Brain, Behavior, and Immunity xxx (2014) xxx–xxx

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Brain, Behavior, and Immunity

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

Role of the IL-1 receptor antagonist in ethanol-induced regulationof GABAergic transmission in the central amygdala

http://dx.doi.org/10.1016/j.bbi.2014.11.0110889-1591/� 2014 Published by Elsevier Inc.

⇑ Corresponding authors at: The Scripps Research Institute, CNAD, SP30-1150,10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Tel.: +1 858 784 7259; fax:+1 858 784 7405 (M. Bajo). The Scripps Research Institute, CNAD, SP30-1160, 10550North Torrey Pines Road, La Jolla, CA 92037, USA. Tel.: +1 858 784 7262; fax: +1 858784 7405 (M. Roberto).

E-mail addresses: mbajo@scripps.edu (M. Bajo), mroberto@scripps.edu(M. Roberto).

Please cite this article in press as: Bajo, M., et al. Role of the IL-1 receptor antagonist in ethanol-induced regulation of GABAergic transmission in theamygdala. Brain Behav. Immun. (2014), http://dx.doi.org/10.1016/j.bbi.2014.11.011

M. Bajo a,⇑, M.A. Herman a, F.P. Varodayan a, C.S. Oleata a, S.G. Madamba a, R.A. Harris b, Y.A. Blednov b,M. Roberto a,⇑a Committee on the Neurobiology of Addictive Disorders, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USAb Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, TX 78712, USA

a r t i c l e i n f o

Article history:Received 2 October 2014Received in revised form 13 November 2014Accepted 24 November 2014Available online xxxx

Keywords:Il1rn knockout miceCeAGABAA

IPSCsKineret

a b s t r a c t

The IL-1 receptor antagonist (IL-1ra), encoded by the Il1rn gene, is an endogenous antagonist of the IL-1receptor. Studies of Il1rn knockout (KO) and wild type (WT) mice identified differences in several ethanol-related behaviors, some of which may be mediated by GABAergic transmission in the central nucleus ofthe amygdala (CeA). In this study we examined phasic (both evoked and spontaneous) and tonic GABAer-gic transmission in the CeA of Il1rn KO and WT mice and the ethanol sensitivity of these GABAergic syn-apses. The mean amplitude of baseline evoked GABAA-inhibitory postsynaptic potentials (IPSPs), and thebaseline frequency of spontaneous GABAA-inhibitory postsynaptic currents (sIPSCs), but not the fre-quency of miniature GABAA-IPSCs (mIPSCs), were significantly increased in KO compared to WT mice,indicating enhanced presynaptic action potential-dependent GABA release in the CeA of KO mice. InKO mice, we also found a cell-type specific switch in the ongoing tonic GABAA receptor conductance suchthat the tonic conductance in low threshold bursting (LTB) neurons is lost and a tonic conductance in latespiking (LS) neurons appears. Notably, the ethanol-induced facilitation of evoked and spontaneous GABArelease was lost in most of the CeA neurons from KO compared to WT mice. Ethanol superfusionincreased the sIPSC rise and decay times in both KO and WT mice, suggesting ethanol-induced postsyn-aptic effects. The pretreatment of CeA slices with exogenous IL-1ra (Kineret; 100 ng/ml) returned sIPSCfrequency in KO mice to the levels found in WT. Importantly, Kineret also restored ethanol-inducedpotentiation of the sIPSC frequency in the KO mice. These results show that IL-1ra regulates baselineGABAergic transmission in the CeA and is critical for the ethanol effects at these synapses.

� 2014 Published by Elsevier Inc.

1. Introduction

The interleukin 1 (IL-1) family is a group of 11 cytokines, thatinduce a complex network of cytokines to initiate and regulateinflammatory responses (Dinarello, 2011). The proinflammatoryactivities of cytokines IL-1a and IL-1b are regulated by an endoge-nous antagonist (IL-1ra), an IL-1 receptor type 1 (IL-1R1), and adecoy receptor (IL-1R2). Specifically, IL-1ra competes with IL-1for binding sites on IL-1R1 and thus prevents activation of down-stream signaling (Garlanda et al., 2013; Krumm et al., 2014).

Initiating intracellular signaling via the IL-1R1 system is a mul-tistep process involving: IL-1a or IL-1b binding to the extracellulardomain of IL-1R1, recruitment of accessory proteins (e.g., the co-receptor IL-1R1 accessory protein (IL-1RAcP)), formation of areceptor heterodimeric complex (comprised of IL-1a or IL-1b, IL-1RI, and IL-1RAcP), and assemblage of intracellular adaptor pro-teins. This leads to the activation of many intracellular signalingpathways and transcription factors, such as NF-jB, c-Jun N-termi-nal kinase, and p38 MAPK (Cohen, 2014).

Gene expression analyses showed alterations in immune/inflammatory response pathways, including the IL-1/IL-1R system,that were associated with a genetic predisposition to high alcoholconsumption in mice (Mulligan et al., 2006). Behavioral studiesalso suggest the involvement of some of these genes in alcoholdrinking and preference (Blednov et al., 2012). Modulation ofGABAA receptors has been shown to alter many ethanol behaviors(Blednov et al., 2013; Blednov et al., 2003; Boehm et al., 2004), andGABAergic transmission in the CeA plays a critical role in a variety

central

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of alcohol-related behaviors elicited by acute and chronic ethanol(Koob and Volkow, 2010; Roberto et al., 2012). Therefore, wehypothesized that disruption of IL-1R1 signaling, by deletion ofits negative regulator Il1rn, leads to a perturbation of GABAergicneurotransmission in brain regions involved in alcohol responses.In this study, we investigated electrophysiologically basal GABAer-gic transmission and ethanol sensitivity in Il1rn KO and WT mice inthe central nucleus of the amygdala (CeA), a brain region criticalfor alcohol-related behaviors and neuroadaptative mechanismsassociated with alcohol dependence (Roberto et al., 2012). Herewe show that both evoked and spontaneous GABAergic transmis-sion are significantly increased in Il1rn KO compared to WT mice,and there is a difference in the cell-type specific tonic GABAA

receptor conductance. Acute ethanol sensitivity of GABAergicresponses is also altered in Il1rn KO compared to WT mice, andsome of the Il1rn KO cellular phenotypes are rescued by applicationof exogenous IL-1ra (Kineret).

2. Materials and methods

2.1. Animal treatment

We obtained male Il1rn KO (n = 32) and wild type (WT; n = 31)mice from the Animal Resources Center at The University of Texasat Austin, and housed them in a temperature- and humidity-con-trolled room on a 12-h light/dark cycle (lights on at 6:00 a.m.) withfood and water available ad libitum. All procedures were conductedin accordance with the National Institutes of Health Guide for theCare and Use of Laboratory Animals and with the Institutional Ani-mal Care and Use Committee policies of The Scripps Research Insti-tute and The University of Texas at Austin.

2.2. Slice preparation

We anesthetized mice (3–6 months; weight: WT, 27.3 ± 0.6 g;Il1rn KO, 23.0 ± 0.4 g) with 3–5% isoflurane and decapitated andquickly removed the brains and placed them in ice-cold oxygen-ated high-sucrose cutting solution gassed with 95% O2 and 5%CO2. Coronal slices (300 lM) containing the CeA were made usinga Leica 1000S vibratome cutter (Campden, Lafayette, IN). The sliceswere then incubated in a gassed N-methyl-D-glucamine (NMDG)-containing recovery solution (in mM: 93 NMDG, 93 HCl, 2.5 KCl,1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 5 sodium ascor-bate, 2 thiourea, 3 sodium pyruvate, 10 MgSO4�7H2O, 0.5 CaCl2-

�2H2O) for 15 min at 31 �C followed by incubation in gassedartificial cerebrospinal fluid (aCSF, in mM: 130 NaCl, 3.5 KCl, 1.25NaH2PO4�H2O, 1.5 MgSO4�7H2O, 2.0 CaCl2�2H2O, 24 NaHCO3, and10 glucose) for 1 h at room temperature. We applied ethanol(44 mM in aCSF) directly onto the slice, and took measurementsbefore ethanol (baseline), during its superfusion (10–15 min), andfollowing washout (20–30 min). Since Kineret’s ability to passthe blood–brain barrier is limited (Gutierrez et al., 1994), we pre-treated the CeA slices with Kineret (100 ng/ml in aCSF; (Clarket al., 2008)) for 30–90 min prior to electrophysiological recordings(Fox et al., 2010; Galea et al., 2011).

2.3. Intracellular recording of evoked responses

We recorded from the medial subdivision of the CeA with sharpmicropipettes filled with 3 M KCl using bridge current-clamp mode(Cruz et al., 2012; Haubensak et al., 2010; Roberto et al., 2004). Mostneurons were held near their resting membrane potential (RMP).We acquired data with an Axoclamp-2A preamplifier (MolecularDevices, Foster City, CA) and stored them for later analysis usingClampfit software (Molecular Devices). We evoked pharmacologi-

Please cite this article in press as: Bajo, M., et al. Role of the IL-1 receptor antagoamygdala. Brain Behav. Immun. (2014), http://dx.doi.org/10.1016/j.bbi.2014.11

cally-isolated GABAA receptor-mediated inhibitory postsynapticpotentials (IPSPs) by stimulating locally within the CeA through abipolar stimulating electrode while superfusing the slices with theglutamate receptor blockers, 6,7-dinitroquinoxaline-2, 3-dione(DNQX; 20 lM) and DL-2-amino-5-phosphonovalerate (DL-AP5;30 lM), and the GABAB receptor antagonist (CGP 55845A; 1 lM).At the end of some recordings, we superfused either 30 lM bicucul-line or 50 lM picrotoxin to confirm the GABAA receptor specificityof the IPSPs. To determine the synaptic response parameters foreach cell, we followed an input–output (I–O) protocol (Robertoet al., 2003; Roberto et al., 2004) consisting of a range of five currentstimulations (50–250 lA; 0.125 Hz), starting at the threshold cur-rent required to elicit an IPSP and ending with the strength requiredto elicit maximum amplitude. These stimulus strengths were main-tained for all I–O protocols throughout the entire duration of theexperiment. To determine changes in synaptic response, we calcu-lated the IPSP amplitude with the stimulus strength adjusted suchthat the amplitude of the IPSP was 50% of maximal determined fromthe I–O relationship. Paired pulse facilitation (PPF) in each neuronwas examined by using paired stimuli at 50- and 100-ms inter-stim-ulus intervals (Roberto et al., 2004). The PPF ratio was calculated asthe second IPSP amplitude divided by the first IPSP amplitude.

2.4. Whole-cell patch-clamp recording

We performed whole-cell patch-clamp recording in the voltageclamp mode as described previously (Bajo et al., 2011; Hermanet al., 2013). We used infrared/DIC visualization (Dodt andZieglgansberger, 1990), followed by digitization and imageenhancement, via an upright, fixed-stage Olympus microscopeand EXi Blue camera (QImaging software) to facilitate visualizationof the CeA neurons. We recorded pharmacologically-isolatedGABAA receptor-mediated spontaneous inhibitory postsynapticcurrents (sIPSCs) by applying blockers of glutamatergic (20 lMDNQX, 30 lM DL-AP5) and GABAB (1 lM CGP 55845A) receptors,and we recorded miniature GABAA-mediated IPSCs (mIPSCs) byadding 0.5 lM tetrodotoxin (TTX) to the bath. We used pipetteswith input resistance 3–7 MX (access resistance <18 MX) filledwith an internal solution (containing in mM: 145 KCl, 10 HEPES,2 MgCl2, 5 EGTA, 2 Na-ATP, and 0.2 Na-GTP, the latter two addedfresh on the day of recording; pH 7.3–7.4, osmolarity 275–290 mOsm). For data acquisition, we used Multiclamp 700B andpClamp 10.2 software (Molecular Devices, Foster City, CA).

2.5. Data analysis and statistics

To analyze data acquired from intracellular and whole-cellrecordings, we used Clampfit 10.2 (Molecular Devices) and Mini-Analysis 5.1 software (Synaptosoft Inc., Leonia, NJ), respectively.All 143 cells were clamped at �60 mV for the duration of therecording. In all experiments, series resistance (<10 MX) was con-tinuously monitored with a 10 mV hyperpolarizing pulse, andexperiments with >20% change in series resistance were rejectedin the final analysis. Frequency, amplitude, and decay of IPSCs wereanalyzed and visually confirmed using a semi-automated thresh-old based detection software (MiniAnalysis, Synaptosoft Inc.). Wedetermined averages of IPSC characteristics from baseline andexperimental drug conditions containing a minimum of 60 sponta-neous events. In voltage clamp recordings, we determined toniccurrents using Clampfit 10.2 (Molecular Devices) and a previouslydescribed method (Glykys and Mody, 2007) in which the meanholding current (i.e., the current required to maintain the�60 mV membrane potential) was obtained by a Gaussian fit toan all-points histogram over a 5-s interval. The all-points histo-gram was constrained to eliminate the contribution of IPSCs tothe holding current. We quantified responses as the difference in

nist in ethanol-induced regulation of GABAergic transmission in the central.011

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holding current between baseline and experimental conditions.We used GraphPad Prism 5.0 (GraphPad Software, San Diego, CA)and Sigma Plot software for all statistical analysis of resultsobtained by whole-cell recordings. We accepted statistical signifi-cance at the p < 0.05 level using one- and two-way ANOVA andStudent’s t-test.

2.6. Drugs

We purchased CGP 55845A, DNQX, and DL-AP5 from Tocris Bio-sciences (Ellisville, MI); GBZ (gabazine), THIP (gaboxadol), and TTXwere from Sigma (St. Louis, MO). Human IL-1ra (Kineret, AmgenInc., Thousand Oaks, CA) was from The University of Texas atAustin Pharmacy (Austin, TX).

Fig. 1. Basal evoked GABAergic transmission is enhanced in the CeA of Il1rn KOcompared to wild type (WT) mice. (A) Input–output curves of mean GABAA IPSP

3. Results

3.1. Baseline evoked CeA GABAergic responses are elevated andethanol-induced increases in these responses are absent in neuronsfrom Il1rn KO compared to WT mice

We first examined if there were any differences in baselineevoked GABA transmission in CeA neurons from Il1rn KO and WTmice. We recorded intracellularly with sharp pipettes from 30 neu-rons. There were no significant differences in voltage–current rela-tionships (data not shown) or membrane properties between KOand WT neurons; thus we combined the data from the two groups,resulting in a mean resting membrane potential (RMP) of�79.8 ± 0.8 mV and a mean input resistance of 140.2 ± 6.5 MX.We pharmacologically isolated GABAA IPSPs and stimulated locallywithin the medial subdivision of the CeA. Baseline IPSP input–out-put curves generated by equivalent stimulus intensities were signif-icantly higher in Il1rn KO compared to WT (Fig. 1A), suggestingincreased GABAergic transmission in the CeA of KO mice.

We next studied the effect of acute superfusion (10–15 min) of44 mM ethanol on evoked GABAergic transmission in CeA neuronsfrom WT and Il1rn KO mice. We used 44 mM ethanol, as we haveshown that this concentration provided a maximum ethanol-induced potentiation of the GABAergic transmission in the CeA(Nie et al., 2004; Roberto et al., 2003, 2004). Ethanol significantlyincreased the peak amplitude by 30% in WT (Fig. 1B; histograminsert), but there was no significant ethanol effect in KO neurons(Fig. 1B). As depicted in the time course of Fig. 1B, the maximumethanol-induced facilitation of GABA responses (evoked usinghalf-maximal strength) occurred within 10–12 min with recoveryupon washout. We examined paired-pulse facilitation (PPF) of theIPSPs at 50- and 100-ms interstimulus intervals to assess pre- ver-sus post-synaptic mechanisms. The ethanol-induced facilitation inCeA of WT mice, but not KO mice, was associated with a significantdecrease in the PPF ratios of IPSPs (Fig. 1C), suggesting increasedGABA release upon ethanol application only in the WT neurons.

amplitudes are significantly increased over all stimulus strengths in Il1rn KO(n = 16) compared with WT (n = 14) neurons (t144 = 2.189; ⁄p < 0.05). Top insert:Representative evoked IPSPs from WT and Il1rn KO mice. (B) Time course depictingchanges in evoked IPSP amplitude (at 50% maximal amplitude determined from theinput–output relationship) upon 44 mM ethanol application and washout in Il1rnKO (n = 13) and WT (n = 11) neurons. Acute application of ethanol significantlyincreases evoked GABAergic transmission in WT, but not KO, with recovery uponwashout of ethanol. Top insert: Representative evoked CeA IPSPs recorded before,during, and after ethanol washout in WT and Ilrn KO mice. Bottom insert:Histograms representing maximal percent increase in mean (±SEM) evoked IPSPamplitude (at half maximal amplitude) with ethanol application; ⁄p < 0.05. (C) Topinsert: Representative recordings of evoked PPF (at 50- and 100-ms interstimulusintervals) of IPSPs in CeA neurons from WT and Il1rn KO mice. Bottom insert:Histograms plotting the PPF ratios of IPSPs before and during ethanol in WT and IlrnKO mice. Acute ethanol significantly (⁄p < 0.05) decreases the PPF ratio of IPSPs fromWT, but not Il1rn KO mice.

3.2. Basal spontaneous GABA release is elevated in the CeA of Il1rn KOcompared to WT mice, and Kineret pretreatment normalizes thisenhanced release

We studied baseline spontaneous inhibitory postsynaptic cur-rents (sIPSCs) mediated by GABAA receptors in CeA of WT and Il1rnKO mice. There was a significantly higher sIPSC frequency in KO(2.7 ± 0.5 Hz) compared to WT (1.0 ± 0.2 Hz) mice, but no signifi-cant differences by genotype in the sIPSC amplitude, rise time, ordecay time (Fig. 2A left panels and B). The increased sIPSC fre-quency suggests enhanced action potential-dependent GABArelease in CeA neurons of Il1rn KO compared to WT mice.

Please cite this article in press as: Bajo, M., et al. Role of the IL-1 receptor antagonist in ethanol-induced regulation of GABAergic transmission in the centralamygdala. Brain Behav. Immun. (2014), http://dx.doi.org/10.1016/j.bbi.2014.11.011

4 M. Bajo et al. / Brain, Behavior, and Immunity xxx (2014) xxx–xxx

Next, we tested the hypothesis that acute application of an exog-enous IL-1R antagonist (Kineret), would reduce the differences insIPSC frequency between the two strains. We pretreated CeA sliceswith 100 ng/ml Kineret for 30–90 min prior to whole-cell recordingsand compared the baseline sIPSCs from untreated and pretreatedslices (Fig. 2 right panels and B). There was a significant main effectof Kineret pretreatment and a significant strain � Kineret interac-tion, with no main effect of strain. Kineret pretreatment significantlyreduced baseline sIPSC frequency in KO mice (from 2.7 ± 0.5 inuntreated to 0.7 ± 0.2 Hz in Kineret pretreated CeA neurons) to thelevels found in WT, without affecting baseline frequency in WT neu-rons (1.0 ± 0.2 Hz for both untreated and Kineret pretreated; n = 13–14). Although Kineret pretreatment had no effect on the baselineamplitude in either WT or KO mice, it significantly increased sIPSCkinetics in both strains. Specifically, we found significant maineffects of Kineret and strain, but no interaction, on the rise time ofsIPSCs in WT (1.6 ± 0.1 and 1.9 ± 0.1 ms for untreated and Kineretpretreated, respectively) and KO mice (1.8 ± 0.1 and 2.3 ± 0.1 ms

Fig. 2. The enhancement of basal GABA release in the CeA of Il1rn KO mice is reversed bWT and Il1rn KO mice. Top panel: sIPSC traces from an untreated (left) and Kineret-pretuntreated neuron from a KO mouse is higher than in the WT mouse; Right, sIPSC trace fsIPSC frequencies, amplitudes, and rise and decay times of individual CeA neurons fromKO (2.7 ± 0.5 Hz, n = 21) compared to WT mice (1.0 ± 0.2 Hz, n = 13), but there were69.6 ± 6 mV; t32 = 0.70, p > 0.05), rise time (WT: 1.6 ± 0.1 ms; KO: 1.8 ± 0.1 ms; t32 = 1.1Kineret (100 ng/ml) pretreatment normalized the higher sIPSC frequency in KO andtransmission in both strains. There were main effects of Kineret (F(1,54) = 5.80, p < 0.05strain on frequency. We also found main effects of Kineret (F(1,54) = 11.93, p < 0.01) andin WT (untreated: 1.6 ± 0.1 ms vs. Kineret pretreated: 1.9 ± 0.1 ms) and KO mice (untreateof Kineret (F(1,54) = 34.24, p < 0.01) and strain (F(1,54) = 5.31, p < 0.05), without inter7.1 ± 0.4 ms; untreated KO: 4.3 ± 0.8 ms vs. Kineret pretreated KO: 9.3 ± 0.9 ms). ⁄ IndicKineret pretreated in Il1rn KO; ^ indicates main effect of Kineret pretreatment (two-way

Please cite this article in press as: Bajo, M., et al. Role of the IL-1 receptor antagoamygdala. Brain Behav. Immun. (2014), http://dx.doi.org/10.1016/j.bbi.2014.11

for untreated and Kineret pretreated, respectively). We also foundsignificant main effects of Kineret and strain, but no interaction, onthe decay time in WT (3.0 ± 0.5 and 7.1 ± 0.4 ms for untreated andKineret pretreated, respectively) and KO mice (4.3 ± 0.8 and9.3 ± 0.9 ms in untreated and Kineret pretreated, respectively). Ourresults indicate that exogenous Kineret can reverse the alterationin basal CeA GABAergic transmission elicited by deletion of theendogenous IL-1ra. However, the changes in sIPSC decay time fol-lowing Kineret pretreatment in both WT and KO mice also suggestthat Kineret may modulate postsynaptic GABAA receptors.

3.3. Deletion of Il1rn decreases the ethanol-induced facilitation ofspontaneous GABAergic transmission in the CeA

We next examined the role of IL-1ra in ethanol facilitation ofGABAergic transmission. Ethanol increased sIPSC frequency inCeA of both strains (WT: 175.5 ± 20.8% of baseline, n = 17; KO:112.1 ± 7.4% of baseline, n = 26); there was a significant main effect

y Kineret pretreatment. (A) Representative sIPSC recordings from CeA neurons fromreated neuron (right) from WT mice. Bottom panel: Left, the sIPSC frequency in anrom a Kineret pretreated neuron from an Il1rn KO mouse. (B) The scatter graphs ofWT and Il1rn KO mice. The mean sIPSC frequency was higher (t32 = 2.36, p < 0.05) inno significant differences by genotype in the amplitude (WT: 77.2 ± 9.9 mV; KO:3, p > 0.05), or decay time (WT: 3.0 ± 0.5 ms; KO: 4.3 ± 0.8 ms; t32 = 1.16, p > 0.05).

increased the rise and decay times of the basal spontaneous GABAA-mediated) and strain � Kineret interaction (F(1,54) = 4.86, p < 0.05), with no main effect of

strain (F(1,54) = 5.23, p < 0.05), without strain � Kineret interaction on the rise timed: 1.8 ± 0.1 ms and Kineret pretreated: 2.3 ± 0.1 ms). Finally, there were main effectsaction on the decay time (untreated WT: 3.0 ± 0.5 ms vs. Kineret pretreated WT:ates t-test comparison of WT vs. Il1rn KO; # indicates comparison of untreated toANOVA followed by Bonferroni post hoc test).

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of ethanol, but no strain or ethanol � strain interaction on sIPSCfrequency (Fig. 3A and B). Although the overall ethanol-inducedincrease in frequency was not dependent on genotype, we didobserve a genotype difference in the proportion of CeA cells show-ing increased sIPSC frequency upon ethanol application. In com-parison to WT, where 16 of 17 (94%) neurons demonstrated anethanol-induced increase (>115% of baseline) in frequency, fewerneurons (13 of 26 or 50%) from Il1rn KO mice demonstrated theethanol-induced increase (p < 0.05, Chi-square test; Fig. 3C). Acuteethanol increased the mean decay time of sIPSCs in most WT(127.2 ± 7.4% of baseline; p < 0.05) and KO (129 ± 9.6% of baseline;

Fig. 3. Deletion of Il1rn reversibly changes the responsiveness of CeA neurons toethanol. (A) Top panel, superfusion of 44 mM EtOH increases sIPSCs in a WT neuron.Bottom panel, EtOH (44 mM) superfusion slightly increases IPSCs in a KO neuron. (B)Summary of ethanol effects on the mean sIPSC frequency, amplitude, and rise anddecay times in CeA neurons from WT and Il1rn KO mice. Ethanol had a significantmain increase in the mean frequency (F(1,41) = 5.37, p < 0.05) and decay time ofsIPSCs (^p < 0.05, two-way ANOVA). (C) The proportion of neurons responding toethanol with an increase in sIPSC frequencies is significantly lower in Il1rn KOcompared to WT mice, but there is no difference between the two strains afterKineret pretreatment for 30–90 min (p < 0.05, Chi-square test). (D) Representativerecordings of CeA neurons after Kineret pretreatment. Top panel: sIPSC frequenciesfollowing application of 44 mM EtOH were still enhanced after Kineret pretreat-ment in a WT neuron. Bottom panel, in the presence of Kineret, 44 mM ethanolsuperfusion significantly increases sIPSCs in an Il1rn KO neuron. (E) In the presenceof Kineret, ethanol significantly increases the mean sIPSC frequency in both WT andIl1rn KO mice. Ethanol does not increase the decay time of the sIPSCs in neuronspretreated with Kineret. (^p < 0.05, two-way ANOVA with Bonferroni post hoc test).

Please cite this article in press as: Bajo, M., et al. Role of the IL-1 receptor antagoamygdala. Brain Behav. Immun. (2014), http://dx.doi.org/10.1016/j.bbi.2014.11

p < 0.05) neurons (Fig. 3B). There were no effects of ethanol onamplitude and rise time of sIPSCs, suggesting that IL-1ra is impor-tant in ethanol-induced modulation of GABAergic transmission insome CeA neurons, mediated predominantly via presynapticmechanisms.

Since Kineret decreased sIPSC frequencies of CeA neurons fromIl1rn KO mice to a level similar to that found in WT, we also exam-ined the effect of Kineret pretreatment on the ethanol-inducedfacilitation of GABAergic transmission (Fig. 3D and E). Kineret pre-treatment increased the proportion of neurons in KO mice thatrespond to ethanol with changes in sIPSC frequencies (p < 0.05;Fig. 3C). Specifically, in the KO mice there were 6 of 8 (75%) CeAneurons showing an ethanol-induced increase (192.7 ± 40.9%) insIPSC frequencies after Kineret pretreatment compared to 50% ofuntreated cells. In WT mice, Kineret had no effect on the propor-tion of CeA neurons responding to ethanol with increased(140.0 ± 7.0%) sIPSC frequencies; thus, 10 of 11 or 91% showedincreased frequencies elicited by acute ethanol. Ethanol did notalter sIPSC amplitude, rise time, or decay time (Fig. 3E). These datasuggest that exogenous application of IL-1ra can restore ethanol’senhancement of GABAergic transmission in KO mice, whereas IL-1ra has limited effects in WT mice.

3.4. Basal vesicular GABA release in the CeA of Il1rn KO is similar tothat in WT mice

We also examined a role for IL-1ra in action potential-indepen-dent (vesicular) GABAergic transmission (as measured by minia-ture IPSCs, mIPSCs, in the presence of TTX) but found nosignificant differences in baseline mIPSCs between WT and Il1rnKO mice (Fig. 4A and B left panel). Ethanol significantly increasedthe frequency of mIPSCs in both genotypes (WT: 140.4 ± 6.5% ofbaseline, n = 6; KO: 121.5 ± 13.6% of baseline, n = 11) (Fig. 4B andC). Similar to effects on the sIPSCs, ethanol increased mIPSC fre-quencies (>115% of baseline) in all of the WT neurons (6 of 6), com-pared to only 6 of 11 (55%) KO neurons (p < 0.05, Chi-square test;data not shown). In contrast to the sIPSCs, we observed a signifi-cant effect of ethanol on mIPSC rise time (WT: 118.8 ± 7.1% of base-line; KO: 114.1 ± 6.7% of baseline), whereas there were no ethanoleffects on mIPSC decay time in WT or Il1rn KO mice (Fig. 4C). Thesedata further support a role for IL-1ra in the ethanol-induced facil-itation of presynaptic vesicular GABA release in CeA.

3.5. Cell type-specific changes in tonic GABAergic signaling in CeAneurons of Il1rn KO compared to WT mice

As our results suggested that IL-1ra is critical in action poten-tial-dependent GABAergic transmission, we assessed ongoing tonicGABAergic signaling because it can dynamically regulate overallinhibitory network activity (Krook-Magnuson and Huntsman,2005; Semyanov et al., 2004). We examined the tonic conductanceusing whole-cell voltage-clamp recordings from 27 Il1rn KO and 29WT neurons. Cell typing was based on spike characteristics, as pre-viously described (Chieng et al., 2006; Dumont et al., 2002). CeAneurons are primarily composed of three main cell types: low-threshold bursting (LTB), late spiking (LS), and regular spiking(RS) (Chieng et al., 2006; Dumont et al., 2002). A GABAA recep-tor-mediated tonic current was defined as the difference in holdingcurrent (i.e., the current required to maintain the neuron at�60 mV) before and after application of the GABAA receptor antag-onist gabazine. Focal application of gabazine (GBZ, 100 lM) pro-duced a significant reduction in holding current in LTB neuronsfrom WT mice (18.1 ± 4.5 pA; Fig. 5A, upper trace and 5B) thatwas not observed in LTB neurons from KO mice (1.8 ± 1.2 pA;Fig. 5A, lower trace and 5B). In contrast, 100 lM GBZ had no effecton holding current in LS neurons from WT mice (0.2 ± 0.7 pA, n = 6;

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Fig. 4. Deletion of Il1rn alters ethanol effects on action potential-independent GABA transmission in CeA. (A) Scatter graphs of mIPSC frequencies and amplitudes of individualCeA neurons from WT (n = 6) and Il1rn KO (n = 11) mice. (B) Representative recordings of mIPSCs from neurons following acute ethanol application. Top panel: traces ofmIPSCs from a WT neuron before (left) and during 44 mM ethanol application (right). Bottom panel: traces of mIPSCs from a KO neuron showing ethanol-induced decrease inthe mIPSC frequency. (C) The comparison of ethanol effects on the mean mIPSC frequency, amplitude, and rise and decay times between CeA neurons from WT and Il1rn KOmice. Two-way ANOVA showed a significant main effect of ethanol (F(1,15) = 6.48; ^p < 0.05) on the mean mIPSC frequencies in WT and Il1rn KO mice and a significant effectof ethanol on mIPSC rise time (F(1,15) = 5.68, p < 0.05).

6 M. Bajo et al. / Brain, Behavior, and Immunity xxx (2014) xxx–xxx

Fig. 5C, upper trace and 5D), but did produce a significant reductionin LS neurons from KO mice (11.4 ± 3.0 pA; Fig. 5C, lower trace and5D). Focal application of gabazine onto RS neurons produced vari-able effects on holding currents (5.5 ± 2.0 and 7.9 ± 2.9 pA in WTand KO mice, respectively). This variability is in line with previousreports on the inconsistent nature of tonic currents in this cell pop-ulation (Herman et al., 2013).

As there were altered tonic conductances in two CeA cell popu-lations from Il1rn KO mice, we determined the role of the d subunitof the GABAA receptor in these conductances (see Herman et al.,2013). In WT mice, LTB neurons displayed a significantly higherbaseline sIPSC frequency compared to LS neurons (Fig. 5A and C);however, this effect was not observed in Il1rn KO mice, suggestingthat the absence of phasic inhibitory tone may contribute to thedecrease in tonic conductance observed in LTB neurons from KOmice. We observed no cell type-specific change in baseline sIPSCfrequency between LS or RS neurons from either genotype. Weused the d subunit-preferring GABAA agonist, gaboxadol (THIP),to determine the contribution of this subunit to the changes intonic signaling. Focal application of 5 lM THIP induced a signifi-

Please cite this article in press as: Bajo, M., et al. Role of the IL-1 receptor antagoamygdala. Brain Behav. Immun. (2014), http://dx.doi.org/10.1016/j.bbi.2014.11

cantly greater increase in holding current in LS (49.2 ± 12.1 pA)compared to LTB (11.2 ± 2.6 pA) neurons from WT mice (Fig. 5E).THIP also increased tonic conductance in LS (46.5 ± 8.2) comparedto LTB (7.2 ± 1.2 pA) neurons from Il1rn KO mice (Fig. 5E), demon-strating cell type differences but no genotype differences. Thesedata suggest that there are no differences in expression or functionof the d subunit-containing GABAA receptors between neuronsfrom WT and Il1rn KO mice.

We next asked if the acute effects of ethanol on tonic signalingdiffered between cell types or genotypes. Consistent with THIPeffects, focal application of EtOH (44 mM) elicited a greaterincrease in holding current in LS compared to LTB neurons fromboth WT (9.1 ± 1.5 vs. 1.5 ± 1.4 pA; p < 0.05; Fig. 5F) and Il1rn KOmice (11.4 ± 1.7 vs. 1.3 ± 1.6 pA; p < 0.05; Fig. 5F), indicating thatthere was no effect of genotype on the cell type-specific effectsof acute ethanol on tonic conductance. Collectively, these data sug-gest that genetic deletion of Il1rn produces functional changes intonic inhibition, which can significantly impact CeA activity.Table 1 provides an overall summary of the effects of ethanoland Kineret on the genotypes and cell types used in our study.

nist in ethanol-induced regulation of GABAergic transmission in the central.011

A B

WT KO0

5

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40 pA5 s

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GBZ 100 µM

GBZ 100 µM

GBZ 100 µM

GBZ 100 µM

Fig. 5. Tonic GABAergic signaling is altered in specific CeA cell types from Il1rn KOcompared to WT mice. (A) Representative recordings from a Low ThresholdBursting (LTB) neuron from a WT (upper trace) and Il1rn KO mouse (lower trace)during focal application of gabazine (GBZ, 100 lM). Dashed lines indicate averageholding current. (B) Summary of the tonic current in LTB neurons from WT (whitebar) and KO mice (black bar) revealed by application of GBZ (n = 7 for bothgenotypes; ⁄p < 0.05, unpaired t-test). (C) Recordings from a Late Spiking (LS)neuron from a WT (upper trace) and Il1rn KO mouse (lower trace) duringapplication of GBZ. Dashed lines indicate average holding current. (D) Summaryof the tonic current in LS neurons from WT (white bar) and Il1rn KO mice (black bar)revealed by application of GBZ (n = 6 (WT), n = 5 (Il1rn KO); ⁄p < 0.05, unpaired t-test). (E) Summary of the tonic current in LTB and LS neurons from WT (white bars)and Il1rn KO mice (black bars) revealed by focal application of 5 lM THIP (n = 6–7;⁄p < 0.05, one-way ANOVA with Bonferroni post hoc comparison). (F) Summary ofthe tonic current in LTB and LS neurons from WT (white bars) and Il1rn KO mice(black bars) revealed by focal application of 44 mM EtOH (n = 5–6; ⁄p < 0.05, one-way ANOVA with Bonferroni post hoc comparison).

M. Bajo et al. / Brain, Behavior, and Immunity xxx (2014) xxx–xxx 7

4. Discussion

In our study, we found that deletion of Il1rn alters CeA GABAer-gic transmission and its sensitivity to acute ethanol. Our resultsshow significant alterations in basal phasic and tonic GABAergictransmission, and a lack of ethanol-induced facilitation of phasicGABAergic transmission in a significant portion of CeA neuronsfrom Il1rn KO mice. Importantly, the changes in the phasic currentsand ethanol responsiveness are reversed or restored by exogenousIL-1ra (Kineret). Thus, both genetic and pharmacological manipula-tions provide convergent evidence for a role of IL-1ra in GABAergicresponses and ethanol potentiation in CeA neurons. We speculatethat the alterations of GABA responses observed in Il1rn KO miceare not isolated to amygdala and likely extend to other brainregions, considering that global knockout of Il1rn affects multiplecentral and peripheral targets.

Please cite this article in press as: Bajo, M., et al. Role of the IL-1 receptor antagoamygdala. Brain Behav. Immun. (2014), http://dx.doi.org/10.1016/j.bbi.2014.11

4.1. IL-1ra is involved in the regulation of GABAergic transmission inthe central amygdala

Increased basal transmission, characterized by increased ampli-tudes of evoked GABAergic responses and sIPSC frequencies, sug-gests a role for IL-1ra in the modulation of action potential-dependent GABA release in the CeA. In contrast, we observed nochanges in the basal mIPSC frequencies between WT and Il1rnKO mice, suggesting that the total number of presynaptic axon ter-minals and the vesicular release at individual synapses are likelyunchanged by Il1rn deletion. We speculate that IL-1ra may beinvolved in the modulation of neuronal or axonal firing rates. Nota-bly, pretreatment with Kineret reversed the increased sIPSC fre-quency observed in Il1rn KO mice, further supporting theimportance of IL-1ra in the regulation of action potential-depen-dent GABA release. IL-1ra inhibits signaling by binding to the IL-1R1 and preventing the recruitment of IL-1RAcP, which is criticalfor the initiation of IL-1R1 signaling (Garlanda et al., 2013;Krumm et al., 2014). We hypothesize that deletion of Il1rn inducesa tonic activation of IL-1R1 signaling in presynaptic axon terminals,mediating the observed increase in action potential-dependentGABA release. Although there were no differences in basal sIPSCkinetics and amplitude between WT and Il1rn KO mice, Kiner-et altered the kinetics in both groups, suggesting that IL-1ra mayalso act postsynaptically in modulating GABAergic transmissionand that postsynaptic IL-1R1s may be regulated differently com-pared to presynaptic IL-1R1s. We propose that the IL-1ra modula-tion of GABAA transmission may depend on synaptic localization ofIL-1R1 and intracellular signaling.

4.2. IL-1ra plays a critical role in the regulation of tonic GABAconductances

Deletion of Il1rn leads to significant enhancement of phasicaction potential-dependent GABAergic transmission, which mayresult from disruptions in local synaptic activity and network func-tion. Tonic inhibition is a dynamic regulator of overall networkactivity and plays a significant role in the fine-tuning of local inhib-itory circuitry that regulates network function (Krook-Magnusonand Huntsman, 2005; Semyanov et al., 2004). In the CeA, distincttypes of tonic inhibition in specific cell populations provide multi-ple levels of inhibitory control that can regulate overall output. Wepreviously showed that the tonic GABA conductance in CeA is celltype-specific and mediated by two populations of GABAA receptorswith different subunit compositions (primarily d or a1 subunits)and distinct functional properties (Herman et al., 2013). In thepresent study, we found a cell type-specific shift in tonic conduc-tance in neurons from Il1rn KO compared to WT mice. In the KOmice, the ongoing tonic conductance in LTB neurons is absentand an ongoing tonic conductance in LS neurons appears. Interest-ingly, the functional ‘switch’ is similar to what we observed in ratschronically treated with ethanol (Herman et al., 2013; Herman andRoberto, 2014). This switch may represent a compensatoryresponse associated with an increase in phasic GABA release and/or disturbance of IL-1R1 signaling.

The mechanisms mediating the switch in tonic conductancesbetween LTB and LS neurons in Il1rn KO mice may include alteredexpression of IL-1R1 and GABAA receptors, trafficking of the recep-tor subunits to the membrane, and/or receptor assembly. Previ-ously, we reported a tonic GABA conductance that is not active inthe basal state and is mediated primarily by d subunits (Hermanet al., 2013). It is sensitive to physiological conditions in whichlocal GABA concentrations are elevated (e.g., blockade of GABAre-uptake, ethanol, etc.). Here we report that, despite the loss ofongoing tonic inhibition in LTB neurons and the presenceof ongoing tonic inhibition in LS neurons in KO mice, the ability

nist in ethanol-induced regulation of GABAergic transmission in the central.011

Table 1Summary of the effects of ethanol and Kineret in CeA neurons from Il1rn WT and KO mice. Ethanol (44 mM) was applied by superfusion for 10–15 min. Kineret (100 ng/ml) wassuperfused for 30–90 min prior to recordings. Abbreviations: GBZ (gabazine; 100 lM), KO (Il1rn KO mice), LS (late spiking neuron), LTB (low-threshold bursting neuron), THIP(gaboxadol; 5 lM), WT (Il1rn WT mice).

Recordings Parameters/pharmacology Baseline Ethanol Kineret baseline Kineret + Ethanol

KO vs. WT WT KO WT KO WT KO

eIPSPs Amplitude " " = n.a. n.a. n.a. n.a.PPF = ; = n.a. n.a. n.a. n.a.

sIPSCs Frequency " " "# = = " "Amplitude = = = = = = =Rise time = = = " " = =Decay time = " " " " = =

mIPSCs Frequency = " "# n.a. n.a. n.a. n.a.Amplitude = = = n.a. n.a. n.a. n.a.Rise time = " " n.a. n.a. n.a. n.a.Decay = = = n.a. n.a. n.a. n.a.

Tonic GBZ KO: LS+/LTB�WT: LS�/LTB+

" " n.a. n.a. n.a. n.a.

THIP = n.a. n.a. n.a. n.a. n.a. n.a.

Symbols: " increase; # decrease in number of cells responding to ethanol compared to WT mice; = no change; ; decrease; n.a. not applicable; + presence of tonic GABAconductance; � lack of tonic GABA conductance.

8 M. Bajo et al. / Brain, Behavior, and Immunity xxx (2014) xxx–xxx

of a d subunit-preferring agonist (THIP) and acute ethanol to fur-ther stimulate tonic inhibition remains unchanged, suggesting thatthere is no change in function or membrane expression of the dsubunit in either cell population or genotype. It is possible thatthere is an alteration of expression/function of other GABAA recep-tor subunits mediating tonic GABA currents, such as a1 and a5subunits. The a1 subunit has been shown to mediate the ongoingtonic conductance driven by action potential-dependent GABArelease in CeA LTB neurons (Herman et al., 2013). The lack of thiscurrent in LTB neurons and its presence in LS neurons raises thepossibility of a switch in the expression and/or function of a-con-taining GABA receptors in these neurons. Whereas GABA receptorscontaining both a1 and d subunits are found in the hippocampus(Glykys et al., 2007), such receptors seem to be missing or are pres-ent in very limited numbers in CeA neurons (Herman et al., 2013).

Previously, we showed no significant role for the a5 subunit inthe tonic GABA conductance in the CeA, although immunohisto-chemical analysis confirms expression of the a5 subunit, especiallyin LS neurons (Herman et al., 2013). In this regard, an IL-1b-induced increase in the surface expression of a5-containing GABAA

receptors and a5-mediated tonic GABA currents in hippocampalneurons (Wang et al., 2012) suggest that a5 subunits may play arole in the tonic GABA conductance in Il1rn KO mice. Assessmentof a role for different a subunits in the tonic GABA conductancein CeA from Il1rn KO mice will be the subject of future studies.

4.3. Kineret restores ethanol’s facilitation of GABAergic transmission inIlrn1 KO mice

Acute ethanol increases GABAergic transmission predominantlyvia presynaptic mechanisms, resulting in increased GABA releasein the CeA of mice (Bajo et al., 2008; Herman et al., 2013; Nieet al., 2004) and rats (Roberto et al., 2010, 2003, 2004). In addition,ethanol potentiates tonic GABA conductance and modulates firingrates in a cell type-specific manner in the murine CeA (Hermanet al., 2013). In WT mice, we verified previous findings that ethanolenhances both evoked and spontaneous GABAergic transmission inCeA by increasing GABA release. This facilitatory effect of ethanolon the evoked IPSP and sIPSC frequency is lost in some Il1rn KOneurons, indicating that IL-1ra may play an important role in medi-ating ethanol potentiation of activity-dependent GABA release.

Application of Kineret restores the basal phasic GABAergictransmission and ethanol effects in Il1rn KO mice, and it also mod-

Please cite this article in press as: Bajo, M., et al. Role of the IL-1 receptor antagoamygdala. Brain Behav. Immun. (2014), http://dx.doi.org/10.1016/j.bbi.2014.11

ulates the kinetics of phasic GABAergic transmission in both WTand KO mice. Furthermore, we demonstrate that Kineret effectivelypenetrates the slices to alter CeA GABA transmission and ethanolresponses.

5. Conclusion

We demonstrate significant increases in action potential-medi-ated GABA release and changes in the tonic GABA conductance ofspecific neurons in Il1rn KO mice that may profoundly impact over-all CeA activity and its GABAergic projection afferents, thusimpacting alcohol-related behaviors. In summary, the IL-1ra sys-tem plays a critical role in basal and ethanol-induced GABAergictransmission in the CeA. We speculate that an undisturbed IL-1Rsignaling system, properly regulated by IL-1ra, modulates GABAer-gic transmission in a cell type-specific manner. The subpopulationsof CeA neurons involved and the neurocircuitry responsible formediating relevant alcohol-related behaviors will be the subjectof future studies. Our electrophysiological analysis provides evi-dence that changes mediated by the IL-1/IL-1R system are criticalfor alcohol responses at the neuronal level in mice.

Author contributions

M.B. and M.R. designed and performed experiments, analyzeddata, prepared figures, and wrote the manuscript. M.A.H., F.P.V.,S.G.M. and C.S.O. performed experiments, analyzed data, preparedfigures, and contributed to writing the manuscript; R.A.H. andY.A.B. provided the mice and edited the manuscript.

Acknowledgements

We thank Dr. George R. Siggins for his critical review and com-ments on the manuscript, George Luu for technical assistance, Dr.Marian L. Logrip for help with statistical analyses, and Dr. JodyMayfield for many helpful comments, critical review, and editingof the manuscript. The Scripps Research Institute’s manuscriptnumber for this paper is 24053.

Supported by NIH/NIAAA INIA West Consortium U01-AA013498 to M.R. AA013520 to Y.A.B. and R.A.H. AA013517,AA006420, AA015566 and AA021491 to M.R.

nist in ethanol-induced regulation of GABAergic transmission in the central.011

M. Bajo et al. / Brain, Behavior, and Immunity xxx (2014) xxx–xxx 9

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