studies, we utilized transgenic mice and theirnon-tg littermate controls to determine if elevated CNS expression

of IL-6 can lead to neuroadaptive changes that alter the effects ofacute ethanol on hippocampal synaptic function. The transgenicmice model the increased CNS expression of IL-6 that would occurwith ethanol use. Expression of elevated levels of IL-6 in the CNS ofthe IL-6 tg mice was achieved through genetic manipulation ofastrocyte expression (Campbell et al., 1993). Astrocytes are themost abundant cell type in the CNS and produce IL-6 both underphysiological and pathological conditions (Gruol and Nelson,1997).Ethanol has been shown to cause increased astrocyte production ofIL-6 protein (Alfonso-Loeches et al., 2010; Sarc et al., 2011). Thus,the consequences of the increased astrocyte production of IL-6 inthe IL-6 tg mice can inform on potential consequences of ethanol-induced increase in astrocyte production of IL-6.

Expression of IL-6 in the IL-6 tg mice is under control of theGFAP promoter. GFAP mRNA expression, which is regulated by IL-6acting through STAT3, and GFAP protein do not become prominentuntil about 1 month postnatal, suggesting that IL-6 production isnot prominent until about 1 month postnatal (Sanz et al., 2008).Thus, results from this model are likely to be most relevant toalcohol use that starts in the juvenile or adolescent stage of life, apattern of alcohol use that has significant risk for developingalcohol dependence and is currently an important societal issue(Foltran et al., 2011; Hingson et al., 2006). Studies in animal modelshave shown that ethanol exposure increases IL-6 levels in the CNSof adolescents as well as adults (Doremus-Fitzwater et al., 2015).

The highest number of transgene-expressing astrocytes in theforebrain region of the IL-6 tg mice occurs in the hippocampus(Vallieres et al., 2002), making it likely that neuroadaptive effectson synaptic transmission will be evident in this CNS region. Thehippocampus expresses one of the highest levels of mRNA for IL-6receptors (IL-6R), which mediate cellular effects of IL-6, suggestingan important role for IL-6 in this CNS region (Gadient and Otten,1994; Schobitz et al., 1993). Both neurons and glia cells of thehippocampus express IL-6R (Schobitz et al., 1993; Vollenweideret al., 2003). Astrocytes are a key regulator of synaptic function(Halassa et al., 2007) and astrocyte produced IL-6 or exogenouslyapplied IL-6 has been shown to alter (i.e., depress) hippocampalsynaptic plasticity at the Schaffer collateral to CA1 neuron synapse(Balschun et al., 2004; Li et al., 1997; Tancredi et al., 2000). Inter-estingly, synaptic plasticity at this synapse is also depressed byacute ethanol (Blitzer et al., 1990; Fujii et al., 2008; Izumi et al.,2005; Schummers et al., 1997; Sinclair and Lo, 1986), and theseactions of ethanol are thought to contribute to the impairment inmemory and learning produced by ethanol.

Our previous studies showed that synaptic transmission at theSchaffer collateral to CA1 pyramidal neuron synapse is enhanced inthe IL-6 tg mice, consistent with the expression of persistent neu-roadaptive changes in synaptic function in the IL-6 tg mice (Nelsonet al., 2012). These neuroadaptive changes in synaptic function maybe related to the enhanced susceptibility of the IL-6 tg mice toNMDA- and kainate-induced seizure activity (Samland et al., 2003).Ethanol exposure also results in increased susceptibility to seizureactivity, as a consequence of transient or persistent ethanol-induced neuroadaptive changes that render the CNS dependenton the presence of ethanol. When ethanol exposure is terminated, asynaptic imbalance is created that results in hyperexcitability andincreased susceptibility to seizure activity (Becker, 2000; Heiliget al., 2010). Thus, neuroadaptive changes in the CNS of the IL-6-tg mice could influence the effects of ethanol on synaptic func-tion and susceptibility to ethanol-induced hyperexcitability/seizureactivity, possibilities that we have assessed in the current studies.Results are consistent with interactions between the neuroadaptivechanges in the CNS of IL-6 tg mice and the effects of ethanol, andsuggest that ethanol-induced astrocyte production of IL-6 may playan important role as a mediator or modulator of CNS actions of

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ethanol, including persistent changes in CNS function thatcontribute to the cognitive dysfunction and development ofdependence.

2. Materials and methods

2.1. Transgenic mice

The studies were carried out in IL-6-tg mice (heterozygote lowexpressor from the 167 line (Campbell et al., 1993)). These miceexpress moderately elevated levels of IL-6 in the CNS throughincreased astrocyte expression. Age-matched littermates that donot express the IL-6 transgene (non-tg) are used as controls. Themethods used to engineer the transgenic mice have been describedpreviously (Campbell et al., 1993). Briefly, IL-6 expression in theCNS was targeted to astrocytes by an expression vector derivedfrom the murine glial fibrillary acidic protein (GFAP) gene. Full-length murine IL-6 cDNA was modified and inserted into theGFAP gene. The genes were then microinjected into fertilized eggsof F1 generation hybrid mice (C57BL/6J � SJL). The IL-6 line hasbeen maintained for many years on a C57BL/6J background and iscongenic. The phenotypic characteristics of themice have remainedstable and are comparable to that originally described (Campbellet al., 1993; Chiang et al., 1994; Giralt et al., 2013).

Transgenic mice with astrocyte-targeted elevated expression ofCCL2 (CCL2 tg) were also used for one set of experiments. Non-transgenic littermates (CCL2 non-tg) served as controls. Construc-tion of the mice has been described previously (Huang et al., 2002).Briefly, the murine CCL2 gene was placed under control of thehuGFAP promoter and purified GFAP-CCL2 fusion gene fragmentwas injected into fertilized eggs of SWXJ (H-21q,s) mice. The linewas later backcrossed onto a C57BL/6J background and maintainedfor several years by breeding heterozygous CCL2-tg micewith wild-type C57BL/6J mice.

All animals were genotyped by PCR analysis of tail DNA usingstandard methods. All animal procedures were performed inaccordance with the National Institutes of Health Guideline for theCare and Use of Laboratory Animals. Animal facilities and experi-mental protocols were in accordance with the Association for theAssessment and Accreditation of Laboratory Animal Care.

2.2. Slice preparation and electrophysiological recordings

Electrophysiological recordings of synaptic function at theSchaffer collateral to CA1 pyramidal neuron synapse of the hippo-campus were performed in vitro as described previously (Nelsonet al., 2012). Non-tg (n ¼ 54) and IL-6 tg (n ¼ 61) mice 3e5months of agewere used (mean age inmonths: 4.5 ± 0.1 for non-tg,n ¼ 31; 4.5 ± 0.1 for IL-6 tg, n ¼ 44). The mice were weighed,anesthetized with isoflurane, and decapitated. Brains were rapidlyremoved and immersed in ice-cold artificial cerebrospinal fluid(ACSF). The ACSF was gassed continuously with 95% O2/5% CO2 (pH7.2e7.4) to maintain cell viability. The composition of the ACSF was(in mM): 130.0 NaCl, 3.5 KCl, 1.25 NaH2PO4, 24.0 NaHCO3, 2.0 CaCl2,1.3 MgSO4, and 10.0 glucose (all chemicals from SigmaeAldrich, St.Louis, MO, USA). After cooling, the hippocampus was removed fromthe brain, cut into 400 mm slices using a McIlwain tissue chopper(Mickle Laboratory Engineering Co. Ltd., Surrey, UK), and the slicesplaced in a gas-fluid interface holding chamber maintained at~33 �C until used. The chamber was superfused continuously withACSF (33� C) at rate of 0.55 ml/min. Slices from the mid-region ofthe hippocampus were used for experiments.

For recordings, a hippocampal slice was transferred to a gas-fluid recording chamber and allowed to stabilize for 30e60 min.The recording chamber was continuously superfused with ACSF or

ACSF plus ethanol (20 or 60 mM) at a rate of 2 ml/min (33 �C). Onlyone concentration of ethanol was tested on each slice studied. Aconcentric bipolar stimulating electrode (FHC Inc., Bowdoin, ME,USA) was placed in the Schaffer collaterals at the border of the CA2and CA1 regions of the hippocampus and synaptic transmissionwas elicited by brief electrical stimulation of the Schaffer collaterals(50 ms duration; S88 Square Pulse Stimulator and PSIU6 StimulusIsolation Unit, Grass Technologies, West Warwick, RI, USA). Theresulting synaptic responses were recorded extracellularly as fieldpotentials. One recordingmicroelectrode (1e3MU) filledwith ACSFwas placed in the stratum radiatum (dendritic region) of the CA1region to record the field excitatory postsynaptic potential (fEPSP)and a second recording microelectrode was placed in the stratumpyramidale (somatic region) of CA1 region to record the populationspike (PS), which was elicited by the fEPSP. The signals wereamplified with an Axoclamp-2A amplifier and acquired using thepClamp software (both from Molecular Devices, Union City, CA,USA).

2.3. Ethanol concentrations tested

Two pharmacologically relevant concentrations of ethanol weretested, 20 mM (92 mg/dl) and 60 mM (276 mg/dl). The ethanolsolutions (in ACSF) were prepared 10e15 min prior to use from 95%ethanol (Remet Alcohols Inc., La Mirada, CA). In humans, a bloodethanol concentration of 80 mg/dl is considered an intoxicatingdose of ethanol, whereas in heavy drinkers and alcoholics bloodethanol concentrations can exceed 300 mg/dl (Perper et al., 1986).These concentrations of ethanol cause cognitive dysfunctionincludingmemory impairments in humans and animal models and,in animal models, altered synaptic function in the hippocampusand other brain regions (reviewed in (Tipps et al., 2014; White,2003; Zorumski et al., 2014)). Brain levels of ethanol have beenshown to correspond to blood levels in animal models (Gilpin et al.,2009; Smolen and Smolen, 1989).

2.4. Assessment of synaptic function

The effect of ethanol on synaptic transmission in the IL-6 tg andnon-tg hippocampal slices was determined by a comparison ofinputeoutput (I/O) relationships for the EPSP or PS under controlconditions (ACSF superfusion) and after exposure to ACSF con-taining ethanol. I/O data were generated by applying a series ofstimuli of increasing intensity to the Schaffer collaterals whilerecording the EPSP and PS. Stimulation, at rate of 0.05 Hz, wasstarted at the threshold intensity required to elicit a fEPSP or PS andincreased in steps (typically 10e20 mA steps from 20 to 240 mA)until the maximum fEPSP or PS was reached. Ethanol was appliedto the hippocampal slices by superfusion (flow rate of 1.0e2.0 ml/min) after baseline control data were collected. I/O measurementsin the presence of ethanol were made ~30 min after the start ofethanol superfusion and new half-max values for the fEPSP and PSwere identified and used in all subsequent protocols. Superfusion ofethanol was continued for the duration of the experiment, whichincluded studies of I/O relationships, and short- and long-termforms of synaptic plasticity. Control data for studies of long-termsynaptic plasticity were provided by an additional series of exper-iments that were carried out in slices exposed to vehicle only(ACSF) using the same protocol as used for the ethanol studies.

The effect of ethanol on short-term synaptic plasticity of thefEPSP and PS was assessed using standard paired-pulse ratio (PPR)protocols. The test stimulus intensities used elicited a response(fEPSP or PS) was equal to 50% of the maximal response determinedfrom I/O protocols. Three paired-pulse responses (1 min betweenacquisitions) were averaged for PPR in each slice. Plasticity of the

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fEPSP was determined by a stimulation protocol applied at paired-pulse intervals of 40, 100, and 200 ms. Plasticity of the PS wasassessed by a paired-pulse stimulation protocol at short intervals(10 and 20 ms). Longer forms of synaptic plasticity elicited by highfrequency stimulation (HFS) were also examined under controlconditions and in the presence of ethanol including post-tetanicpotentiation (PTP), short-term potentiation (STP), and long-termpotentiation (LTP) of the fEPSP slope. The HFS protocol consistedof 100 pulses for 1 s (100 Hz) repeated a total of three times with a20 s interval between trains. A test stimulus intensity that elicited afEPSP equal to 50% of the maximal fEPSP, as determined from I/Oprotocols, was used. A slicewas considered to exhibit successful LTPif the slope of the fEPSP remained at an elevated level of �25% ofbaseline for �60 min following HFS.

2.5. Analysis of electrophysiological data

Data analyses were conducted off-line using AxoGraph softwareprogram (Axograph Scientific, axograph.com). For I/O data, themagnitude of the fEPSP was quantified by the slope of the initialrising phase of the negative deflection over the 40e60% range ofthe peak. The magnitude of the PS was measured as the peakamplitude of the negative deflection from a baseline value. Baselinevalue was estimated from a tangent line fitted across the twopositive peaks of the synaptic response recorded in the somaticregion. To construct I/O curves, mean values for fEPSP slope or PSamplitude were plotted as a function of stimulus intensity.Threshold stimulus intensity for the fEPSP or PS varied across slicesfor both the IL-6 tg and non-tg hippocampus under baseline con-ditions. Therefore, for data analysis the stimulus intensity thatelicited a threshold fEPSP (e.g., slope of ~0.15 mV/ms) or PS (e.g.,amplitude of ~0.70 mV) under baseline conditions was standard-ized, by setting the threshold stimulus intensity for the fEPSP or PSunder baseline conditions to the same value for all slices (e.g., 70 mAfor fEPSP, 100 mA for PS). To construct graphs reflecting the I/O re-lationships, stimulus intensities values were incremented (e.g.10 or20 mA intervals) from the standardized threshold value according tothe increments used during the experiment. Stimulus intensitiesfor I/O curves generated in the presence of ethanol were correlatedto stimulus intensities for I/O curves generated under baselineconditions in the same slice to retain baseline/ethanol I/Orelationships.

For statistical analyses of I/O data, the data were first normal-ized. This normalization adjusted for differences in the amplitudeof the synaptic responses within a genotype under baseline con-ditions. For normalization, fEPSP or PS values in the presence ofethanol were divided by fEPSP or PS values under baseline condi-tions for the same stimulus intensity measured in the same slice(Wei et al., 2004). The normalized data were compared statisticallyusing repeated measures ANOVA to identify significant genotypiceffects. In addition, a mean value for the normalized fEPSP or PScollapsed across the stimulus intensity range (e.g., 100e200 mA)was calculated for each slice. The mean normalized values werecompared statistically to identify significant within genotype ef-fects of treatment (i.e., baseline vs. ethanol, one group t-test) anddose (i.e., 20 vs. 60 mM ethanol, unpaired t-test). For this and allother statistical comparisons, p < 0.05 was taken to indicate asignificant difference.

For studies of short-term plasticity assessed by paired-pulsestimulation protocols, PPRs were determined from the ratio ofthemagnitude of the response to the second stimulation divided bythe magnitude of the response to the first stimulation for eachpaired-pulse interval. Mean PPRs were compared statistically byANOVA or the paired t-test. For studies of long-term plasticity, LTP,PTP, and STP, the slope of the fEPSP was expressed as a percentage

of the mean for five baseline (pre-HFS) fEPSP slopes. Mean PTP wasdetermined for each slice by averaging the first 3 measurementsafter HSF. Mean STP was determined for each slice by averaging themeasurements from 15 to 25 min after HSF. Mean LTP was deter-mined for each slice by averaging measurements from 50 to 60 minafter HSF. Mean values for PTP, STP and LTP in IL-6 tg and non-tgslices were compared statistically by ANOVA. Compiled data areexpressed as the mean ± SEM. Both females and males were usedfor these studies. Results showed no consistent gender differencesand data from males and females were combined. For electro-physiological studies n ¼ number of slices studied (one slice/animal).

2.6. IL-6 and signal transduction

The relative level of IL-6 and activation of signal transductionmolecules utilized by IL-6 was determined by ELISA or Westernblot, respectively, in control and ethanol exposed hippocampalslices. Both the left and right hippocampi were obtained from thebrain of each animal and cut into 400 mm slices following the sameprotocol as used for electrophysiological recordings. Approximately6 slices were obtained from each hippocampus. The slices werepooled according to the hippocampus from which they were ob-tained. One set of hippocampal slices from each animal served asthe control sample and the second set was used for ethanol treat-ment. The source (i.e., left or right hippocampus) for the control andethanol samples was reversed in different experiments. The sliceswere placed in two recording chambers maintained at ~33 �C andwere continuously superfused with oxygenated ACSF at rate of1 ml/min. The slices were incubated for 30e60 min to allow themto recover, and then one set of slices was superfused with 60 mMethanol for 30 min and the second set of slices was superfused withACSF. After 30 min, control and ethanol-treated slice sets weretransferred to eppendorf tubes, snap frozen and stored at �80 �C.

Protein samples for ELISA or Western blot were prepared fromthe IL-6 tg and non-tg hippocampal slices following protocolsdescribed previously (Gruol et al., 2014; Nelson et al., 2012). Pro-teins were extracted from the hippocampal samples by sonicationin cold lysis buffer containing 50 mM TriseHCl, pH 7.5, 150 mMNaCl, 2 mM EDTA,1% Triton X-100, 0.5% NP-40, a Complete ProteaseInhibitor Cocktail Tablet (Roche Diagnostics, Mannheim, Germany),and a cocktail of phosphatase inhibitors (Naþ pyrophosphate, b-glycerophosphate, NaF, Naþ orthovanadate; all from Sigma-eAldrich). The samples were incubated on ice for 30 min, centri-fuged at 13,860g for 30 min at 4 �C, and the supernatants werecollected. Protein concentration in the supernatants was deter-mined using the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA).Protein aliquots were stored at �80 �C.

IL-6 levels in protein samples were determined by ELISA usingthe Mouse IL-6 ELISA Ready-SET-Go! Kit (eBioscience, Inc., SanDiego, CA). Levels of other proteins were determined by Westernblot. For Western blot, equal amounts of protein samples (25 mg)were subjected to SDS-PAGE using 4e12% Novex NuPAGE Bis-Trisgels (Invitrogen Life Technologies, Grand Island, NY). IL-6 tg andnon-tg protein samples were run on the same gel. Samples wererun in duplicate. Proteins were transferred to Immobilon-P mem-branes (Millipore, Billerica, MA) and uniform transfer was assessedby Ponceau S staining (Pierce, Rockford, IL). Membranes werewashed and blocked in a 5% casein solution (Pierce) and thenincubated in primary antibody overnight (4 �C). After washing, themembranes were incubated (room temperature) in secondaryantibody coupled to horseradish peroxidase (HRP). Protein bandswere visualized by chemiluminescence and quantified by densi-tometry measurements using NIH Image software (http://rsb.info.nih.gov/nih-image/). Membranes were stripped and reprobed for

R.V. Hernandez et al. / Neuropharmacology 103 (2016) 27e43 31

b-actin. To adjust for possible loading errors, the density of eachband was normalized to the density of the band for b-actin in thesame lane. Normalized data from IL-6 tg slices were then normal-ized to the average normalized value for non-tg slices run on thesame gel. Data were combined according to genotype and treat-ment and reported as mean ± SEM.

The following antibodies were used for Western blot studies: amonoclonal antibody to b-actin (#AC-15, 1:5000; Sigma, St. Louis,Missouri); a rabbit polyclonal antibody raised against p42/p44mitogen-activated protein kinase (MAPK) (#61-7400; 1:5000,Zymed, Carlsbad, CA, USA); a purified rabbit polyclonal antibodyraised against a synthetic phospho-peptide (KLH-coupled) corre-sponding to residues around Thr202/Tyr204 of human p42/p44MAPK (#9101; 1:500; Cell Signaling Technologies, Danvers, MA;pp42/44 MAPK); a purified rabbit polyclonal antibody raisedagainst a synthetic peptide (KLH-coupled) corresponding to thesequence of mouse signal transducer and activator of transcription3 (STAT3) (AB#9132; 1:1000; Cell Signaling Technologies), a puri-fied rabbit polyclonal antibody raised against a synthetic phospho-peptide (KLH-coupled) corresponding to the residues surroundingTyr705 of mouse STAT3 (AB#9131; 1:1000; Cell SignalingTechnologies).

2.7. Behavioral studies of acute ethanol withdrawalhyperexcitability

The ability of a single high dose of ethanol to induce a hyper-excitable state during the period of declining blood ethanol levelswas assessed by scoring handling-induced convulsions and byrecording the electroencephalograph activity. Male IL-6 tg and non-tg mice were used for these studies. n ¼ number of animals tested.Compiled data are expressed as the mean ± SEM.

2.7.1. Ethanol treatmentAll animals were ethanol naive before acute ethanol adminis-

tration. A standard protocol for ethanol exposure was used, whichinvolves a single i.p. injection of 4 g/kg ethanol (20%w/v in 0.9%saline) administered to mice two hours into the dark phase of thecircadian rhythm. This protocol has been shown to increase CNSexcitability during the period of declining ethanol levels followingthe acute high dose of ethanol (Crabbe, 1998; Crabbe et al., 1983;McQuarrie and Fingl, 1958; Roberts et al., 1992).

2.7.2. Recovery of righting reflex and blood ethanol levelsPotential differences in the pharmacokinetics of ethanol be-

tween IL-6 tg and non-tg mice were assessed by measurements ofrecovery of righting reflex and blood ethanol levels. The time torecovery of righting reflex reflects the sedative effects of ethanoland total sleeping time for each animal. Righting reflex wasassessed by placing the animal on its back after the ethanoladministered and recording the duration (in minutes) before itrighted itself (all 4 paws touching the floor). Retro-orbital bloodsamples (50 ml) to determine blood ethanol levels were taken at thetime of recovery of righting reflex. Plasma (5 ml) was used for bloodethanol measurement using an Analox GM 7 analyzer (Analox In-struments LTD, Lunenberg, MA). The reaction is based on theoxidation of ethanol by alcohol oxidase in the presence of molec-ular oxygen (ethanol þ O2 / acetaldehyde þ H2O2). The rate ofoxygen consumption is directly proportional to the ethanol con-centration. Single point calibrations are done for each set of sam-ples with reagents provided by Analox Instruments (0.025e0.400 g%). A statistically significant difference was determined by the un-paired t-test.

2.7.3. Handling-induced convulsions (HIC)HIC were quantified using a scale developed by Goldstein and

Pal (Goldstein and Pal, 1971), which is used extensively in the field(e.g., (Crabbe et al., 1980; Farook et al., 2008; Finn et al., 2007;Ghozland et al., 2005; Metten et al., 2010; Olive and Becker,2008)). Briefly, this procedure involves lifting the mouse by thetail and observing it for possible convulsions. If none occur, themouse is gently spun 180� by rubbing the tail between the thumband forefinger. Convulsions are scored on a 6 point scale rangingfrom facial grimace to severe tonic-clonic convulsions (highernumbers reflect more severe behavioral signs). Baseline HIC wasassessed prior to ethanol injection. Mice were scored for HIC at 2, 4,6, 8, 12 and 24 h following ethanol administration by a singleexperimenter blind to the animals’ experimental history. A statis-tically significant genotypic difference was determined by repeatedmeasures ANOVA.

2.7.4. Electroencephalogram (EEG)EEG activity was measured in a separate cohort of animals

before and after 4 g/kg ethanol administration (i.p.) to assess morequantitatively neuronal activity changes across the same timecourse used in HIC studies. EEG analysis was performed as previ-ously described (Hedlund et al., 2005; Huitron-Resendiz et al.,2005, 2004). EEG data were recorded from stainless steel screwelectrodes implanted on the frontal and parietal bone over thehippocampus (coordinates: 2.0 mm posterior and 2.0 mm lateral tobregma according to The Mouse Brain in Stereotaxic Coordinatesfrom Franklin and Paxinos, 1997). A fourth EEG electrode wasimplanted over the cerebellum and used to ground the animal toreduce signal artifacts. Insulated leads from the EEG electrodeswere crimped to male pins (220-P02) and then cemented to theskull with dental acrylic. Following surgical implantation undergeneral anesthesia (1e1.5% isoflurane), mice were allowed 2 weeksto recover prior to the study.

To record EEG, mice were connected to commutators (Plasti-cOne) with flexible recording cables allowing their unrestrictedmovements within the cage and habituated to the recording cagesfor 24 h. Ethanol was administered as in HIC tests (4 g/kg, i.p.) andthe EEG was recorded for an additional 12 h. Electomyograph(EMG) signals were also recorded. EEG and EMG signals wereamplified in a Grass Model 7D polygraph in a frequency range of0.3e10 KHz. The EEG and EMG are displayed on a computermonitor and storedwith a resolution of 128 Hz in the hard drive of acomputer for the off-line spectral analysis and investigation ofpossible seizure activity using software supplied by Kissei Comptec.EEG power, a measure of EEG amplitude as a function of frequency(microvolts squared per Hz), was analyzed using 4-s epochs at delta(0.5e4.0 Hz), theta (4e9 Hz), alpha (6e12 Hz) and gamma(30e45 Hz and 45-1-100 Hz) frequencies. At some time points,records were inspected using 15-s epochs to obtain the total timespent in wakefulness, slow wave sleep (SWS) and rapid eyemovement (REM) sleep. A statistically significant genotypic differ-ence was determined by repeated measures ANOVA.

3. Results

3.1. Acute ethanol reduces synaptic responses in non-tg but not IL-6tg hippocampal slices

To determine if the effect of acute ethanol on synaptic responseswas altered in the IL-6-tg hippocampus, I/O relationships for thefEPSP (Fig. 1) and PS (Fig. 2) elicited by Schaffer collateral stimu-lation were studied in the CA1 region of IL-6-tg and non-tg hip-pocampal slices. Measurements of I/O relationships were madeunder baseline control conditions and in the presence of ethanol.

Fig. 1. Ethanol differently altered the slope of the fEPSP in hippocampal slices from IL-6 tg vs. non-tg mice. (A1) Representative traces of the fEPSP before and after acute applicationof 20 mM ethanol (EtOH) in non-tg and IL-6 tg hippocampal slices. For each genotype and treatment, two traces are overlaid showing fEPSP at low intensity (#1) and high intensity(#2) stimulation (applied at the arrow) within the respective I/O protocol. (A2) Representative graph showing I/O relationships for fEPSP slopes (mean ± SEM) over the range ofstimulus intensities tested in non-tg hippocampal slices before (open symbols) and after (filled symbols) application of 20 mM ethanol. (B) Graph showing mean (±SEM) normalizedvalues (ethanol/baseline) for fEPSP slopes at various stimulus intensities from 80 to 200 mA for non-tg (triangle) and IL-6 tg (circle) slices exposed to 20 mM (open symbols) or60 mM (closed symbols) ethanol. Dashed line indicates control values (i.e., 1) for IL-6 tg and non-tg slices. (C) Mean (±SEM) values for normalized fEPSP slopes averaged on anindividual slice basis over the range of stimulus intensities used for studies of 20 mM (stripped bar) and 60 mM (solid bar) ethanol in non-tg and IL-6 tg hippocampal slices. Dashedline indicates control values (i.e., 1) for IL-6 tg and non-tg slices. In non-tg slices, 20 mM and 60 mM ethanol significantly depressed the fEPSP slope. In the IL-6 tg slices, 20 ethanolhad no effect on the fEPSP slope, whereas 60 mM ethanol enhanced the fEPSP slope. *Significant difference between IL-6 tg and non-tg slices for the same ethanol concentration(Repeated measures ANOVA, p < 0.05). @Significant difference from baseline values (i.e., 1) for the same genotype (one sample t-test, p < 0.05). #Significant difference between20 mM vs. 60 mM ethanol for the same genotype (unpaired t-test, p < 0.05).

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Analysis involved a within slice comparison after normalization ofthe I/O data. For comparisons within a genotype, mean normalizeddata (collapsed across stimulus intensity) were used.

Results showed a significant genotypic difference (repeated-measures ANOVA) in the normalized fEPSP slopes for both20 mM (F(1,36) ¼ 15.02, p ¼ 0.0004) and 60 mM ethanol(F(1,14)¼ 13.996, p¼ 0.0022), with ethanol producing a depressionof the fEPSP slope in the non-tg slices, and an enhancement of thefEPSP slope in the IL-6 tg slices (Fig. 1A and B). Analysis of meannormalized data showed that the small depressions of the fEPSPslope produced by both 20 mM (~10%) and 60 mM (~7%) ethanol inthe non-tg slices were significantly different from the respectivebaseline control values (i.e., 1; one group t-test, 20 mM, p¼ 0.0004;60 mM, p ¼ 0.0076) (Fig. 1C). The depressions of the fEPSP slopeproduced by 20 mM and 60 mM ethanol were not significantlydifferent in the non-tg slices (p ¼ 0.43, unpaired t-test), perhapsbecause the effect was maximal at 20 mM (Fig. 1C). In contrast, inthe IL-6 tg slices 20mM ethanol did not significantly alter the fEPSPslopes relative to control values (i.e., 1; p ¼ 0.26, one group t-test),while 60 mM ethanol significantly increased (~17%) the fEPSPslopes relative to control values (p ¼ 0.028, one group t-test)(Fig. 1C). The effects of 20 mM and 60 mM ethanol on the fEPSPslopes were significantly different in the IL-6 tg slices (p ¼ 0.013,unpaired t-test) (Fig. 1C).

A significant genotypic difference (repeated measures ANOVA)was also observed for the effect of 20 mM (F(1,36) ¼ 11.91,p¼ 0.0014) and 60 mM (F(1,16)¼ 18.29, p¼ 0.0006) ethanol on thenormalized PS amplitude, with ethanol producing a depression ofthe PS in the non-tg slices and an enhancement of the PS in the IL-6tg slices (Fig. 2A,B). Analysis of mean normalized data showed thatin the non-tg slices, 20 mM ethanol did not significantly alter PSamplitude (~3% depression, p ¼ 0.46, one group t-test) relative tocontrol values (i.e., 1), whereas 60 mM ethanol produced a signif-icant depression (~20%, p ¼ 0.003, one group t-test) (Fig. 2C). Theeffects of 60 mM vs. 20 mM ethanol on the PS amplitude weresignificantly different in the non-tg slices (p ¼ 0.042, unpaired t-test) (Fig. 2C). In contrast, in the IL-6 tg slices ethanol significantlyenhanced the PS amplitude relative to control values (i.e., 1) at both20 mM (p ¼ 0.003, one group t-test) and 60 mM (p ¼ 0.03, onegroup t-test) (Fig. 2C). The enhancement of the PS amplitude pro-duced by 20 mM (~38%) and 60 mM (~23%) ethanol were notsignificantly different in the IL-6-tg slices (p ¼ 0.41, unpaired t-test)(Fig. 2C). Thus, the depressive effect of acute ethanol on hippo-campal synaptic responses in the non-tg slices was transform intoan enhancement in the IL-6 tg slices.

Fig. 2. Ethanol depresses the PS in non-tg hippocampal slices but enhances the PS in IL-6 hippocampal slices. (A1) Representative traces of PS evoked by Schaffer collateralstimulation (applied at the arrow) before and after acute application of 20 mM ethanol (EtOH) in non-tg and IL-6 tg slices. (A2) Representative graph showing I/O relationships forPS amplitudes mean (±SEM) over the range of stimulus intensities tested in IL-6 tg slices before (open symbols) and after (filled symbols) application of 20 mM ethanol. (B) Graphshowing normalized (ethanol/control) values (mean ± SEM) for PS amplitudes for the range of stimulus intensities studied (100e180 mA) for non-tg and IL-6 tg slices. Dashed lineindicates control values (i.e., 1) for IL-6 tg and non-tg slices. (C) Mean normalized values (±SEM) for PS amplitudes averaged on an individual slice basis over the range of stimulusintensities used for studies of 20 (stripped bar) and 60 mM (solid bar) ethanol. Dashed line indicates control values (i.e., 1) for IL-6 tg and non-tg slices. Ethanol significantlydepressed the PS amplitude in non-tg slices at 60 mM ethanol but not 20 mM ethanol, whereas the PS amplitude in IL-6 tg slices was increased by both 20 mM and 60 mM ethanol.*Significant difference between IL-6 tg and non-tg slices for the same ethanol concentration (Repeated measures ANOVA, p < 0.05). @Significant difference from baseline values (i.e.,1) for the same genotype (one sample t-test, p < 0.05). #Significant difference between 20 mM and 60 mM ethanol for the same genotype (unpaired t-test, p < 0.05).

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3.2. Effects of ethanol on synaptic plasticity are altered in the IL-6tg hippocampus

Experience-dependent changes in synaptic transmission,referred to as synaptic plasticity, play an important role in hippo-campal functions such as learning and memory, and are known tobe altered by ethanol (reviewed in (McCool, 2011; Zorumski et al.,2014)). Such changes can be identified experimentally by alter-ations in the magnitude of synaptic responses elicited by specificpaired-pulse or high frequency stimulation protocols. Synapticplasticity elicited by paired-pulse stimulation was determined us-ing a standard protocol and quantified by the PPR. In both IL-6 tgand non-tg hippocampal slices, PPR of the fEPSP slope was greaterthan 1 (mean PPRs ~ 1.8 to 1.3) at all paired-pulse intervals tested(40, 100 and 200 ms) under both baseline and ethanol conditions,indicating paired-pulse facilitation (PPF). PPF is thought to resultfrom an increased probability of transmitter release from presyn-aptic terminals (Zucker and Regehr, 2002). PPF was comparable inthe IL-6 tg and non-tg slices in the presence and absence of 20 mMor 60 mM ethanol (not shown), indicating a lack of effect of ethanolor genotype on this form of synaptic plasticity.

A second form of plasticity assessed by paired-pulse stimulation(at 10 or 20 ms interpulse interval) is measured in the somaticregion and reflects the level of excitability of the somatic region(Fig. 3). Excitability in the somatic region is a function of the actionsof somatic/dendritic ion channels that mediate excitability incombination with the effects of recurrent GABAergic inhibitorysynaptic transmission in the vicinity of the pyramidal cell layer. Inour studies, PPR of the PS under baseline conditions was �1 forsome hippocampal slices from both the IL-6 tg and non-tg mice,indicating no effect or a facilitation of the PS by paired-pulse

stimulation in these slice (Fig. 3B). These data imply that inhibi-tory influences, which would produce inhibition of the second PS ofthe pair (i.e., PPR<1), were not prominent in those IL-6 tg and non-tg slices. However, genotypic differences in the effect of ethanol onPPR of the PS were independent of whether or not PPR of the PSshowed inhibition or facilitation under baseline conditions. Facili-tation vs. inhibition for the PPR of the PS presumably results fromthe angle at which the slices were cut, although regional differencesin level of synaptic inhibition in the hippocampus could alsocontribute (Papatheodoropoulos et al., 2002; Petrides et al., 2007).

Exposure to both 20 mM and 60 mM ethanol produced a sig-nificant increase in the PPR for the PS in the non-tg slices at boththe 10 and 20 ms paired-pulse intervals, indicating ethanol pro-duced increased excitability during repetitive stimulation (Fig. 3).20mM ethanol also increased the PPR for the PS in the IL-6 tg slices,but only at the 20 ms paired-pulse interval (Fig. 3). There was noeffect of 60 mM ethanol on PPR for the PS at either at 10 ms or20 ms paired-pulse intervals in the IL-6 tg slices (Fig. 3B). Thus,ethanol consistently enhanced PPR of the PS in the non-tg slices,whereas the IL-6 tg slices weremore resistant to this ethanol effect.

We also examined the effect of ethanol on long-term synapticplasticity of the fEPSP induced by HFS of the Schaffer collaterals.HFS produced a prolonged increase in the fEPSP slope in both IL-6tg and non-tg slices characterized by an initial peak (i.e., PTP) thatoccurred immediately after the termination of the HFS protocol andlasted for 2e3min, a subsequent declining phase of the fEPSP slopethat lasted ~30 min (i.e., STP), followed by a stable, enhancement ofthe fEPSP slope that lasted up to 60 min following the inductionprotocol (i.e., LTP) (Fig. 4). PTP is thought to result from a presyn-aptic enhancement of synaptic vesicle release from the reserve poolthat lasts for seconds to minutes after HFS (Zucker, 1996). Both

Fig. 3. The PPR for the PS amplitude showed genotypic differences. (A) Representative traces show PS evoked by 20 ms interval paired stimulation in non-tg and IL-6 tg hippo-campal slices under control conditions and in the presence of 20 mM ethanol. (B) Graph shows mean values for the PS PPR for 10 and 20 ms paired pulse intervals under controlconditions and in the presence of ethanol. Dashed line indicates control values (i.e., 1) for IL-6 tg and non-tg slices. In the non-tg hippocampal slices, 20 mM and 60 mM ethanolproduced a small but significant increase in the PS PPR at 10 and 20 ms stimulus intervals. In the IL-6 tg hippocampal slices, 20 mM ethanol increased the PS PPR but only at the20 ms stimulus interval, whereas 60 mM ethanol did not alter the PS PPR. 1st ¼ response to first stimulus of the paired stimulation; 2nd ¼ response to the second stimulus of thepaired stimulation. *Significantly different from control PS PPR of the same genotype (paired t-test, p < 0.05).

Fig. 4. Ethanol significantly depressed long-term synaptic plasticity of the fEPSP in the non-tg hippocampal slices but not in the IL-6 tg hippocampal slices. (A1,B1) Representativetraces of fEPSPs evoked by Schaffer collateral stimulation (at the arrow) from non-tg (A1) and IL-6 tg (B1) hippocampal slices before HSF was applied and 60 min after HFS, when LTPwas established, under control conditions (0 mM EtOH) and in the presence of 60 mM ethanol. (A2, B2). Graphs show mean values for the fEPSP slopes during the LTP experimentsin non-tg (A2) and IL-6 tg (B2) hippocampal slices under control conditions and in the presence of 20 or 60 mM ethanol. fEPSP slopes are expressed as a percentage of mean baselinefEPSP slope prior to HSF. HSF was delivered at the arrow. (A3,B3) Mean values for PTP, STP and LTP in the non-tg (A3) and IL-6 tg (B3) hippocampal slices under control conditionsand in the presence of ethanol. Mean PTP was determined for each slice by averaging the first 3 measurements after HSF. Mean STP was determined for each slice by averaging themeasurements from 15 to 25 min after HSF. Mean LTP was determined for each slice by averaging measurements from 50 to 60 min after HSF. *Significantly different from 0 ethanolvalues (ANOVA followed by Fisher's PLSD, p < 0.05).

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presynaptic and postsynaptic mechanisms appear to be involved inSTP (Erickson et al., 2010; Lauri et al., 2007), which is thought toplay a role in short-term memory (Erickson et al., 2010). LTP pri-marily results from increased membrane expression of AMPA re-ceptors and is considered to be an important cellular model for

memory and learning (Miyamoto, 2006; Peng et al., 2011).Under control conditions (ACSF superfusion), the increase in the

fEPSP slope induced by HFS was similar for IL-6 tg and non-tg slicesfor time periods that reflect PTP, STP and LTP. In non-tg slices,exposure to acute ethanol (20 mM and 60 mM) significantly

R.V. Hernandez et al. / Neuropharmacology 103 (2016) 27e43 35

reduced LTP in a dose-dependent manner (Fig. 4A), as expectedbased on previous studies of ethanol actions on synaptic plasticity(reviewed in (McCool, 2011; Zorumski et al., 2014)). For example,60 mM ethanol completely blocked LTP in the non-tg slices,consistent with studies by others (Ramachandran et al., 2015).Exposure to 60 mM ethanol also significantly reduced PTP (~45%)and STP (~40%) in the non-tg slices (Fig. 4A2 and A3). In contrast,these concentrations of ethanol had no effect on LTP, PTP or STP inthe IL-6 tg slices (Fig. 4B). Thus, in the IL-6 tg slices several forms ofsynaptic plasticity mediated by presynaptic or postsynapticmechanisms showed a resistance to the effects of ethanol. Resultsfrom these electrophysiological studies are summarized in Table 1,and support the proposal that increased expression of astrocyteproduced IL-6 can lead to altered effects of ethanol on synapticfunction.

3.3. Acute ethanol does not result in STAT3 or p42/44 MAPKactivation

Acute ethanol (4 g/kg, i.p.) has been reported to increase (~350%)IL-6 mRNA in the hippocampus of rats when measured 3e9 h postethanol administration (earlier times not measured) (Doremus-Fitzwater et al., 2014). Acute ethanol (100 mM, 24 h treatment)has also been reported to induce secretion of IL-6 (~5 fold) from ratcortical astrocytes in primary cultures, from ~50 pg/mg cell proteinunder control conditions to ~ 250 pg/mg cell protein (Sarc et al.,2011). These data raised the possibility that ethanol exposure ofthe hippocampal slices induced IL-6 (i.e., acute IL-6) production byastrocytes or other cell types, in which case differences in effects ofthe acute IL-6 could contribute to the observed genotypic differ-ences in the effects of ethanol on synaptic function reported above.The acute IL-6 would presumably activate IL-6Rs on nearby cellsand cause functional changes through downstream signaling. IL-6Rs are expressed on several cell types in the hippocampus,including neurons and glia (Gruol, 2015). To assess this possibilitywe carried out two types of experiment: (a) we measured by ELISAthe levels of IL-6 in a separate group of IL-6 tg and non-tg hippo-campal slices treated with control saline (ACSF) or 60 mM ethanolfor 30 min, and (b) we measured the relative level of activation ofdownstream signal transduction molecules activated by IL-6.

As reported previously, levels of IL-6 in slices studied in vitrowere higher than in samples from whole hippocampus immedi-ately snap frozen after dissection (Gruol et al., 2014). In addition,levels of activated p42/44MAPKwere not higher in the IL-6 tg slicescompared with the non-tg slices, as was observed for the samplesfrom whole hippocampus (Nelson et al., 2012). These differencescould result from the in vitro protocol used to prepare the slices(Jankowsky et al., 2000). To adjust for potential effects of thein vitro protocol on IL-6 levels in the slices and potential differencesin the levels of IL-6 across animals of the same genotype, IL-6 levelsin ethanol-treated slices were normalized to IL-6 levels in controlslices obtained from the same animal and exposed to ACSF rather

Table 1Summary of effects of ethanol on electrophysiological properties.

Parameter measured Non-tg

20 mM EtOH 60 m

fEPSP slope Y Y

PS No D Y

PPF No D No DPPR [ [

PTP No D Y

STP No D Y

LTP Y Y

than ethanol. IL-6 levels varied across the slices for both IL-6 tg andnon-tg slices, ranging from 12 to 80 pg/ml for both IL-6 tg and non-tg slices under control conditions. Normalization (ethanol/control)showed that in the non-tg slices, ethanol exposure significantlyincreased (one sample t-test) IL-6 levels by approximately 50%(mean normalized value ¼ 1.50 ± 0.11, n ¼ 3) compared to non-tgslices exposed to control saline. A similar increase was producedby ethanol in the IL-6 tg slices (mean normalizedvalue ¼ 1.49 ± 0.09, n ¼ 3) compared to IL-6 tg slices exposed tocontrol saline. These results are consistent with studies showingthat ethanol causes an increase in IL-6 levels in CNS cells.

IL-6 produces its biological effects through several differentsignal transduction pathways linked to IL-6R, the primary pathwaybeing STAT3 but also p42/44 MAPK. Measurement of the activatedforms of these proteins (i.e., phosphorylated forms; pSTAT3, pp42/44 MAPK) in hippocampal slices exposed to control saline orethanol (60 mM) for 30 min showed no significant effect of ethanolin either the non-tg or IL-6 tg slices (Fig. 5). These results make itunlikely that the differences in the effects of ethanol on synapticfunction between IL-6 tg and non-tg hippocampal slices are due todifferences in the actions of ethanol-induced acute IL-6 productionbetween the IL-6 tg and non-tg hippocampus. However, the po-tential involvement of other ethanol-induced factors in the differ-ential effects of ethanol in IL-6 tg and non-tg hippocampus cannotbe eliminated at this time.

3.4. Ethanol withdrawal hyperexcitability is enhanced in the IL-6 tgmice

The enhanced synaptic responses in the IL-6 tg hippocampalslices produced by acute ethanol and the increased susceptibility ofthe IL-6 tg mice to seizure activity (Campbell et al., 1993; Sallmannet al., 2000), raised the possibility that IL-6 tg mice may be moresusceptible to ethanol-induced withdrawal symptoms such as hy-perexcitability than the non-tg mice. To test this possibility, weexamined the behavioral effects of a single exposure to a high dose(4 g/kg, i.p.) of ethanol, an ethanol exposure protocol that canproduce a mild state of ethanol dependence that increases sus-ceptibility to a hyperexcitable state as blood ethanol levels declinetoward zero.We used both the HIC test (Crabbe et al., 1991) and EEGrecordings, which provided more quantitative data, to assess theeffects of ethanol exposure/withdrawal on CNS activity in the IL-6tg and non-tg mice. A high dose of ethanol (4 g/kg, i.p.) has beenassociated with increased seizure thresholds 8 h following ethanolinjection (McQuarrie and Fingl, 1958) and increased HIC, albeitmild, in C57BL/6J mice 6e12 h following ethanol injection (Crabbe,1998; Crabbe et al., 1983; Roberts et al., 1992). A high dose ofethanol (4 g/kg, i.p.) has also been shown to produce elevated levelsof IL-6 mRNA (~100% increase) in the hippocampus at 3e9 hfollowing ethanol injection (Doremus-Fitzwater et al., 2014).

IL-6 tg

M EtOH 20 mM EtOH 60 mM EtOH

No D [

[ [

No D No D[ No DNo D No DNo D No DNo D No D

Fig. 5. Level of activation of signal transduction molecules associated with IL-6R activation was not altered by acute exposure of hippocampal slices to ethanol (60 mM, 30 min).Graph shows mean (±SEM) values for the relative level of the activated form and total (activated plus non-activated) STAT3 and p42/44 MAPK measured by Western blot in non-tgand IL-6 tg hippocampal slices. For each gel, measurements were normalized to the mean value for the non-tg hippocampus run on the same gel. Normalized values were thenaveraged according to genotype and protein. Representative Western blots are shown above the corresponding bars in the graph. Six slices were studied for each condition. Dottedline shows mean values for non-tg hippocampal slices (i.e., 1).

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3.4.1. PharmacokineticsAdministration of the high dose of ethanol (4.0 g/kg, i.p.) caused

a period of sedation in both the IL-6 tg and non-tg mice charac-terized by a loss of righting reflex. Time to recovery of rightingreflex was similar (unpaired t-test) for the IL-6 tg and non-tg mice(IL-6 tg¼ 107 ± 17 min, n¼ 8; non-tg¼ 94 ± 12 min, n¼ 13). Bloodethanol levels were also similar (unpaired t-test) for the IL-6 tg(408 ± 10 mg/dl, n ¼ 7) and non-tg (411 ± 10, n ¼ 9) mice. Thesemeasures indicate that the pharmacokinetics of ethanol, whichcould influence results, were comparable in the IL-6 tg and non-tgmice. Blood ethanol levels were estimated to return to zeroapproximately 8e12 h after ethanol administration.

3.4.2. HICIL-6 tg and non-tg mice were scored for HIC during a baseline

period and at 2, 4, 6, 8, 12 and 24 h following administration of asingle high dose of ethanol (4.0 g/kg, i.p.). HIC scores at baselineprior to ethanol administration were not significantly different forIL-6 tg and non-tg mice (unpaired t-test). HIC scores followingethanol administration were modestly increased for both the IL-6

Fig. 6. IL-6 tg mice showed a higher level of CNS excitability during withdrawal from acutemeasured over a 24 h period in IL-6 tg (closed circles) and non-tg (open circles) mice and Cdose of ethanol (4 g/kg, i.p.) and tested at several different time points. The IL-6 tg mice shdifference was observed for the CCL2 tg and non-tg mice. Baseline HIC scores were similar foadministration (arrow). *Significantly different from non-tg mice (Repeated measure ANOV

tg and non-tg mice (Fig. 6). The increases at 6e24 h were signifi-cantly larger for IL-6 tg than the non-tg mice (repeated measuresANOVA, F(1,19) ¼ 4.928, p ¼ 0.038), indicating a higher level of CNSactivity in the IL-6 tg mice. A significant increase (unpaired t-test)above baseline occurred at the 8 and 12 h in IL-6 tgmice, but only at8 h in the non-tg mice (Fig. 6), consistent with a higher level of CNSactivity in the IL-6 tg mice.

We also measured HIC in similar studies of CCL2 transgenic(CCL2 tg) mice and their non-transgenic littermate control (CCL2non-tg) mice. Like the IL-6 tg mice, the CCL2 tg mice expresselevated levels of CCL2 through increased astrocyte expression. TheCCL2 tg and non-tg mice showed no genotypic difference in theeffects of a single high dose of ethanol (4.0 g/kg, i.p.) on HIC scores(Fig. 6). These results indicate specificity for the effects of IL-6 onethanol-induced CNS activity during ethanol withdrawal.

3.4.3. EEGCNS activity in EEG recordings (Fig. 6A) was quantified by EEG

power at delta (0.5e4.0 Hz), theta (4e9 Hz), alpha (6e12 Hz), andgamma (30e45 Hz and 45.1e100 Hz) frequencies. In general, the

ethanol as measured by HIC assay. Graph showing mean values (±SEM) for HIC scoresCL2 tg (closed triangle) and CCL2-non-tg (open triangle) mice. Mice were given a highowed significantly higher peak HIC scores than their non-tg littermates. No genotypicr all genotypes, as were the scores for initial time points (2,4, and 6 h) following ethanolA with post hoc Fisher's PLSD, p < 0.05).

R.V. Hernandez et al. / Neuropharmacology 103 (2016) 27e43 37

slow-wave activities (1e4 Hz) of the EEG are associated with sleep,less attentive conditions and drowsiness, while the faster-waveactivities (12e50 Hz) are associated with arousal and attentivestates (Pian et al., 2008).

Potential differences in baseline EEG activity were evaluatedduring a 12 h period (in the dark) one day prior to testing the effectsof ethanol. There were no significant differences between the IL-6tg and non-tg mice in the EEG power for delta, theta and alphafrequency ranges during the baseline period (Fig. 7B1eD1). Incontrast, a significant increase in EEG power for the 30e45 Hzgamma frequency range (~38% increase; repeated measuresANOVA, F(1,8) ¼ 12.798, p ¼ 0.007) was observed during thebaseline period for IL-6 tg mice compared to the non-tg mice(Fig. 7E1). EEG power for the 45.1e100 Hz frequency range was notsignificantly increased in the IL-6 tg mice, although it did show atrend (0.1 > p > 0.05) for an increase (repeated measures ANOVA,F(1,8) ¼ 3.914, p ¼ 0.08) (Fig. 7F1).

On the following day, EEG power was measured at hourly in-tervals during a 12 h period (in the dark). After 2 h of baselinerecording, ethanol was administered (4 g/kg, i.p.). EEG power for allmeasured frequencies declined precipitously after ethanol admin-istration, to a similar extent for IL-6 tg and non-tg mice(Fig. 7B2eF2). Similar administration of saline does not alter EEGpower (unpublished). Recovery of EEG power occurred during thenext 12 h that EEG activity was recorded, with the IL-6 tg miceshowing faster recovery from the depressive effects of ethanol thannon-tg mice (Fig. 7B2eF2). Repeated measures ANOVA confirmed asignificant main effect of genotype for EEG power in the alpha(F(1,8) ¼ 6.306, p ¼ 0.033) and gamma (30e45 Hz, F(1,8) ¼ 11.891,p ¼ 0.009; 45.1e100 Hz, F(1,8) ¼ 8.453, p ¼ 0.02) frequency rangesof the EEG, and also a significant time � genotype interaction forEEG power in the alpha (F(1,11) ¼ 3.363, p ¼ 0.0007) and gamma(30e45 Hz, F(1,11)¼ 3.089, p¼ 0.002; 45.1e100 Hz, F(1,11)¼ 2.306,p ¼ 0.016) frequency ranges. No significant genotype effect wasobserved for EEG power in delta (F(1,8) ¼ 0.487, p ¼ 0.51) or theta(F(1,8) ¼ 4.784, p ¼ 0.06) frequency ranges, although there was asignificant genotype � time interaction for EEG power for both thedelta (F(1,11) ¼ 2.476, p ¼ 0.009) and theta (F(1,11) ¼ 3.571,p ¼ 0.0004) frequency ranges. Also, EEG power for the delta andtheta frequency ranges exceeded baseline levels (paired t-test) atsome recovery time points in the IL-6 tgmice, an effect that was notobserved in non-tg mice (Fig. 7B2 and C2).

When differences in baseline levels of EEG power were takeninto consideration, by expressing the EEG activity at time pointsafter ethanol administration as a percent of the average baselinevalue before ethanol administration for each mouse, EEG power forthe gamma frequency ranges no longer showed a significantgenotypic effect (repeated measures ANOVA for hours 4e12;30e45 Hz, F(1,8) ¼ 0.689, p ¼ 0.43; 45.1e100 Hz, F(1,8) ¼ 0.570,p ¼ 0.47) or genotype � time interactions (30e45 Hz,F(1,11) ¼ 1.245, p ¼ 0.29; 45.1e100 Hz, F(1,11) ¼ 0.992, p ¼ 0.45).These results indicate that the effects of ethanol on EEG gammapower was proportional to baseline levels, which were higher inthe IL-6 tg mice. Differences between the IL-6 tg and non-tg mice inthe timecourse of recovery of EEG power, and the increase in EEGpower over baseline levels observed for other EEG frequencies werenot evident for EEG gamma frequencies (Fig. 7B2eF2). Takentogether, these results show that IL-6 tg mice differ significantlyfrom non-tg mice with respect to both baseline activities of CNScircuits as well as the effect of acute ethanol on these circuits.

To determine if differences in wakefulness contribute to geno-typic differences in EEG power, an analysis of sleep timewas carriedout for a one hour interval starting at the 10 h time point, whensignificant differences were observed in EEG power between the IL-6 tg and non-tg mice. This time point correlates with the 8 h time

point in the HIC studies. There was no significant difference in thepercent of time spent in wakefulness (~60% for IL-6 tg vs. 44% fornon-tg), slow wave sleep (~39% for IL-6 tg vs. 54% for non-tg) orREM sleep (~1% for IL-6 tg vs. 2% for non-tg) between the IL-6 tg andnon-tg mice (p > 0.05, unpaired t-test). Thus, differences in wake-fulness or sleep time during ethanol withdrawal do not appear tocontribute to the genotypic differences in EEG power observedbetween the IL-6 tg and non-tg mice.

4. Discussion

Results from the current studies show that transgenic micegenetically engineered to produce increased astrocyte expressionof IL-6 show altered responses to acute ethanol. The altered re-sponses were demonstrated in electrophysiological studies on theeffects of acute ethanol on synaptic transmission and plasticity inhippocampal slices from the IL-6 tg and non-tg mice, and inbehavioral studies of ethanol withdrawal hyperactivity in the CNSof IL-6 tg and non-tg mice. These results raise the possibility thatethanol consumption will produce similar neuroadaptive changesin the CNS as a consequence of ethanol-induced increases in as-trocytes production of IL-6, an action that could result in persistentalterations in CNS function, contribute to mechanisms involved inthe development of alcohol dependence, and confound disordersthat are co-morbid with alcohol dependence.

4.1. Ethanol and synaptic responses

Our previous studies identified neuroadaptive changes in thehippocampus of IL-6 tg mice that enhanced the fEPSP and the PS(Gruol et al., 2014; Nelson et al., 2012). Here we show that neuro-adaptive changes in the IL-6 tg hippocampus also alter the effects ofacute ethanol on these synaptic responses. Thus, in the IL-6 tghippocampal slices ethanol enhanced the fEPSP and PS, whereas innon-tg hippocampal slices ethanol reduced the fEPSP and PS,consistent with the known depressive actions of ethanol. Incontrast, neuroadaptive changes in synaptic plasticity were notevident in ethanol naïve IL-6 tg hippocampus but were revealed byethanol. Thus, there were no significant differences in the PPR ofthe PS or the magnitude of PTP, STP and LTP between the non-tgand IL-6 tg hippocampal slices from ethanol naïve mice (Nelsonet al., 2012). Acute ethanol enhanced PPR of the PS in the non-tghippocampal slices, but the IL-6 tg slices were more resistant tothis effect of ethanol. Ethanol reduced PTP, STP, and LTP in a dose-dependent manner in the non-tg slices, consistent with the knowndepressive effects of acute ethanol on these forms of synapticplasticity (McCool, 2011; Zorumski et al., 2014). However, In the IL-6 tg hippocampal slices, there was no effect of ethanol on PTP, STP,or LTP.

The neuroadaptive changes that are responsible for the differ-ences in the effects of ethanol on synaptic function between the IL-6 tg and non-tg hippocampus remain to be determined in futurestudies. However, several sites of ethanol action could be involved.For example, the magnitude of the PPR of the PS is a function of theaction of recurrent GABAergic inhibition to the pyramidal neuronand somatic/dendritic ion channels that mediate pyramidal neuronexcitability. Ethanol has been shown to alter recurrent GABAergicinhibition of CA1 neurons (Weiner and Valenzuela, 2006). Inhibi-tory interneurons that provide recurrent GABAergic inhibition toCA1 neurons and express parvalbumin (PAV), a Ca2þ binding pro-tein, show morphological alterations at 2 months of age and cellloss at 6 months of age in the IL-6 tg mice (Campbell et al., 1993;Heyser et al., 1997; Samland et al., 2003). Therefore, functionalchanges in recurrent GABAergic inhibition in the IL-6 tg hippo-campus could contribute to differences between non-tg and IL-6 tg

Fig. 7. EEG analysis revealed differences in power spectra between IL-6 tg and non-tg mice before and after ethanol administration. (A) Representative EEG recordings from IL-6 tgand non-tg mice under baseline conditions, after ethanol administration and during the phase of declining blood ethanol levels (Recovery). (B1eF1). Graphs show mean values(±SEM) of EEG power for different frequency ranges recorded in IL-6 tg (closed circle) and non-tg (open circle) mice during a baseline control period. The IL-6 tg mice showed asignificantly higher level of EEG power for the gamma frequency ranges (E1,F1) than the non-tg mice. This difference was significant for the 30e45 Hz frequency band (E1). (B2eF2)Graphs showing mean values (±SEM) of EEG power at different frequencies obtained during a 12 h recording period that included a baseline period (2 h), injection of a high dose ofethanol (arrow; 4 g/kg, i.p.) and during the period of declining blood ethanol levels (4e12 h). The IL-6 tg mice consistently showed higher EEG power than the non-tg mice for all

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hippocampus in the effects of ethanol on the PPR of the PS.Neuroadaptive changes that affect the activity/expression of

ethanol-sensitive ion channels could also contribute to differencesin the effects of ethanol between the IL-6 tg and non-tg hippo-campus. For example, ethanol has been shown to reduce the ac-tivity/expression of Kþ channels (e.g., KCa2, Kv4), an effect thatincreases excitability (Mulholland et al., 2009, 2015). Kþ channelscontrol excitability in both the somatic and dendritic region ofhippocampal neurons by setting the resting membrane potentialand repolarizing the membrane potential following the depolariz-ing phase of synaptic responses and action potentials. If functionalexpression of ethanol-sensitive Kþ channels was altered in the IL-6tg hippocampus, this difference could contribute to differences inthe effects of ethanol on fEPSP, PS and/or PPR of the PS. Neuro-adaptive changes in NMDA receptor (NMDAR)-mediated synapticresponses could also contribute to the altered effects of ethanol inthe IL-6 tg hippocampus. Our previous studies showed that thepeak amplitude of the NMDAR-mediated fEPSP was larger in the IL-6 tg slices than in the non-tg slices (Nelson et al., 2012). Ethanol isknown to reduce responses mediated by NMDARs (Lovinger et al.,1990; Wirkner et al., 1999). A larger NMDAR component of thesynaptic response in the IL-6 tg slices could lead to a smaller neteffect of ethanol, both with respect to the fEPSP and LTP. Interest-ingly, our studies of cultured cerebellar granule neurons showedthat chronic treatment with IL-6 results in reduced levels of Kv4.2protein (Gruol et al., 2011) and increased NMDAR-mediated re-sponses (Qiu et al., 1995, 1998) in the granule neurons. Thesefindings raise the possibility that astrocyte production of IL-6 in theIL-6 tg hippocampus could result in similar changes in the CA1pyramidal neurons and, consequently, enhanced excitability andaltered response to ethanol. Alternatively or in addition, neuro-adaptive effects of IL-6 could alter the sensitivity of NMDAR- orKv4.2-mediated events to ethanol, a possibility to be pursued infuture studies.

Neuroadaptive changes in the level or activation of signaltransduction molecules utilized by IL-6 including STAT3 and p42/44 MAPK could also play a role in the altered effects of acuteethanol on synaptic plasticity in the IL-6 tg hippocampus. STAT3and p42/44 MAPK are known to be involved in the induction ofhippocampal synaptic plasticity (Nicolas et al., 2012; Sweatt, 2001).Ethanol has been shown to alter p42/44 MAPK and STAT3 activa-tion in CNS tissue, effects that can vary across brain regions(Bachtell et al., 2002; Chandler and Sutton, 2005; Fujita et al., 2003;Kalluri and Ticku, 2002; Roberto et al., 2003). Thus, multipleethanol-sensitive targets could be involved in the neuroadaptivechanges produced in the IL-6 tg CNS and, consequently, in thealtered effects of ethanol.

4.2. Acute ethanol-induced IL-6 production

Measurement of IL-6 levels in hippocampal slices showed thatacute ethanol increased the level of IL-6 in both the IL-6 tg and non-tg hippocampal slices. However, there were no correspondingchanges in the levels of activated STAT3 and p42/44 MAPK, sug-gesting that IL-6 signal transduction was not activated in the hip-pocampal slices, at least at the 30 min time period tested. Previousstudies have shown that effects of IL-6 on these signal transductionmolecules evident with a 30 min IL-6 exposure period (Schumannet al., 1999; Tancredi et al., 2000). Studies of cultured astrocytesshowed that exposure to 50 mM ethanol (24 h exposure) did not

EEG frequencies during the period of declining blood ethanol levels. However, significant dgamma frequency bands when the data were expressed as a percent of baseline (not shownwith post hoc Fisher's PLSD, p < 0.05). @Significant increase over baseline level for the sam

induce increased secretion of IL-6 protein from the astrocytes (Sarcet al., 2011). These results make it unlikely that ethanol-inducedproduction/release of acute IL-6 contributed to the differences inthe effects ethanol on synaptic function between the IL-6 tg andnon-tg slices. Consistent with this interpretation, acute applicationof IL-6 does not alter the fEPSP or PS evoked by Schaffer collateralstimulation in CA1 pyramidal neurons, (Li et al., 1997; Tancrediet al., 2000), making it unlikely that acute IL-6 contributed to theeffects of ethanol on the fEPSP or PS in the IL-6 tg or non-tg slices.Acute application of IL-6 does reduce long-term synaptic plasticity(PTP, STP and LTP) (Li et al., 1997; Tancredi et al., 2000). Thus, IL-6could have played a role in the depressive effects of ethanol onlong-term synaptic plasticity in the non-tg slices but not the IL-6 tgslices, which did not show a similar ethanol-induced depression oflong-term synaptic plasticity.

Ethanol effects on IL-6 signaling could explain the lack ofchanges in the level of activation of STAT3 or p42/44 MAPK in ourstudies of hippocampal slices. The effect of acute ethanol on STAT3activation has not been reported for CNS tissue, but in other celltypes (i.e., liver cells, monocytes) acute ethanol reduces IL-6 acti-vation of STAT3 (Chen et al., 1999; Norkina et al., 2008). Acuteethanol has been reported to inhibit p42/44 MAPK activation incortical and hippocampal tissue (Kalluri and Ticku, 2002, 2003;Spanos et al., 2012).

Although ethanol-induced production of acute IL-6 did notappear to contributed to differences in the effects ethanol on syn-aptic function between the IL-6 tg and non-tg hippocampal slices,acute IL-6 could play a role in genotypic differences observed In thebehavioral studies (HIC and EEG). In these studies, blood ethanollevels were in the 400 mg/dl range (~90 mM), a range where as-trocytes have been shown to secrete IL-6 (Sarc et al., 2011). A highdose of ethanol (4 g/kg, i.p.) has been shown to produce elevatedlevels of IL-6 mRNA (~100% increase) in the hippocampus at 3e9 hfollowing ethanol injection (Doremus-Fitzwater et al., 2014).Therefore, ethanol-induced release of IL-6 from astrocytes couldplay a role in the effects of ethanol on the EEG activity in the IL-6and non-tg mice.

4.3. Ethanol and HIC

The action of acute ethanol to increase the fEPSP and PS in theIL-6 tg hippocampus could extended to other CNS regions andcontribute to the greater CNS excitability of the IL-6 tg miceobserved in the HIC test and EEG recordings. The increased ethanolwithdrawal excitability was specific to the IL-6 tg mice in thatsimilar studies of CCL2 transgenic (CCL2-tg) mice and their non-transgenic littermate control (CCL2-non-tg) mice showed nogenotypic difference in the effects of a single high dose of ethanol(4.0 g/kg, i.p.) on HIC scores. Like the IL-6 tg mice, the CCL2-tg miceexpress elevated levels of CCL2 in the CNS through increasedastrocyte expression (Bray et al., 2013; Huang et al., 2002). Also likeIL-6 tg mice, CCL2 levels are increased in the CNS by ethanol (Heand Crews, 2008; Kane et al., 2013). HIC scores for CCL2 null micewere similar to scores for wildtypemicewhen themicewere testedafter exposure to ethanol in a two-bottle choice drinking paradigm(Blednov et al., 2005), supporting a lack of involvement of CCL2 inethanol withdrawal hyperexcitability.

ifferences in EEG power between IL-6 tg and non-tg mice were not observed for the). *Significant difference between IL-6 tg and non-tg mice (Repeated measures ANOVAe genotype (paired t-test, p < 0.05). Dotted lines indicate baseline levels.

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4.4. Ethanol and EEG activity

Acute ethanol-induced alterations in CNS activity duringethanol withdrawal were also demonstrated by a comparison ofEEG activity in the IL-6 tg and non-tgmice. EEG power for theta andalpha frequencies showing a faster recovery from the depressiveeffects of ethanol, and the delta and theta frequencies showed anovershoot of baseline levels in the IL-6 tg mice compared with non-tg mice, consistent with greater activity in these frequency rangesin the IL-6 tg mice. Thus, neuroadaptive effects in the IL-6 tg mice,although not evident under baseline conditions, were revealed forthe delta, theta, and alpha frequency range when ethanol was onboard.

In contrast, a genotypic difference was observed for EEG powerin the gamma frequency range during baseline recordings and afterethanol administration. However, no genotypic difference ingamma frequency levels was observed after ethanol administrationwhen adjustments were made for baseline differences in gammafrequency levels. Thus, the effect of acute ethanol on activity in thegamma frequency range was proportional to baseline levels, whichwere higher in the IL-6 tg mice. In addition, the timecourse of acuteethanol action for the gamma frequency range was similar for theIL-6 tg and non-tg mice. These results suggest that neuroadaptiveeffects in the IL-6 tg mice and actions of acute ethanol overlap tosome extent with mechanisms responsible for generation orregulation of gamma frequency activity.

The higher EEG power in the gamma frequency range in the IL-6tg mice under baseline conditions indicates a higher level of CNSactivity than in the non-tg mice, a condition that could contributeto the increased susceptibility of the IL-6 tg mice to kainate- andNMDA-induce seizure activity (Samland et al., 2003). Consistentwith this possibility, systemic administration of kainate has beenshown to cause generalized non-convulsive discharges character-ized by increased gamma activity (30e40 Hz) in the hippocampusand neocortex of rats prior to the expression of seizure activity(Medvedev et al., 2000).

4.5. Gamma frequency activity

Gamma frequency activity is observed in many brain areasduring different behavioral states and is thought to arise throughlocal circuit activity involving excitatory, glutamate containingpyramidal neurons, and inhibitory, GABAergic interneurons thatexpress PAV (Buzsaki and Wang, 2012; Ferando and Mody, 2013;Mann and Mody, 2010). Thus, neuroadaptive effects on local cir-cuit activity could contribute the increased gamma frequency ac-tivity observed under baseline conditions in the IL-6 tg mice. Thealterations in GABAergic interneurons that express PAV observed inthe hippocampus and cortex of IL-6 tg mice are consistent withaltered local circuit activity in the IL-6 tg hippocampus (Campbellet al., 1993; Heyser et al., 1997; Samland et al., 2003). Also, ourWestern blot analysis of the level of GAD65/67, the syntheticenzyme for GABA, showed reduced levels in the IL-6-tg hippo-campus compared to the non-tg hippocampus, although no geno-typic difference was observed in PAV levels (Gruol et al., 2014;Nelson et al., 2012). Thus, effects on inhibitory interneurons couldcontribute to the increased EEG power in the gamma frequencyrange in the IL-6 tg mice.

4.6. IL-6 expression in the IL-6 tg mice

Although the neuroadaptive changes in the IL-6 tg mice thatunderlie the altered response to ethanol have yet to be identified,several lines of evidence indicate that the changes are directly orindirectly related to the biologic actions of transgene derived IL-6.

For example, the highest number of astrocytes expressing thetransgene in the forebrain region of the IL-6 tg mice occurs in thehippocampus (Vallieres et al., 2002), where IL-6 transgeneexpression in astrocytes was demonstrated by expression of thelacZ reporter gene and immunohistochemical detection of b-gal(Vallieres et al., 2002). Studies of supernatant from astrocytescultured from the CNS of IL-6 tg mice showed that IL-6 tg astrocytessecrete significant IL-6 protein (Campbell et al., 1993). Immuno-histochemical studies showed increased levels of activated STAT3(i.e., pSTAT3), primarily in astrocytes and microglia of the IL-6 tgCNS, indicative of activation of IL-6R and consistent with elevatedlevels of IL-6 in the IL-6 tg CNS (Sanz et al., 2008). The IL-6 tg CNSshows increase expression of IL-6 regulated genes (e.g., GFAP, eb22,Socs3), increased levels of GFAP, and increased levels of activatedSTAT3 (pSTAT3), the signal transduction molecule through whichIL-6 acts to increase GFAP (Gruol et al., 2014; Herrmann et al., 2008;Nelson et al., 2012; Sanz et al., 2008; Shu et al., 2011). The increasedsensitivity to seizure activity observed in the IL-6 tg mice wasspecific for IL-6 in that a similar sensitivity was not observed intransgenic mice that express elevated levels of TNF-a in the CNSthrough astrocyte expression (Samland et al., 2003). However, thelink to IL-6 could be indirect and result from actions of secondaryeffectors such as other cytokines under the control of IL-6 or otherdownstream factors activated by IL-6.

Although astrocytes are a primary producer of IL-6 in the IL-6 tgCNS, microglia or other cell types that are responders and/or pro-ducers of IL-6 could also play a secondary role. Astrocytes arepresent at a higher density than microglia in the hippocampus(~50% fewer microglia) (Long et al., 1998; Savchenko et al., 2000),making it likely that they play a more prominent role than micro-glia as IL-6 producers in the IL-6 tg hippocampus. Moreover,microglial production of IL-6 is typically associated with prolongedexposure to high doses of ethanol rather than the acute ethanolexposure used in our studies (Marshall et al., 2013; Ward et al.,2014; Zhang et al., 2014; Zhao et al., 2013).

4.7. Summary

Our studies show that neuroadaptive changes in the IL-6 tgmicesignificantly alter the CNS actions of ethanol at both the synapticand behavioral levels. The neuroadaptive changes resulted in anethanol-induced increase in hippocampal synaptic responsesrather than a depression, a resistance of long-term synaptic plas-ticity to the depressive effects of ethanol, and enhanced ethanolwithdrawal hyperexcitability. Although it is unknown if the neu-roadaptive changes in the IL-6 tg mice result from direct or indirecteffects of IL-6, these results support the idea that ethanol-inducedincreases in the levels of IL-6 in the CNS could play a critical role inthe CNS actions of ethanol and in the severity of ethanol-inducedwithdrawal symptoms, which is a contributing factor to the riskfor developing alcohol dependence (Becker and Mulholland, 2014).A role for IL-6 as a risk factor for developing alcohol dependencehas also been noted in other studies. Blednov et al. (Blednov et al.,2012) showed that mice that lack IL-6 (IL-6 null) exhibited lowerethanol preference and consumption thanwildtype control mice ina two-bottle choice behavioral paradigm, suggesting a role for IL-6in the regulation of ethanol consumption. Genomic studies ofalcohol-preferring rats and mice also identified IL-6 signaling as acandidate contributor to alcohol preference (Mulligan et al., 2006).

Interestingly, chronic ethanol exposure/withdrawal reduces thenumber of neurons that immunostain for PAV (Smiley et al., 2015;Udomuksorn et al., 2011; Vongvatcharanon et al., 2010), increasesexpression of GFAP (Chiang et al., 1994; Vongvatcharanon et al.,2010), increases EEG gamma power in the CNS (Cheaha et al.,2014), increases NMDAR-mediated synaptic responses (Carpenter-

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Hyland et al., 2004; Hendricson et al., 2007; Nelson et al., 2005),and reduces Kv4.2 expression (Mulholland et al., 2015). Thus,chronic ethanol exposure/withdrawal can produce some of thesame characteristics as observed in ethanol-naïve IL-6 tg mice orCNS cultures chronically treated with IL-6. These similarities raisethe possibility that ethanol-induced IL-6 production could, directlyor indirectly, play a role in these neuroadaptive effects of chronicethanol exposure/withdrawal, in which case the long-term effectsof IL-6 are an important consideration and could provide alterna-tive targets for therapeutic intervention. An understanding of in-teractions between neuroadaptive effects of IL-6 and ethanol is alsoan important consideration for conditions where CNS injury (e.g.,traumatic brain injury (Teng and Molina, 2014)) or disease (e.g.,depression (Neupane et al., 2014)), is comorbid with alcohol usedisorders, as both conditions produce increased levels of IL-6 in theCNS.

Conflict of interest

The authors have no conflict of interest to declare.

Acknowledgments

Supported by NIAAA Grant AA019261 and the IntegratedNeuroscience Initiative on Alcoholism (INAI)-West grantAA020893.

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