In summary, these data indicate that repeated cycles of CIE exposure disrupt glutamate homeostasis in the NAc, an important brain region implicated in motivated behaviors such as ethanol drinking. Specifically, in a model of ethanol dependence that produces escalated drinking, significant (twofold) elevation in basal extracellular glutamate concentrations in NAc was observed. Using an *ex vivo* assay to evaluate glutamate transporter function, CIE exposure did not alter Na+-dependent and -independent transporter functions. That is, CIE-induced elevation of basal glutamate levels in the NAc does not appear related to alterations in transport mechanisms responsible for bringing glutamate back into cellular compartments (i.e., Na+-dependent processes; EAATs) or mechanisms that release glutamate into the extracellular space (i.e., Na+-independent processes; system Xc −). Given the importance of group II receptors in regulating extracellular glutamate concentrations, including pre-synaptic release, ongoing work is investigating the role of group II receptors on regulating extracellular glutamate as well as ethanol drinking in the CIE model.

#### **ACKNOWLEDGMENTS**

NIAAA P50 AA010761, T32 AA007474, F32 AA021321, and VA Medical Research.

#### **REFERENCES**

sion in rat central amygdala: an in vitro and in vivo analysis. *J. Neurosci.* 24, 1594–1603. doi: 10.1523/JNEUROSCI.5077-03.2004

**Conflict of Interest Statement:** The Guest Associate Editor Justin Gass declares that, despite being affiliated to the same institution as authors William C. Griffin, Vorani S. Ramachandra, and Howard C. Becker, the review process was handled objectively and no conflict of interest exists. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 15 October 2014; accepted: 02 February 2015; published online: 23 February 2015.*

*Citation: Griffin WC, Ramachandra VS, Knackstedt LA and Becker HC (2015) Repeated cycles of chronic intermittent ethanol exposure increases basal glutamate in the nucleus accumbens of mice without affecting glutamate transport. Front. Pharmacol. 6:27. doi: 10.3389/fphar.2015.00027*

*This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology.*

*Copyright* © *2015 Griffin, Ramachandra, Knackstedt and Becker. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Ceftriaxone and cefazolin attenuate the cue-primed reinstatement of alcohol-seeking

*Ana Weiland1, Steven Garcia2 and Lori A. Knackstedt 2\**

*1 Department of Neuroscience, Medical University of South Carolina, Charleston, SC, USA, 2 Department of Psychology, University of Florida, Gainesville, FL, USA*

Alcohol consumption and the reinstatement of alcohol-seeking rely on glutamate and GABA transmission. Modulating these neurotransmitters may be a viable treatment strategy to prevent alcohol relapse. *N*-acetylcysteine (NAC) and the antibiotic ceftriaxone (CEF) alter the glial reuptake and release of glutamate while the antibiotic cefazolin (CEFAZ) modulates GABA signaling without affecting glutamate. Here, we used the extinction-reinstatement model of relapse to test the ability of these compounds to attenuate the reinstatement of alcohol-seeking. Male Sprague-Dawley rats were trained to self-administer 20% (v/v) alcohol in the home cage using an intermittent schedule (24 h on, 24 h off) for 12 sessions. Subsequently, animals self-administered alcohol during daily 45-min operant sessions for 26 sessions, followed by extinction training. We tested whether chronic administration of NAC, CEF, or CEFAZ attenuated the cue-primed reinstatement of alcohol-seeking. CEF and CEFAZ attenuated cueprimed reinstatement of alcohol-seeking while NAC had no effect. We subsequently investigated whether CEF and CEFAZ alter the self-administration of sucrose and chow pellets and if CEFAZ attenuates the reinstatement of cocaine-seeking. The operant selfadministration of regular chow and sucrose was not altered by either CEF or CEFAZ. CEFAZ had no effect on cocaine reinstatement, a behavior that has been strongly tied to altered glutamate homeostasis in the nucleus accumbens. Thus the ability of CEFAZ to attenuate alcohol reinstatement likely does not involve the glial modulation of glutamate levels. The dampening of GABA transmission may be a common mechanism of action of cefazolin and ceftriaxone.

#### Keywords: glutamate, alcohol, GABA, cocaine, addiction, relapse, *N*-acetylcysteine, ceftriaxone

## Introduction

Alcoholism or alcohol dependence is a chronic, progressive disease which results from an inability to regulate drug-seeking behavior. Approximately 17 million Americans are dependent on alcohol (NIH, 2006), resulting in significant legal and medical costs to society totaling an estimated \$184 billion per year (Kenna et al., 2004). At present, only disulfiram, naltrexone, and acamprosate are approved by the Food and Drug Administration to treat alcoholism. However, the ability of these medications to reduce the risk of relapse is modest (Heilig and Egli, 2006; Soyka and Roesner, 2006) and thus there is a need to develop more targeted pharmacological treatments for alcohol addiction.

#### *Edited by:*

*M. Foster Olive, Arizona State University, USA*

#### *Reviewed by:*

*George V. Rebec, Indiana University, USA Jamie Peters, Medical University of South Carolina, USA*

#### *\*Correspondence:*

*Lori A. Knackstedt, Department of Psychology, University of Florida, 114 Psychology Building, P.O. Box 112250, Gainesville, FL 32611-2250, USA<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology*

> *Received: 23 January 2015 Accepted: 19 February 2015 Published: 10 March 2015*

#### *Citation:*

*Weiland A, Garcia S and Knackstedt LA (2015) Ceftriaxone and cefazolin attenuate the cue-primed reinstatement of alcohol-seeking. Front. Pharmacol. 6:44. doi: 10.3389/fphar.2015.00044*

Animal models of relapse permit the identification of the underlying neurobiology of alcohol relapse in humans and thus the targeted development of pharmacotherapies. Relapse is modeled in animals with the reinstatement paradigm, where animals are trained to self-administer drug in an operant chamber. The drug-seeking response is extinguished and reinstated with one of the stimuli known to cause relapse in humans such as stress, cues associated with drug delivery, or the drug itself (Epstein et al., 2006). It has proven challenging to get laboratory rats to voluntarily consume doses of alcohol relevant to human alcohol addiction. Various procedures have been attempted to increase oral consumption of alcohol in rodents, such as sucrose fading (Samson, 1986), food and waterdeprivation (Meisch and Thompson, 1972), alcohol vapor exposure (Walker and Koob, 2007; Walker et al., 2008), and the use of rat strains selectively bred for high preference to alcohol (Samson et al., 1998; Vacca et al., 2002). Since its introduction in the 1980s, sucrose fading was largely adopted as the chief method of inducing rodents to self-administer alcohol in an operant setting (Samson, 1986). While sucrose fading yields high alcohol consumption in the presence of sucrose, drinking drops sharply when the sweetener is removed (Samson, 1986; Koob and Weiss, 1990; Samson et al., 1999; Carrillo et al., 2008). Additionally, sucrose has addictive properties itself (Colantuoni et al., 2002; Avena et al., 2008), possibly contributing to the motivation to consume sweetened alcohol. One method of inducing high amounts of unsweetened alcohol consumption in rodents is the intermittent access to alcohol (IAA) procedure in which rodents are provided 24 h access to alcohol followed by a 24 h period with no access (Wayner and Greenberg, 1972; Wise, 1973; Pinel and Huang, 1976). Simms et al. (2008) recently resurrected this model and induced levels of drinking (20% alcohol v/v) in the 5–8 g/kg/day range in Wistar and Long-Evans strains. Furthermore, Simms et al. (2010) adapted the IAA paradigm to an operant self-administration model and engendered high levels of alcohol-seeking in the operant chamber after using 14 h operant sessions on intermittent days (MWF), followed by shorter (30–45 min) daily sessions in the operant chamber. Here, we modified this method to eliminate the use of 14 h operant sessions and trained animals in the operant chamber only during daily 45-min sessions for 26 sessions (MTWRF).

We then sought to test the ability of translational compounds which modulate glutamate and/or GABA transmission to attenuate the cue-primed reinstatement of alcohol-seeking. Glutamate transmission in general and specifically in the nucleus accumbens (NAs) and amygdala is strongly implicated in cue-induced alcohol reinstatement. During the cue-primed reinstatement of extinguished operant alcohol-seeking, synaptic glutamate release increases in the NA and the amygdala (Gass et al., 2011). Thus, it follows that both systemic and intra-NA or intra-amygdala infusion of antagonists to post-synaptic glutamate receptors attenuate alcohol reinstatement (e.g., Backstrom and Hyytia, 2004; Backstrom et al., 2004; Sanchis-Segura et al., 2006; Sinclair et al., 2012).

The pharmacological manipulations which prevent relapse to alcohol-seeking also prevent the reinstatement of cocaine-seeking (for review see Kalivas et al., 2009; Knackstedt and Kalivas, 2009). Following cocaine self-administration and withdrawal, evidence exists for altered glutamate homeostasis, defined as the homeostatic regulation of synaptic and extrasynaptic glutamate levels and signaling. Glutamate homeostasis in the accumbens is complex and regulated by multiple mechanisms, including by the major glutamate transporter GLT-1 and by system xc-. System xc- exchanges extracellular cystine for intracellular glutamate, thereby contributing the majority of basal extrasynaptic glutamate in the accumbens (Baker et al., 2002). The catalytic subunit of system xc- is the protein xCT and the hallmarks of altered glutamate homeostasis after cocaine include a reduction in expression and function of both GLT-1 and xCT/system xc- (Knackstedt et al., 2010; Trantham-Davidson et al., 2012). Based on the widespread involvement of various types of glutamate receptors in alcohol relapse, it is likely that glutamate homeostasis is altered following alcohol consumption as well.

Modulation of GABA transmission also impacts alcohol consumption and reinstatement. GABAA antagonists and inverse agonists decrease alcohol consumption (see review by McBride and Li, 1998) and operant self-administration of alcohol (Samson et al., 1987).The GABAB (autoreceptor) agonist baclofen decreases self-administration in non-dependent rats (Janak and Michael Gill, 2003). Conversely, allopregnanolone, an endogenous neurosteroid which has also been shown to be a potent positive modulator of GABAA receptors, dose-dependently induces the reinstatement of alcohol-seeking (Nie and Janak, 2003; Finn et al., 2008).

We hypothesized that glutamate homeostasis is altered postalcohol administration, and restoring homeostasis would reverse alcohol-induced plasticity and thereby attenuate the reinstatement of alcohol-seeking. It is possible to restore glutamate homeostasis and reverse cocaine-induced pathologies in synaptic plasticity following with *N*-acetylcysteine (NAC; Moussawi et al., 2009, 2011) or ceftriaxone (CEF; Trantham-Davidson et al., 2012) and here we tested their ability to attenuate cue-primed reinstatement of alcohol-seeking. NAC serves as a cystine prodrug that drives system xc- to export more glutamate while CEF is an antibiotic that upregulates the expression of both GLT-1 (Lee et al., 2008) and xCT (Lewerenz et al., 2009) at the transcriptional level. We also hypothesized the antibiotic cefazolin (CEFAZ), which has been demonstrated to inhibit GABAA transmission (Yamazaki et al., 2002; Sugimoto et al., 2003), would attenuate cue-primed alcohol reinstatement. In order to investigate a possible effect of CEFAZ on glutamate homeostasis, we tested its ability to attenuate the reinstatement of cocaine-seeking, a behavior that is dependent on altered glutamate release, clearance and basal levels (for review see Knackstedt and Kalivas, 2009).

## Materials and Methods

### Animals and Housing

Adult male Sprague-Dawley rats (*n* = 84), weighing 250–275 g upon arrival were individually housed in ventilated Plexiglas cages in a climate-controlled room on a 12-h reverse light/dark cycle (lights off at 9 AM) and given at least 1 week to acclimate to the individual housing conditions and handling. Rats were provided ad libitum water throughout the experiment and were food-deprived to ∼85% of free-feeding weight with a daily allowance of 20 g of food. Food-restriction generally increases responding without altering reinstatement relative to non-restricted controls (Bongiovanni and See, 2008). Experiment 1 was conducted at the Medical University of South Carolina and Experiments 2–4 were conducted at the University of Florida. All procedures were preapproved by the Medical University of South Carolina and University of Florida Institutional Animal Care and Use Committees and were in accordance with National Institutes of Health *Guide for the Care and Use of Laboratory Animals*.

### Operant Chambers and Training

Prior to beginning operant self-administration (Experiments 1–4), animals underwent a single, overnight food-training session in the operant chambers where presses on the active lever resulted in the delivery of food pellets. Operant chambers (Med Associates) were equipped with two retractable levers, stimulus lights, and tone generators. On the day following food training, animals were trained to self-administer alcohol, cocaine, or food pellets in the operant chamber. For the food self-administration study (Experiment 3), presses on the active lever delivered food pellets. For drug self-administration studies, presses on the active lever activated a pump which dispensed drug. For alcohol studies (Experiments 1 and 2), 0.1 mL alcohol (20% v/v) was dispensed into a dipper tray; infrared sensors verified head entry into the trough area to consume the alcohol. For the cocaine study (Experiment 4), 0.25 mg cocaine HCl (kindly provided by NIDA) in 0.1 mL 0.9% saline was delivered per infusion. For both cocaine and alcohol studies, drug delivery was accompanied by the presentation of discrete cues (a light and 2900 Hz tone) and for cocaine self-administration, was followed by a timeout (20 s) during which time presses on the active lever did not yield drug. Inactive lever presses had no consequences, but were recorded. For alcohol self-administration, the trough was blotted with a tissue at the end of the session to verify that all alcohol was consumed. Rats who habitually did not consume alcohol despite lever presses were eliminated from the experiment (*n* = 3 for Experiment 1; *n* = 2 for Experiment 2). Additionally, three rats that were in ill-health were removed from Experiments 1 and 2. For both alcohol and cocaine selfadministration studies, animals self-administered drug for a pre-determined number of days in the operant boxes (26 for alcohol; 12 for cocaine) regardless of whether drug intake was stable or not. This method was chosen because we wanted to prevent animals from self-administering drug for varying amounts of time which could lead to a wide range of drug intake. The amount of cocaine consumed during selfadministration has been correlated with later drug-seeking during reinstatement tests (e.g., Deroche et al., 1999; Baker et al., 2001).

## Alcohol Self-Administration, Extinction Training, and Reinstatement Testing

All rats that later self-administered alcohol in the operant chambers were first trained to drink with the IAA (e.g., Simms et al., 2008). This paradigm provides rats with 24-h access to unsweetened 20% alcohol on alternating days (3 days/weeks) without water-deprivation. Animals experienced 12 sessions (over 4 weeks) of the intermittent-access drinking paradigm.

Following exposure to alcohol using the IAA, animals were trained to self-administer 20% alcohol (v/v) using daily 45 min training sessions on an FR-1 schedule of reinforcement for 6 days followed by an FR-3 schedule for 20 daily sessions. This procedure followed that of Simms et al. (2010), with the exception of using 45 min FR-3 operant sessions in place of 30 min sessions and replacing the 12 overnight operant sessions with 12 IAA sessions. Extinction training began following the 20th FR-3 session. During extinction training, presses on the previously active lever no longer provided drug and cue presentation. Extinction training lasted a minimum of 10 sessions and until animals reached the criteria of less than 20 lever responses per session. Once this criteria was reached, animals underwent a 45 min cue-primed reinstatement test during which presses on the previously active lever once again yielded presentation of discrete cues that had been paired with alcohol delivery (stimulus light, tone and the sound of the syringe pump activating). In addition, 2 mL of 20% EtOH was sprinkled on the bedding of the operant chamber to provide an olfactory cue.

## Experiment 1

Here, we tested the ability of chronically administered NAC to attenuate the cue-primed reinstatement of alcohol-seeking. *N*acetylcysteine (Sigma, 30 or 60 mg/kg IP) or vehicle (VEH; saline 0.3 mL IP) was administered 2 h prior to the last 6–8 extinction sessions and prior to the reinstatement test (see **Figure 1A**). The dose and timing of injections was based on previous studies demonstrating the ability of 60 mg/kg NAC to restore basal glutamate levels after cocaine and attenuate cocaine reinstatement when given both acutely prior to testing (Baker et al., 2003) and chronically during extinction training (Amen et al., 2011) while a lower dose (33 mg/kg) only partially inhibits cocaine-seeking (Moussawi et al., 2009). NAC works acutely, within 2 h of administration, to increase basal glutamate by serving as an exogenous source of cysteine, which drives system xc- to export more glutamate (Baker et al., 2003), but also alters glutamate homeostasis when administered daily for 5 or more days (Knackstedt et al., 2010; Amen et al., 2011). After reaching extinction criteria, the responding of the NAC-treated animals during a cue- primed reinstatement trial was compared with VEH-treated animals. Animals were assigned to receive NAC or VEH treatment in a counterbalanced manner to ensure no group differences in drug self-administration. One animal was excluded from reinstatement testing (and all data analysis) due to a failure to reach extinction criteria.

## Experiment 2

This experiment was carried out in an identical manner to Experiment 1, with the exception that CEF (Hospira, 200 mg/kg

IP), CEFAZ (Sigma, 100 mg/kg IP), or VEH (0.9% saline) were administered immediately following the last 6–8 extinction sessions, but not on test day (see **Figure 1A**). After reaching extinction criteria, the responding of the CEF and CEFAZtreated animals during a cue- primed reinstatement trial was compared with VEH -treated animals. The dose and timing of CEF injections was based on previously published reports showing that 200 mg/kg effectively attenuates cocaine reinstatement and restores glutamate homeostasis when given chronically *after* extinction sessions for at least 5 days (Knackstedt et al., 2010; Trantham-Davidson et al., 2012). The dose of CEFAZ was based on allometric calculations from the maximum human dose recommended by the manufacturer to the equivalent rat dose of 100 mg/kg maximum and was given after extinction sessions akin to the administration of CEF.

### Food and Sucrose Self-Administration (Experiment 3)

Animals self-administered standard chow pellets (45 mg; Bioserv) in the operant chamber using an FR-1 schedule of reinforcement for 2 h/day for 12 days. On the 13th day, sucrose pellets (45 mg; Bio-serv) replaced the standard chow pellets. Animals received CEF, CEFAZ, or VEH in an identical manner as described for Experiment 2, with the exception that injections began after the operant session on Day 5 of food self-administration (see **Figure 1B**).

## Cocaine Self-Administration, Extinction, and Reinstatement (Experiment 4)

For the implantation of catheters, rats were anesthetized with ketamine HCl (87.5 mg/kg, IM) and xylazine (5 mg/kg, IM). Ketorolac (3 mg/kg, IP) was administered before surgery to provide analgesia. Catheter construction and surgical implantation is described in detail elsewhere (Knackstedt et al., 2014). Rats recovered 6 days after surgery prior to beginning selfadministration training. Cocaine self-administration was conducted using an FR-1 schedule; sessions lasted 2 h/day and are described in more detail above. Self-administration continued until subjects had attained 12 days with a minimum of 10 cocaine infusions and was followed by extinction training. Animals were either treated with CEFAZ or VEH in manner identical to that in Experiment 2, immediately following the last 6–8 extinction sessions (see **Figure 1C**). Once extinction criteria was attained, animals were tested for cue- and cocaineprimed reinstatement (each test lasting 2 h), with tests separated by 3 days of extinction training. During cue-primed reinstatement, presses on the previously active lever once again yielded the discrete cues paired with cocaine delivery but no drug was delivered. During cocaine-primed reinstatement, animals were injected with 10 mg/kg cocaine (IP) immediately prior to being placed into the operant chamber; presses on the previously active lever had no consequences during this test.

#### Data Analysis

The alpha level was set at *p <* 0.05 for all statistical tests, which were conducted using SPSS software. Active lever presses during self-administration and extinction components of the experiment were analyzed with mixed-factorial 2-way analysis of variances (ANOVAs) test, with time as the repeated measure. Reinstatement tests were planned comparisons (extinction vs. test day) and thus were analyzed with paired-sample *t*-tests. Independent-sample *t*-tests were used to compare active lever presses during reinstatement tests between treatment groups. Group differences in food self-administration were examined using one-way ANOVAs. Outliers were defined as values more than two standard deviation +/– the mean and were excluded from further analysis. Groups with outlier values are mentioned in the Results section.

## Results

### The Effects of NAC on Cue-Primed Reinstatement of Alcohol-Seeking (Experiment 1)

VEH- and NAC-treated groups did not differ in the amount of alcohol self-administered (**Figure 2A**). A two-way ANOVA with repeated measures on Day was computed on the active lever press data during self-administration and found no significant effect of Day [*F*(19,475) = 1.480, n.s.], or Group [*F*(2,25) = 0.080, n.s.]. There was a significant Day × Group interaction [*F*(19,475) = 2.020, *p <* 0.01]. Similarly, a two-way ANOVA was computed on the lever presses during extinction training (**Figure 2B**) and revealed a significant effect of Day [*F*(9,225) = 6.977, *p <* 0.001], but not Group [*F*(2,25) = 0.240, n.s.]. There was not a significant Day × Group interaction [*F*(18,225) = 0.809, n.s.]. We used paired-sample *t*-tests to analyze the data presented in **Figure 2C** to compare active lever presses during cue-primed reinstatement to those during the average of the last 3 days of extinction. Significant differences were found between extinction and cue-primed reinstatement for all treatment groups [Veh: *t*(1,7) = 4.036, *p <* 0.01; NAC60: *t*(1,8) = 4.302, *p <* 0.01; NAC30: *t*(1,9) = 3.651, *p <* 0.05], indicating that neither dose of NAC was able to prevent cue-primed reinstatement of alcohol-seeking.

### The Effects of CEF and CEFAZ on Cue-Primed Reinstatement of Alcohol-Seeking (Experiment 2)

Vehicle, CEF, and CEFAZ groups did not differ in the amount of active lever presses during self-administration (**Figure 3A**). A two-way ANOVA with repeated measures on Day was computed on the active lever presses during alcohol self-administration (**Figure 3A**) and revealed a significant effect of Day [*F*(19,380) = 2.612, *p <* 0.01], but not Group [*F*(2,20) = 0.370, n.s.]. There was not a significant Day × Group interaction [*F*(19,380) = 1.277, n.s.]. Similarly, a two-way ANOVA was computed on the number of lever presses during extinction training (**Figure 3B**) and revealed a significant effect of Day [*F*(9,180) = 8.050,

*p <* 0.001], but not Group [*F*(2,20) = 0.989, n.s.]. There was not a significant Day × Group interaction [*F*(2,180) = 1.249, n.s.]. We used paired-sample *t*-tests to compare active lever presses during cue-primed reinstatement to those during the

last 3 days of extinction (**Figure 3C**). A significant difference between extinction and cue-primed reinstatement was only observed for vehicle-treated animals [*t*(1,8) = 2.892, *p <* 0.05], indicating that CEF [*t*(1,6) = 1.3998, n.s.] and CEFAZ [*t*(1,6) = 2.090, n.s.] attenuated cue-primed reinstatement of alcohol-seeking.

## CEF and CEFAZ Did Not Alter Food and Sucrose Self-Administration or Weight (Experiment 3)

Because CEF and CEFAZ treatment commenced following the session on Day 5, statistical analyses were conducted only on the data during the period of antibiotic (or vehicle) treatment (Days 6–12). One animal was excluded from data analysis because his lever pressing on Day 13 was greater than two standard deviations from the mean value. A one-ANOVA was computed on active lever presses during self-administration and revealed no effect of treatment group [*F*(2,17) = 2.128, n.s.; **Figure 4A**]. We also conducted a two-way ANOVA with repeated measures on Day and this analysis revealed no significant effects [Day: *F*(6,102) = 1.963, n.s.; Day × Group: *F*(12,102) = 1.375, n.s.; Group: *F*(2,17) = 2.361, n.s.]. A oneway ANOVA was used to determine if there were group differences in the number of sucrose pellets earned on Day 13 of the experiment (**Figure 4B**) and found no effect [*F*(2,17) = 1.068, n.s.]. There was also no difference in weight over time between the three treatment groups (**Figure 4C**). A two-way RM ANOVA found an effect of Day [*F*(11,102) = 9.145, *p <* 0.001] but no Day × Group interaction [*F*(12,102) = 2.507, n.s.] or effect of Group [*F*(2,17) = 1.924, n.s.] on animal weight.

## CEFAZ Did Not Attenuate the Reinstatement of Cocaine-Seeking (Experiment 4)

A two-way ANOVA computed on the amount of cocaine intake (**Figure 5A**) revealed no group differences [*F*(1,12) = 0.357, n.s.], no effect of Day [*F*(11,132) = 1.562, n.s.], and no Day × Group interaction [*F*(12,132) = 0.453, n.s.]. There were also no group differences in extinction training [*F*(1,12) = 0.472, n.s.] and no Day × Group interaction [*F*(12,108) = 0.696, n.s.] but there was an effect of Day [*F*(9,108) = 19.076, *p <* 0.001] as both groups decreased lever pressing during the course of training (**Figure 5B**). Paired-sample *t*-tests revealed significant differences between extinction and cue-primed reinstatement for both CEFAZ [*t*(1,5) = 2.834, *p <* 0.05] and vehicletreated [*t*(1,7) = 4.453, *p <* 0.01] animals (**Figure 5C**). Paired-sample *t*-tests revealed significant differences between extinction and cocaine-primed reinstatement for both CEFAZ [*t*(1,5) = 2.834, *p <* 0.05] and vehicle-treated [*t*(1,7) = 2.614, *p <* 0.05] animals (**Figure 5D**). Thus, CEFAZ did not attenuate cue- or cocaine-primed reinstatement. Furthermore, an independent sample *t*-test showed no lever pressing differences between vehicle- and CEFAZ-treated groups during the cue [*t*(1,12) = 0.272, n.s.] and cocaine tests [*t*(1,12) = 0.269, n.s.].

FIGURE 4 | Ceftriaxone and cefazolin did not alter the motivation to self-administer regular chow or sucrose pellets. (A) CEF and CEFAZ did not alter the self-administration of regular chow pellets. (B) CEF and CEFAZ did not alter the mean number of sucrose pellets self-administered. (C) Mean body weight did not differ between vehicle-, CEF- or CEFAZ-treated rats.

## Discussion

Here, we demonstrated that training Sprague-Dawley rodents to self-administer unsweetened alcohol in the operant chamber can be accomplished using daily training sessions without lengthy (14 h; e.g., Simms et al., 2010) operant sessions on alternating days. This is the first demonstration of such an effect in the Sprague-Dawley strain. Augier et al. (2014) recently demonstrated the same effect in Wistar rats. Using the procedure outlined here (**Figure 1A**) reduced the time to complete the experiment by 4 weeks relative to a procedure utilizing intermittent (MWF) operant sessions (e.g., Simms et al., 2010). This represents a significant conservation of lab resources.

We used this operant procedure to investigate the ability of NAC, CEF, and CEFAZ to attenuate cue-primed reinstatement. Five days of NAC treatment (30 or 60 mg/kg) did not attenuate cue-primed reinstatement (**Figure 2C**). We found that chronic CEF (200 mg/kg) and CEFAZ (100 mg/kg) attenuated cue-primed reinstatement of alcohol-seeking (**Figure 3C**). Both NAC and CEF attenuate cocaine and heroin reinstatement (Baker et al., 2003; Zhou and Kalivas, 2008; Knackstedt et al., 2010; Reichel et al., 2011; Sondheimer and Knackstedt, 2011; Shen et al., 2014). Furthermore, both compounds have been documented to similarly restore glutamate homeostasis in the NAc following cocaine (see **Table 1**). Both compounds increase GLT-1 and system xc-/xCT expression and function (Knackstedt et al., 2010; Trantham-Davidson et al., 2012), increase basal glutamate

levels (Baker et al., 2003; Trantham-Davidson et al., 2012), and normalize potentiated glutamate synapses (Moussawi et al., 2011; Trantham-Davidson et al., 2012). Because CEF and NAC modulate post-cocaine glutamate homeostasis in similar manner, and the present results reveal a dissociation between the ability of the compounds to attenuate alcohol reinstatement, CEF and CEFAZ likely attenuate alcohol reinstatement via a glutamateindependent mechanism.

Cefazolin has been shown to reduce GABAA transmission (Yamazaki et al., 2002; Sugimoto et al., 2003). Positive allosteric modulation of GABAA enhances reinstatement of alcoholseeking (Finn et al., 2008; Ramaker et al., 2014) and thus a reduction in GABA transmission by CEFAZ may be the mechanism of action by which it reduces alcohol reinstatement. CEF has not been fully evaluated for its ability to alter GABA transmission although one report exists showing that it does not alter GABAA receptor binding in the hippocampus or frontal and parietal cortices (Inui et al., 2013). CEFAZ has not been demonstrated to alter glutamate transmission: it does not alter NMDA function (Yamazaki et al., 2002; Sugimoto et al., 2003) and was not determined to potently induce GLT-1 expression as do CEF and penicillin (Rothstein et al., 2005). The data presented in **Figures 5C,D** provide evidence that CEFAZ does not alter cue- or cocaineprimed reinstatement of cocaine-seeking. Thus, the mechanism by which CEFAZ attenuates alcohol reinstatement likely does not involve modulation of glutamate neurotransmission in the NAs.

Ceftriaxone and CEFAZ are antibiotics and have the potential to produce gastrointestinal distress upon chronic administration. Oral alcohol self-administration in rodents and even alcohol-seeking during cue-primed reinstatements tests may be impacted by this side-effect. Thus we sought to determine the effects of these antibiotics on the operant self-administration of regular chow and sucrose pellets. We found no effect of either antibiotic on the self-administration of chow (**Figure 4A**) or sucrose (**Figure 4B**) pellets or on body weight during the course of CEF and CEFAZ injections (**Figure 4C**). These findings are in agreement with those of Ward et al. (2011) who found no effect of CEF on the self-administration of sweet food in mice. Interestingly, the chronic administration of NAC, which did not attenuate alcohol-seeking here, has been demonstrated to reduce operant self-administration of regular chow (Ramirez-Niño et al., 2013). Thus, we can conclude that the ability of CEF and CEFAZ to attenuate cue-primed alcohol-seeking is likely not dependent on any gastrointestinal side-effects of these compounds.

Because CEFAZ had no impact on cocaine reinstatement (**Figure 5C**), it may not be capable of restoring glutamate homeostasis after cocaine. Thus, this is not likely the mechanism by which CEF and CEFAZ attenuate cue-primed reinstatement of alcohol-seeking. In support of this, NAC, which does alter glutamate homeostasis, did not attenuate cue-primed alcohol-seeking. This conclusion is surprising in light of the evidence supporting glutamate signaling in this behavior. In alcohol-preferring (P) rats, CEF reduces the consumption of alcohol acutely and after a period of withdrawal (Sari et al., 2013; Alhaddad et al., 2014). In P rats, the ability of CEF to modulate alcohol-seeking was accompanied by increases in GLT-1 and xCT (Sari et al., 2013; Alhaddad et al., 2014). These results indicate that the modulation of glutamate homeostasis may be the mechanism by which CEF attenuates alcohol consumption, at least in P rats. However, the increased expression of these proteins was not shown to be the mechanism by which CEF exerted its behavioral effects. An alternative explanation for the failure of NAC to attenuate alcohol reinstatement in the present manuscript is that the operationalization of the alcohol-seeking response may have altered the underlying neurobiology of alcohol-seeking, rendering the manipulation of GLT-1 and xCT (and thereby glutamate levels) ineffective at preventing reinstatement.

The lack of an effect of NAC on alcohol-seeking here does not discount the involvement of glutamate in alcohol reinstatement. Antagonism of the post-synaptic mGluR5 and AMPA receptors attenuates cue-induced reinstatement (Backstrom and Hyytia, 2004; Backstrom et al., 2004; Sanchis-Segura et al., 2006; Sinclair et al., 2012). Stimulating the pre-synaptic glutamate autoreceptor mGluR2/3 also attenuates cue-induced reinstatement of alcohol-seeking (Zhao et al., 2006), presumably by decreasing pre-synaptic glutamate release. While both cocaine and alcohol reinstatement are driven by synaptic glutamate release in the NA core during the reinstatement event (McFarland et al., 2003; Gass et al., 2011) and NAC and CEF attenuate that increase during *cocaine* reinstatement (Baker et al., 2003; Trantham-Davidson et al., 2012), NAC (and potentially CEF) may not be able to attenuate glutamate release during *alcohol* reinstatement. The reason for this may be that cocaine and alcohol have opposite effects on basal glutamate levels in the NA core. Cocaine decreases (Baker et al., 2003) while alcohol increases (Melendez et al., 2005; Griffin Iii et al., 2013) basal glutamate levels. Both NAC and CEF increase basal glutamate levels after cocaine but not in drug-naïve animals (Baker et al., 2003; Trantham-Davidson et al., 2012). After NAC treatment, this increased basal glutamate derived from system xc- restores tone on mGluR2/3 autoreceptors (Moussawi et al., 2011), thereby restoring the ability of these receptors to exert negative feedback on glutamate release during reinstatement. It is possible that increased basal glutamate following alcohol also causes a failure in mGluR2/3 signaling that is insensitive to NAC treatment. The effects of CEF on mGluR2/3 function have not yet been investigated (see **Table 1**). Future experiments examining basal glutamate levels 2–3 weeks after cessation of alcohol self-administration in combination with NAC and CEF would provide valuable data regarding the potential value in manipulating basal glutamate to treat alcohol relapse.

Overall, these results indicate that training Sprague-Dawley rats to self-administer alcohol in the operant chamber

## References

can be accomplished without sucrose fading and by using daily training sessions without lengthy operant sessions. We found that modulating glutamate homeostasis likely will not represent a successful treatment strategy for preventing relapse to alcohol-seeking. Future studies should investigate GABA modulation as a potential mechanism of action for CEF and as a method of attenuating alcohol relapse.

## Acknowledgments

This project was funded by a pilot award from the Charleston Alcohol Research Center (P50 AA010761) and NIDA R01 DA033436, both awarded to LK.

expression and glutamate uptake in primary human astrocytes. *J. Biol. Chem.* 283, 13116–3123. doi: 10.1074/jbc.M707697200

**Conflict of Interest Statement:** The Reviewer Jamie Peters declares that, despite being affiliated to the same institution as the author Ana Weiland, the review process was handled objectively and no conflict of interest exists. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Weiland, Garcia and Knackstedt. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## IL-1 interacts with ethanol effects on GABAergic transmission in the mouse central amygdala

*Michal Bajo1\*, Florence P. Varodayan1, Samuel G. Madamba1, Amanda J. Roberts 2, Lindsey M. Casal 2, Christopher S. Oleata1, George R. Siggins 2 and Marisa Roberto1*

*1 Committee on the Neurobiology of Addictive Disorders, The Scripps Research Institute, La Jolla, CA, USA, 2 Molecular and Cellular Neuroscience Department, The Scripps Research Institute, La Jolla, CA, USA*

Neuroinflammation is hypothesized to enhance alcohol consumption and contribute to the development of alcoholism. GABAergic transmission in the central amygdala (CeA) plays an important role in the transition to alcohol dependence. Therefore, we studied the effects of interleukin-1β (IL-1β), a proinflammatory cytokine mediating ethanol-induced neuroinflammation, and its interaction with ethanol on CeA GABAegic transmission in B6129SF2/J mice. We also assessed ethanol intake in B6129SF2/J mice. Intake with unlimited (24 h) ethanol access was 9.2–12.7 g/kg (3–15% ethanol), while limited (2 h) access produced an intake of 4.1 ± 0.5 g/kg (15% ethanol). In our electrophysiology experiments, we found that recombinant IL-1β (50 and 100 ng/ml) significantly decreased the amplitude of evoked inhibitory postsynaptic potentials (eIPSPs), with no significant effects on paired-pulse facilitation (PPF). IL-1β (50 ng/ml) had dual effects on spontaneous miniature inhibitory postsynaptic currents (mIPSCs): increasing mIPSC frequencies in most CeA neurons, but decreasing both mIPSC frequencies and amplitudes in a few cells. The IL-1β receptor antagonist (IL-1ra; 100 ng/ml) also had dual effects on mIPSCs and prevented the actions of IL-1β on mIPSC frequencies. These results suggest that IL-1β can alter CeA GABAergic transmission at pre- and postsynaptic sites. Ethanol (44 mM) significantly increased eIPSP amplitudes, decreased PPFs, and increased mIPSC frequencies. IL-1β did not alter ethanol's enhancement of the eIPSP amplitude, but, in IL-1β-responsive neurons, the ethanol effects on mIPSC frequencies were lost. Overall, our data suggest that the IL-1 system is involved in basal GABAergic transmission and that IL-1β interacts with the ethanol-induced facilitation of CeA GABAergic transmission.

Keywords: IL-1**β**, central amygdala, GABAA, IPSCs, eIPSPs, interleukin, cytokine, IL-1ra

## Introduction

Studies of human alcoholic brains and animal models have shown a link between the neuroimmune system and the brain changes associated with acute and chronic alcohol exposure (Crews and Vetreno, 2011; Crews et al., 2011; Harris and Blednov, 2012; Szabo et al., 2012; Szabo and Lippai, 2014). In particular, the interleukin-1 (IL-1) system has emerged as an important player in alcohol drinking and the development of alcohol dependence, and as a key regulator of alcohol-induced neuroimmune responses (Crews and Vetreno, 2011;

#### *Edited by:*

*M. Foster Olive, Arizona State University, USA*

#### *Reviewed by:*

*Ciaran J. Faherty, Cadence Pharmaceuticals, USA Jennifer L. Cornish, Macquarie University, Australia*

#### *\*Correspondence:*

*Michal Bajo, Committee on the Neurobiology of Addictive Disorders, The Scripps Research Institute, SP30-1150, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology*

> *Received: 08 November 2014 Accepted: 28 February 2015 Published: 19 March 2015*

#### *Citation:*

*Bajo M, Varodayan FP, Madamba SG, Roberts AJ, Casal LM, Oleata CS, Siggins GR and Roberto M (2015) IL-1 interacts with ethanol effects on GABAergic transmission in the mouse central amygdala. Front. Pharmacol. 6:49. doi: 10.3389/fphar.2015.00049* Crews et al., 2011; Harris and Blednov, 2012; Szabo et al., 2012; Szabo and Lippai, 2014). The IL-1 system includes the cytokines IL-1α and IL-1β, the receptor IL-1R1, the IL-1R accessory protein (IL-1RAcP), and two negative regulators (a decoy receptor IL-1R2 and the IL-R1 antagonist: IL-1ra). The proinflammatory activities of the cytokines IL-1α and IL-1β are initiated by their binding to the IL-1 receptor (IL-1R1) and formation of a receptor heterodimeric complex with IL-1RAcP. After recruitment of the myeloid differentiation primary response gene 88 (MyD88) adaptor to the IL-1R/IL-1RAcpP complex, signaling pathways, including NF-κB, c-Jun N-terminal kinase (JNK) and p38 MAPK, are activated (Garlanda et al., 2013; Krumm et al., 2014).

 Several studies have reported changes in the expression of genes encoding components of the IL-1R1 signaling pathways in the brains of mice with a genetic predisposition to alcohol consumption (Mulligan et al., 2006; Blednov et al., 2012). Additionally, polymorphisms in the genes encoding the IL-1R antagonist (IL-1ra; *Il1rn*) and IL-1β (*Il1b*), but not IL-1α (*Il1a*) and IL1-R1 (IL-1R1 type 1; *Il1r*), have been associated with a susceptibility to alcoholism or ALD (alcohol liver disease) in Spanish men (Pastor et al., 2005). Behavioral studies indicate a reduction in alcohol drinking and/or preference in *Il1rn* knockout mice (Blednov et al., 2012) and suggest an important role of the IL-1 system in alcohol's effects. IL-1β levels are increased in alcoholics, as well as animal models of chronic alcohol exposure (Valles et al., 2004; Qin et al., 2008; Lippai et al., 2013a,b), and intracerebroventricular administrations of IL-1β potentiate alcohol withdrawal-induced anxiety (Breese et al., 2008). Conversely, administration of IL-1ra prevented and protected against alcohol-induced neuroinflammation (Lippai et al., 2013b), and reduced alcohol-induced sedation and motor impairment recovery time in mice (Wu et al., 2011).

As the central nucleus of the amygdala (CeA) plays a critical role in mediating alcohol-related and anxiety-like behaviors (Gilpin et al., 2014), it is likely that the IL-1 signaling system modulates ethanol's effects on CeA function. In fact, we reported recently that the IL-1ra regulates baseline GABAergic transmission in the CeA and is critical for the effects of ethanol at these synapses (Bajo et al., 2014a). Additionally, immune challenges, such as systemic IL-1β or LPS administration, are known to activate the CeA (Dayas et al., 2001; Frost et al., 2001; Konsman et al., 2008). Moreover, IL-1R1 is expressed in the amygdala under basal conditions (Frost et al., 2001), while both IL-1β and IL-1ra are induced in the CeA by excitotoxic stimuli or systemic immune challenge (Eriksson et al., 2000; Konsman et al., 2008).

As neuroinflammation plays an important role in alcohol use disorders and other psychiatric disorders (e.g., depression, PTSD; Jones and Thomsen, 2013), there are concerted efforts to develop new therapeutic strategies using compounds with anti-inflammatory properties to treat these disorders. Therefore, understanding the molecular and cellular mechanisms that mediate normal and pathological neuroimmune responses in the key brain regions involved in the pathogenesis of psychiatric disorders is critical for the evaluation of potential candidate drugs and their clinical use. Here, we examined the effects of IL-1β on GABAergic transmission in the CeA, as well as its actions on ethanol-induced facilitation of GABAergic transmission. We recorded from B6129SF2/J mice because they have been used previously as a control for *Il1r* KO mouse studies assessing the role of IL-1R1 in various biological phenomena1 (for list of publications). Because alcoholrelated behaviors in these mice have not been studied, we also characterized the B6129SF2/J strain for alcohol drinking and preference.

## Materials and Methods

### Animal Treatment

Male B6129SF2/J (*n* = 80; 29.5 ± 0.3 g) mice were housed in a temperature- and humidity-controlled room on a 12-h light/dark cycle (lights on at 6:00 pm) with food and water available *ad libitum*. We conducted all care procedures in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with the Institutional Animal Care and Use Committee policies of The Scripps Research Institute.

### Slice Preparation

The mice (10–16 weeks old at the time of electrophysiological recordings) were anesthetized with 3% isoflurane, decapitated, and the brains quickly removed and placed in ice-cold artificial cerebrospinal fluid (ACSF: composition in mM: NaCl, 130; KCl, 3.5; NaH2PO4, 1.25; MgSO4.7H2O, 1.5; CaCl2, 2.0; NaHCO3, 24; glucose, 10) and ice-cold oxygenated high-sucrose cutting solution (composition (in mM): sucrose, 206; KCl, 2.5; CaCl2, 0.5; MgCl2, 7; NaH2PO4, 1.2; NaHCO3, 26; glucose, 5; HEPES, 5; pH7.3–7.4) gassed with 95% O2 and 5% CO2. We cut coronal slices containing the CeA using a Leica 1000S vibratome cutter (Campden, Lafayette, IN, USA).

### Intracellular Recordings

We incubated the slices (400 μm) in an interface configuration for 30 min, and then completely submerged and continuously superfused (flow rate of 2–4 ml/min) them with warm (31◦C), O2/CO2-gassed ACSF. We added drugs to the ACSF from

1http://jaxmice.jax.org/strain/003018.html

stock solutions to obtain known concentrations in the superfusate. We recorded from CeA neurons with sharp micropipettes containing 3 M KCl (65–80 M resistance) using currentclamp mode. Data were acquired with an Axoclamp-2B preamplifier (now Molecular Devices, Sunnyvale, CA, USA) and stored for offline analysis via pClamp 10.2 software (Molecular Devices). We evoked pharmacologically isolated GABAAergic IPSPs by stimulating locally within the medial subdivision of the CeA with a bipolar stimulating electrode, while continuously superfusing the glutamate receptor blockers 6,7 dinitroquinoxaline-2,3-dione (DNQX, 20 μM) and DL-2-amino-5-phosphonopentanoic acid (DL-AP5, 30 μM), and the GABAB blocker CGP 55845A (1 μM).

We held the CeA neurons near their resting membrane potentials (RMPs ranging from –65 to –85 mV (mean: –78.6 + 0.7 mV, *n* = 73), and applied hyperpolarizing and depolarizing current steps (200 pA increments, 750 ms duration) to generate voltage-current curves. To determine half-maximal IPSP amplitudes, we examined input/output (I/O) curves by measuring evoked IPSP amplitudes at five stimulus strengths ranging from the threshold to maximum stimulation. Subsequent analyses were done with averages of two IPSPs evoked with the half-maximal stimuli. We measured the IPSP amplitudes before (baseline), during (up to 20 min) and after (washout for 10– 25 min) drug application, and determined the percent change in IPSP amplitude at each stimulus intensity using the equation: (Vdrug/Vcontrol) ∗ 100.

We examined paired-pulse facilitation (PPF) using 100 ms interstimulus intervals and the stimulus strength was adjusted so that the amplitude of the first IPSP was 50% of the maximal determined from the I/O relationship. We calculated the PPF using the equation: (2nd IPSP amplitude/1st IPSP amplitude) ∗ 100. We took PPF measurements before drug superfusion (baseline), during (10–20 min) and after drug washout (10–25 min).

#### Whole-cell Patch-Clamp Recording

After cutting, the slices (300 μm) were incubated in O2/CO2 gassed ACSF for 30 min at 32◦C, followed by incubation for 30 min at room temperature. We performed whole-cell patchclamp recording in voltage clamp mode, as described previously (Bajo et al., 2011). Briefly, we used infrared/DIC visualization of CeA neurons (Dodt and Zieglgansberger, 1990), followed by digitization and image enhancement via an upright, fixed-stage Olympus microscope. We used micropipettes with an input resistances of 3–6 M*-* (access resistance *<*20 M*-*, compensated 60–80%) filled with an internal solution (composition in mM: KCl, 145; EGTA, 5; MgCl2, 5; HEPES, 10; Na-ATP, 2; Na-GTP, 0.2; the latter two added fresh on the day of recording), pH 7.3–7.4. We isolated spontaneous miniature GABAA-mediated IPSCs (mIPSCs) pharmacologically by applying blockers of glutamatergic (20 μM DNQX, 30 μM DL-AP5) and GABAB receptors (1 μM CGP 55845A), and adding 0.5 μM tetrodotoxin (TTX) to the bath. We used the Multiclamp 700B and pClamp 10.2 software (Molecular Devices) for data acquisition. Recombinant mouse IL-1β, recombinant IL-1ra and ethanol were added to the ACSF from stock solutions in known concentrations. We took

all measures before drug (baseline) and during drug superfusion (12–15 min).

### Ethanol Drinking Procedure

This procedure was adapted from that of Blednov et al. (2005). Mice were allowed to acclimate for 1 week to individual housing. Two drinking tubes were continuously available, Monday– Friday, to each mouse and fluid consumption was measured daily. One bottle of water was available across weekends. Food was available *ad libitum* and mice were weighed each week. After 4 days of water consumption (on Monday, off Friday; water in both tubes), mice were offered 3% ethanol (v/v) versus water on the following Monday–Friday. We changed tube positions every day to control for position preferences. Over the following 4 weeks mice received 6, 9, 12, and 15% ethanol in this same manner. Following this, mice received 15% ethanol for 2 h per day (starting 3 h after lights off) for 5 days in order to examine limited access two bottle-choice (2BC) drinking. The quantity of ethanol consumed (g/kg body weight/24 h or 2 h) was calculated for each mouse and averaged across each 4–5 day measurement period.

## Data Analysis and Statistics

To analyze data acquired from intracellular and whole-cell recordings, we used Clampfit 10.2 (Molecular Devices) and MiniAnalysis 5.1 software (Synaptosoft, Leonia, NJ, USA), respectively. We used GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) software for all statistical analysis. We accepted statistical significance at the *p <* 0.05 level using oneway ANOVA and *t*-tests. The data are presented as percentile changes in mean ± SEM.

### Drugs

We purchased CGP 55845A, DNQX, and DL-AP5 from Tocris Biosciences (Ellisville, MI, USA), recombinant mouse IL-1β from Biolegend (San Diego, CA, USA), recombinant human IL-1ra from Peprotech (Rocky Hill, NJ, USA), and TTX from Calbiochem (San Diego, CA, USA). We obtained ethanol from Remet (La Mirada, CA, USA).

## Results

## Ethanol Drinking and Preference of B6129SF2/J Mice

Although the B6129SF2/J mice have been used previously as controls for *Il1r* KO mouse studies assessing the role of IL-1R1 in various biological phenomena, their ethanol drinking and preference behavior is unknown. Therefore, we used 2BC tests with unlimited (24 h) and limited (2 h) ethanol access to determine their voluntary drinking and preference. The average daily ethanol intake with 24 h access, measured for a period of 5 days, ranged from 9.24 to 12.65 g/kg for the ethanol concentrations tested (3, 6, 9, 12, and 15%; **Figure 1A**). There were no significant differences in ethanol intake between the tested concentrations, nor was there a correlation between the intake and ethanol concentration (*R*2 = 0.7). During 2 h limited access, the average ethanol (concentration 15%) intake was 4.1 ± 0.5 g/kg

FIGURE 1 | Ethanol drinking behavior of B6129SF2/J mice. The ethanol intake of B6129SF2/J mice was tested using unlimited (24 h) and limited (2 h) 2-bottle choice (2BC) paradigms. (A) The intake of 3, 6, 9, 12, and 15% ethanol was measured by 2BC with unlimited access to ethanol. On average, the mice consumed 11.6 ± 0.9 g/kg/day of 3% ethanol solution, 12.7 ± 1.8 g/kg/day of 6% ethanol solution, 10.7 ± 1.1 g/kg/day of 9% ethanol solution, 9.2 ± 1.1 g/kg/day at 12% ethanol solution, and 9.3 ± 1.0 g/kg/day of 15% ethanol solution. There was significant main difference in ethanol intake between the ethanol concentrations [*F(*4*,*19*)* = 2.5, *n* = 20], but Tukey *post hoc* analysis did not reveal significant differences between specific ethanol concentrations. (B) For limited access measurements of ethanol consumption, we used 15% ethanol solution and intake was measured for 2 h daily (starting 3 h after lights off) for a period of 5 days. Consumption was 3.3 ± 0.6 g/kg of ethanol on day 1; 4.2 ± 0.4 g/kg of ethanol on day 2; 0.4.2 ± 0.5 g/kg on days 3 and 4; 4.4 ± 0.5 on day 5. Repeated measure one-way ANOVA showed no significant difference in ethanol intake between testing days [*F(*4*,*17*)* = 0.7, *n* = 18]. (C) Ethanol intake of individual mice is plotted as a function of preference. The average ethanol preference ratio (volume of ethanol consumed/total volume of fluid consumed) was 0.56 ± 0.05. There was no significant correlation (*R*2 = 0.01) between ethanol preference and intake in B6129SF2/J mice.

(*n* = 18; **Figure 1B**) and the average ethanol preference ratio was 0.56 ± 0.05 (volume of ethanol consumed/total volume of fluid consumed). We did not observe a significant correlation between ethanol preference and ethanol intake (*R*2 = 0.01) in the individual B6129SF2/J mice (**Figure 1C**). These data indicate that B6129SF2/J mice drink a substantial amount of ethanol and have a modest preference for ethanol.

### IL-1**β** Decreased eIPSP Amplitudes in the CeA

We tested the effects of recombinant mouse IL-1β (5, 50, and 100 ng/ml) on GABAA receptor-mediated eIPSPs in the CeA. None of the tested concentrations significantly altered the current–voltage relationships, resting membrane potentials, or resistance (data not shown). In the majority of CeA neurons, high IL-1β concentrations (50 and 100 ng/ml) significantly decreased eIPSP amplitudes, by 27.8 ± 6.0% (*n* = 11; *t*-test: *p <* 0.01) and by 21.6 ± 6.7% (*n* = 5; *t*-test: *p <* 0.05), respectively (**Figure 2A**). However, 5 ng/ml IL-1β had no significant effect on the mean amplitudes of the eIPSPs (to 89.8 ± 5.0% of baseline, *n* = 7). The significant decreases in eIPSPs were not associated with changes in the PPF ratio, although there was a trend toward an increase in the PPF ratio by IL-1β at 100 ng/ml (50 ng/ml: 103.7 ± 7.2% of baseline, *n* = 8; 100 ng/ml: 130.1 ± 19.4% of baseline, *n* = 4; **Figure 2B**). Thus, these results indicate that IL-1β reduces GABAergic transmission, likely via postsynaptic mechanisms.

## IL-1**β** had Dual Effects on mIPSC Frequencies and Decreased mIPSC Amplitudes in CeA Neurons

We performed whole-cell recordings of mIPSCs in CeA neurons while superfusing 50 ng/ml of IL-1β. Here, we present the combined results of all experiments where IL-1β was applied to the naïve slice for 12–15 min (**Figure 3**). We found that IL-1β had dual effects ( *>* 15% from baseline) on mIPSC frequencies and amplitudes, and so we examined its effects on mIPSC frequencies and amplitudes separately. We found that IL-1β significantly increased the mean mIPSC frequency by 50.7 ± 10.1% in 13 of 21 CeA neurons (**Figure 3A**). In 6 of 21 cells, we observed a significant decrease in the mean mIPSC frequency by 44.1 ± 9.5%, which was associated with a significant decrease in the mean mIPSC amplitude (76.7 ± 6.2% of baseline) and an increase in mIPSC rise time (114.4 ± 4.7% of baseline; **Figure 3B**). In the remaining 2 neurons, IL-1β had no effect on mIPSC frequency (data not shown). Using the changes in mIPSC amplitude as the parameter for the division of the data, we found a significant decrease in the mIPSC amplitude in 8 of 21 cells (72.2 ± 6.2% of baseline) and no effects in 10 of 21 cells. In the remaining three CeA neurons, IL-1β increased both the mIPSC amplitude by 46.0 ± 14.7% and frequency by 62.8 ± 26.7% (data not shown). Since changes in mIPSC frequencies suggest an altered probability of vesicular transmitter release, and changes in mIPSC amplitudes may reflect modulation of postsynaptic GABAA receptors (De Koninck and Mody, 1994; Otis et al., 1994), our data indicate that IL-1β alters spontaneous action potential-independent GABA transmission through both presynaptic and postsynaptic mechanisms of action. Importantly, the parallel changes in mIPSC frequencies and amplitudes of individual CeA neurons suggest that acute IL-1β acts in a cell-specific manner.

### IL-1ra Modulates CeA mIPSCs and Blocks the Effects of IL-1**β** on mIPSCs

To examine the role of IL-1R1 in the effects of IL-1β in the CeA, we used an IL-1R1 antagonist (recombinant IL-1ra) to block IL-1β's actions on GABAergic transmission. Here, we present the results of experiments where IL-1ra (100 ng/ml) was applied to the naïve slice for 12–15 min, and the subset of these experiments where IL-1β was subsequently co-applied for 12–15 min (**Figure 4**). We observed transient IL-1ra effects with maximal cellular responses within 9–15 min of drug application. Similar to the IL-1β effects, the IL-1ra-induced changes in mIPSC frequency and/or amplitude varied among individual CeA neurons. In the majority (67%) of CeA cells, IL-1ra decreased significantly the mean mIPSC frequency by 31.3 ± 2.1% (**Figure 4A**). In the remaining cells, IL-1ra significantly increased the mIPSC frequency by 34.1 ± 7.7% (**Figure 4A**). These changes in the mIPSC frequencies were not associated with significant changes in mIPSC amplitudes or kinetics. On the other hand, when we used the change in mIPSC amplitude ( *>* 15%) as the criterion for cell grouping, we found that IL-1ra increased significantly the mean mIPSC amplitude by 27.9 ± 4.5% in 39% of the cells and decreased the mean mIPSC amplitude by 21.7 ± 5.9% in 28% of CeA neurons (**Figure 4B**). These changes in mIPSC amplitudes were not associated with significant changes in mIPSC frequencies or kinetics. In the rest of the cells (33%), IL-1ra did not alter significantly the mean mIPSC frequency, amplitude or kinetics. These results indicate that IL-1R1 plays a role in basal GABAergic transmission in the CeA.

We also examined whether IL-1ra prevents the IL-1β-induced changes in mIPSCs. In order to do this, we grouped the neurons into two groups based on their IL-1ra-induced changes in mIPSC frequencies, and compared their mean mIPSC characteristics to the average mIPSC characteristics observed after 12–15 min of IL-1ra and IL-1β co-application. Co-application with IL-1β did not induce significant differences in the mIPSCs compared to IL-1ra alone (**Figure 4C**). These results suggest that IL-1β modulates mIPSCs via IL-1R1.

### Ethanol Increased eIPSPs and mIPSCs in the CeA Via a Predominantly Presynaptic Mechanism

Ethanol (44 mM) had no significant effects on the intrinsic membrane properties (resting membrane potential, the current– voltage relationship, resistance) of CeA neurons (data not shown; see also Roberto et al., 2003). Superfusion of 44 mM ethanol increased the mean eIPSP amplitude by 22.5 ± 5.9% in CeA neurons and significantly decreased the PPF ratio (82.2 ± 4.0% of baseline), suggesting that ethanol acts via presynaptic mechanisms (**Figure 5A**). This finding is supported by ethanol's facilitation of the mean mIPSC frequency by 40.7 ± 17.5% (**Figure 5B**). Although ethanol had no effect on the mean mIPSC amplitude, it significantly increased the mIPSC rise (by 14.7 ± 4.2%) and decay (by 25.4 ± 7.0%) times.

### Co-Application of Ethanol Reversed the IL-1**β**-Induced Decrease in the Mean eIPSP Amplitude

We then examined the interaction between IL-1β and ethanol on GABAergic transmission in the CeA, by superfusing IL-1β (50 ng/ml) for 15–20 min, followed by co-application of IL-1β and ethanol (44 mM) for an additional 15–20 min. Using

intracellular recording, we found no significant changes in the membrane properties induced by IL-1β or co-application of IL-1β and ethanol (data not shown). IL-1β alone significantly decreased the mean eIPSP amplitude (84.8 ± 4.7% of baseline), whereas co-application with ethanol reversed the IL-1β-induced decrease in the mean eIPSP amplitude back to 115.4 ± 5.3% of the original baseline (**Figures 6A,B**). Ethanol co-application significantly increased the mean eIPSP amplitude in comparison to the IL-1β effect, but not with respect to the baseline level (**Figure 6C**). In addition, we did not observe significant changes in the PPF ratio following superfusion with IL-1β alone or co-application of IL-1β and ethanol (**Figure 6D**). These results indicate that IL-1β and ethanol modulate CeA eIPSPs via different mechanisms, though the occlusion of ethanol's PPF effects suggest that IL-1β may interfere with the downstream mechanisms mediating ethanol-facilitated GABA release.

### IL-1**β** Occluded Ethanol Effects on CeA mIPSCs

Finally, we investigated the potential interaction between IL-1β and ethanol on action potential-independent vesicular GABA release. In the majority of CeA neurons (6 of 10 cells), IL-1β alone, as well as its co-application with ethanol, significantly increased the mean mIPSC frequency (to 145.9 ± 14.6% and 142.4 ± 8.9% compared to baseline, respectively; **Figure 7A**). There were no significant changes in the mean mIPSC amplitudes or kinetics with IL-1β or co-application of IL-1β and ethanol in these cells (**Figure 7A**). However, in 3 of 10 cells, IL-1β alone decreased the mIPSC frequency by 53.5 ± 11.3%, and subsequent co-application of ethanol did not alter this IL-1β-induced decrease in mIPSC frequency (remained at 59.2 ± 7.9% of baseline; **Figure 7B**). Although there was a trend toward a decrease in mIPSC amplitudes by IL-1β and co-application of IL-1β and ethanol (82.15 ± 9.7% and 88.14 ± 11.8 of baseline, respectively) in these three neurons, it did not reach statistical significance. Finally, there was one CeA cell that showed no IL-1β effect on mIPSC frequency, but co-application of ethanol increased the mIPSC frequency to 127.6% of baseline. In this neuron, the mIPSC amplitude was decreased by IL-1β alone (by 30.8%) and also by ethanol co-application (by 32.1%).

## Discussion

In the present study, we investigated cytokine IL-1β modulation of GABAergic transmission and its interaction with ethanolinduced facilitation of GABA signaling in the CeA of B6129SF2/J mice. Behaviorally, B6129SF2/J mice have a moderate preference for alcohol and consume a substantial amount of alcohol. At the cellular level, IL-1β modulation of CeA GABAergic transmission is characterized by a reduction of evoked IPSPs, mediated predominantly by postsynaptic mechanisms, and by predominantly presynaptic dual effects on spontaneous miniature IPSCs in a cell-specific manner. The IL-1β effects on mIPSCs appear to be mediated by IL-1R1. Moreover, IL-1R1 regulates basal mIPSCs in

IL-1**β**-induced modulation of mIPSCs in the CeA. IL-1ra-induced changes in mIPSC frequencies and/or amplitudes vary among CeA neurons, indicating cell-specific differences in IL-1ra modulation of GABAergic transmission. (A) Dual IL-1ra-induced changes in mIPSC frequencies. (Top) Representative recordings of two CeA neurons showing a decrease (left column) or increase (right column) in mIPSC frequencies following IL-1ra application (100 ng/ml). (Bottom) Summary bar graph showing IL-1ra decreased significantly (*t*-test, *p <* 0.01) the mean mIPSC frequency by 31.3 ± 2.1% in 12 of 18 (67%) neurons. In the remaining CeA cells (6 of 18), IL-1ra increased the mIPSC frequency by 34.1 ± 7.7% (*t*-test, *p <* 0.05). The changes in mIPSC frequencies were not associated with significant changes in mIPSC amplitudes or kinetics. (B) The IL-1ra induced changes in the mIPSC amplitudes were also variable. (Top) Representative recordings of two cells responding to IL-1ra with increased (left column) or decreased (right column) mIPSC amplitudes. (Bottom) Summary bar graph showing IL-1ra increased (*t*-test, *p <* 0.01) mIPSC amplitudes by 27.9 ± 4.5% in 7 of 18 cells (39%) and decreased (*t*-test, *p <* 0.01) by 21.7 ± 5.9% in 5 of 18 cells

and kinetics across all cell groups. The statistical significance (∗*p <* 0.05) and (∗∗*p <* 0.01) was calculated by *t*-test. (C) To examine the effects of IL-1ra on the IL-1β-induced modulation of mIPSCs, we compared the mIPSC parameters recorded within 9–15 min of 100 ng/ml IL-1ra and 50 ng/ml IL-1β co-application to the last 6 min (9–15 min) of IL-1ra application alone. We divided the CeA neurons into two groups according to their cellular responses (mIPSCs frequency) to IL-1ra alone: the cells that responded to IL-1ra with decreased mIPSC frequency [by 31.8 ± 3%; *F(*2*,*23*)* = 7.5, *p <* 0.05; *n* = 7] and the cells that responded to IL-1ra with increased mIPSC frequency [by 27.9 ± 7%; *F(*2*,*8*)* = 1.7, *p <* 0.05; *n* = 3]. (Left) Representative recordings from two CeA neurons responding to IL-1ra with decreased (left column) or increased (right column) mIPSCs frequencies. (Right) IL-1ra prevented the IL-1β-induced modulation of mIPSCs, as there were no significance differences in mIPSCs after co-application of IL-1ra and IL-1β compared to IL-1ra alone. The statistical significance was set at (∗*p <* 0.05) and was calculated by repeated measurement one-way ANOVA followed by a Tukey *post hoc* test.

FIGURE 5 | Ethanol potentiates CeA GABAergic transmission. (A) Ethanol potentiated eIPSPs via a presynaptic mechanism in CeA neurons. (Top) Representative recordings of eIPSPs from a CeA neuron showing an ethanol-induced increase in eIPSP amplitude that is reversed upon drug washout. (Bottom) On average, 44 mM ethanol significantly increased the mean eIPSP amplitude by 22.5 ± 5.9% (left column: *n* = 8; *t*-test: *p <* 0.05) and decreased the PPF ratio to 82.2 ± 4.0% of baseline in six of eight neurons (right column: *n* = 6; *t*-test: *p <* 0.05), indicating that ethanol-induced eIPSP potentiation is mediated by increased GABA release. (B) Ethanol increases spontaneous miniature GABA transmission in the CeA

by both pre- and postsynaptic mechanisms. (Top) Representative mIPSC recordings from a CeA neuron showing an ethanol-induced increase in frequency. (Bottom) Superfusion of 44 mM ethanol induced a significant increase in the mean mIPSC frequency (140.7 ± 17.5% of baseline), but had no effect on the mean amplitude (102.6 ± 9.5% of baseline; *n* = 4, *t*-test: *p <* 0.05), supporting the finding that ethanol's mechanism of action is predominantly presynaptic. However, ethanol significantly altered mIPSC kinetics, with a 14.7 ± 4.2% increase in the rise time and a 25.4 ± 7.0% increase in the decay time, indicating additional postsynaptic changes (*t*-test: ∗*p <* 0.05 and ∗∗*p <* 0.01).

Statistical significance [*p <* 0.05; ∗(comparisons to baseline) and #(comparison of the effects of ethanol plus IL-1β co-application to IL-1β alone or washout)] was calculated by repeated measurement one-way ANOVA followed by a Tukey *post hoc* test. (D) There were no significant effects on the PPF ratio (100 ms interstimulus interval) of IL-1β alone, or when it was co-applied with ethanol [*F(*2*,*23*)* = 0.24].

the CeA. The interaction of IL-1β and ethanol is likely to occur presynaptically, and is characterized by the occlusion of ethanol's facilitation of vesicular GABA release by IL-1β.

B6129SF2/J mice have been used previously as controls for *Il1r* KO mice (#003018, Jackson Laboratories) in studies characterizing the role of IL-1R1 in various physiological and pathological processes2 (for a list of publications). Alcohol drinking behavior in B6129SF2/J mice has not been determined, despite the fact that different mice strains exhibit a range of alcohol drinking behaviors in terms of alcohol consumption and preference (Rhodes et al., 2007; Yoneyama et al., 2008). Since the genetic background of B6129SF2/J mice is based on C57BL/6J and 129S1/SvImJ mice, we expected to find similarities in the alcohol drinking phenotype of B6129SF2/J mice to those two strains, particularly the C57BL/6J mice. Our behavioral data showed that ethanol intake and ethanol preference of B6129SF2/J mice are similar to the values reported for C57BL/6J mice (Yoneyama et al., 2008).

Cytokines, including IL-1, play an important role in the regulation of both excitatory and inhibitory neurotransmission in the central nervous system (Camacho-Arroyo et al., 2009). IL-1R1 is expressed on glial cells and neurons, and thus, the overall effect of IL-1 on synaptic transmission is a combination of the direct effects of IL-1 binding to neuronal IL-1R1 and the indirect effects mediated by other signaling molecules generated and released by both neurons and glia in response to IL-1/IL-1R1 binding (e.g., cytokines, chemokines, ATP, etc.). The IL-1β effects on GABAergic transmission appear to be brain region specific, as IL-1 increases GABAergic transmission in some regions (e.g. hypothalamus, hippocampus; Miller et al., 1991; Plata-Salaman et al., 1998; Tabarean et al., 2006) and decreases it in others (e.g., basolateral amygdala, cerebellum; Yu and Shinnick-Gallagher, 1994; Pringle et al., 1996).

In our study we determined a concentration response curve for IL-1β, and found that only higher concentrations of IL-1β (*>*5 ng/ml) were effective in the modulation of CeA GABAergic transmission. The fact that the effective doses in our study are higher than in other brain regions (often in pg/ml range) may be caused by regional differences in the IL-1 system, especially in the expression of IL-1R1 (Wong and Licinio, 1994; Yabuuchi et al., 1994; Ericsson et al., 1995). To examine the role of IL-1R1 in the IL-1β effects, we used a recombinant IL-1R1 antagonist

2http://jaxmice.jax.org/strain/003018.html

(IL-1ra). IL-1ra blocks the effects of IL-1β on mIPSCs, indicating that IL-1R1 mediates the IL-1β-induced modulation of CeA GABAergic transmission. We also observed a transient modulation of mIPSCs by IL-1ra alone, indicating that IL-1R1 regulates basal mIPSCs. In agreement with this finding, we reported recently an important role of IL-1ra and the IL-1 system in basal CeA GABAergic transmission. In that study, we observed an increase in the frequency of the spontaneous action potentialdependent IPSCs in IL-1ra deficient mice, but mISPC frequencies (action potential-independent IPSCs) were not affected (Bajo et al., 2014a). In addition to the compensatory mechanism associated with knockout technology and the different strains of mice used in the two studies (B6129SF2/J vs. C57Bl6J), the transiency of the IL-1ra effects on mIPSC in the current study may explain the lack of differences between baseline mIPSC frequencies of IL-1ra deficient mice and wild-type controls. Overall, both studies indicate that IL-1ra and the IL-1 system are involved in the regulation of basal GABAergic transmission in the mouse CeA. In the hippocampus, IL-1R1 also plays a critical role in baseline neuronal activity, (Hellstrom et al., 2005), while IL-1ra alone had no effects in neurons from the paraventricular nucleus of the hypothalamus (Ferri and Ferguson, 2003) or the spinal cord (Liu et al., 2013). Collectively, these findings further support that the regional specificity of the IL-1 system-dependent regulation of neuronal activities may underlie the brain region differences in the neuropathology associated with neuroinflammation.

Additionally, in our study, the IL-1β and IL-1ra effects on GABAergic transmission occurred in a majority of CeA neurons, and the effects were characterized by a duality of responses in individual CeA neurons. Other groups have reported a similar duality in their results, with electrophysiological studies revealing that IL-1β only affects synaptic transmission in a portion of neurons in the amygdala, cerebellum, hippocampus, and hypothalamus (Miller et al., 1991; Yu and Shinnick-Gallagher, 1994; Pringle et al., 1996; Plata-Salaman et al., 1998; Tabarean et al., 2006). It is therefore plausible to speculate that the cell-specific IL-1β and IL-1ra effects that we observed are determined by neuronal type, especially given the considerable heterogeneity of CeA neurons in terms of their biochemical and electrophysiological properties (Chieng et al., 2006; Herman et al., 2013) and their likely IL-1R1 expression. We classified each CeA neuron according to the electrophysiological criteria used in our previous studies on cell-type specific tonic GABA conductance in the CeA (Herman et al., 2013; Herman and Roberto, 2014), but did not observe any correlation between the IL-1β or IL-1ra effects and cell-type (low-threshold bursting, late spiking and regular spiking CeA neurons). Instead, it is likely that the cell-specificity of the IL-1β and IL-1ra effects are determined by the CeA neuronal expression of IL-1R1 (pre- versus postsynaptic expression), signaling pathways and/or other biochemical properties. Although our findings strongly indicate that the IL-1β effects on GABAergic transmission are mediated by IL-1R1, we cannot rule out completely that some of the IL-1β effects in the CeA may be caused by indirect actions of IL-1β via other signaling molecules (Camacho-Arroyo et al., 2009).

In this study, we have corroborated in B6129SF2/J mice our previous findings on ethanol's facilitation of GABAergic transmission in the mouse CeA (mostly C57BL6/J; Bajo et al., 2008, 2014b; Kang-Park et al., 2009; Cruz et al., 2011; Herman et al., 2013). In CeA slices from B6129SF2/J mice, ethanol potentiated both evoked and spontaneous CeA GABAergic transmission predominantly via presynaptic mechanisms, but also had limited postsynaptic effects. To investigate the potential interaction between ethanol and IL-1β, we pretreated slices with IL-1β, and then co-applied ethanol and IL-1β. Ethanol, in the presence of IL-1β, was still able to potentiate evoked GABAergic transmission, despite the IL-1β-induced reduction in evoked IPSP amplitudes. However, the effects of ethanol co-application with IL-1β were not significantly different when compared to the original baseline levels. These data suggest that the mechanisms of action of IL-1β and ethanol on evoked GABA transmission are different, in line with our findings that IL-1β acts via predominantly postsynaptic mechanisms, whereas ethanol acts presynaptically. In the case of spontaneous GABAergic transmission in the CeA, IL-1β has dual effects, either increasing or decreasing vesicular GABA release. Notably, ethanol co-application with IL-1β did not facilitate further vesicular GABA release in the neurons that had previously responded to IL-1β with an increase in mIPSC frequency. Ethanol also failed to increase GABA release in the neurons that responded to IL-1β with decreased mIPSC frequency, suggesting an occlusion of ethanol's effects by IL-1β pretreatment. The differences in eIPSP and mIPSC findings on the interaction of IL-1β and ethanol are likely to originate from differences in the forms of GABA release involved in each kind of synaptic transmission (Mathew et al., 2008; Fredj and Burrone, 2009). Specifically, mIP-SCs are recorded in the presence of TTX to block Na+ channels and consequently, the generation of action potentials. In contrast, evoked IPSPs require stimulation of a synaptic network, and thus, action potential-dependent release (Farrant and Nusser, 2005).

## Conclusion

Our data collectively demonstrate that B6129SF2/J mice show an ethanol phenotype similar to that of C57BL6J mice, both behaviorally and electrophysiologically, in the CeA. IL-1β modulation of CeA GABAergic transmission is complex and characterized by dual and cell-specific modulations of presynaptic GABA release and postsynaptic GABAA receptor activity. With regard to the IL-1β effects on ethanol-induced facilitation of CeA GABAergic transmission, our data indicate that IL-1β interacts with ethanol presynaptically to occlude ethanol's enhancement of GABA signaling. Understanding these complex interactions of acute ethanol with IL-1 on GABAergic transmission are critical for shedding light on the potential role of the IL-1 neuroimmune system in the development of alcohol dependence and addiction.

## Acknowledgments

We thank Dr. Marian L. Logrip for help with statistical analyses. This work was supported by NIH/NIAAA INIA West Consortium U01-AA013498 to MR and GRS, U01-AA013498- 14S1 to MR, AA013517 to MR and MB, and AA020893 to AR.

## References

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Bajo, Varodayan, Madamba, Roberts, Casal, Oleata, Siggins and Roberto. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

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**3. Solution by Adomian Decomposition Method**

In this section, the Adomian solution is determined for the velocity field. According to the Adomian decomposition method, Equation (12) can be written in the operator form as

$$L\_r w = \frac{\mathrm{d}p}{\mathrm{d}z} + \mathrm{M}^2 w \tag{14}$$

where the differential operator *Lr* is defined in the form

$$L\_r = \frac{1}{r} \frac{\partial}{\partial r} \left( r\mu(r) \,\,\frac{\partial}{\partial r} \right) \tag{15}$$

and the inverse operator *L*−1 *r* is defined by

$$L\_r^{-1}(.) = \int \left[ \frac{1}{r\mu(r)} \int r(.) \,\mathrm{d}r \right] \mathrm{d}r \tag{16}$$

Applying the inverse operator, Equation (12) takes the form

$$\begin{array}{l} w(r,z) = L\_r^{-1} \left[ \frac{\mathrm{d}p}{\mathrm{d}z} + M^2 w \right] + c\_1 r + c\_2\\ w(r,z) = \frac{\mathrm{d}p}{\mathrm{d}z} I(r) + L\_r^{-1} \left( M^2 w \right) + c\_1 r + c\_2 \end{array} \tag{17}$$

in which

$$L\_r^{-1} \left(\frac{\mathrm{d}p}{\mathrm{d}z}\right) = \int \left[\frac{1}{r\mu(r)} \int r \left(\frac{\mathrm{d}p}{\mathrm{d}z}\right) \mathrm{d}r\right] \mathrm{d}r = \frac{\mathrm{d}p}{\mathrm{d}z} I(r) \tag{17a}$$

and *I*(*r*) is given by

$$I(r) = \int \frac{r}{2\mu(r)} \mathrm{d}r \tag{18}$$

According to Adomian decomposition, it can be written as

$$w = \sum\_{n=0}^{\infty} w\_n \tag{19}$$

Using the Adomian decomposition method, the solution *w*(*r*, *z*) can be elegantly computed by the recurrence relation

$$\begin{array}{rcl} w\_0 &=& c\_1 r + c\_2\\ w\_1 &=& \frac{\mathrm{d}p}{\mathrm{d}z} I(r) + M^2 L\_r^{-1}(w\_0) \\ w\_{n+2} &=& M^2 L\_r^{-1}(w\_{n+1}), \; n \ge 0 \end{array} \tag{20}$$

The above equations give

$$w\_n = M^{2n-2} \left(\frac{\mathrm{d}p}{\mathrm{d}z} + M^2 c\_2\right) \left(L\_r^{-1}\right)^{n-1} I(r) + M^{2n} c\_1 \left(L\_r^{-1}\right)^{n-1} I\_1(r), \ n \ge 1\tag{21}$$

in which

$$I\_1(r) = \int \frac{r^2}{3\mu(r)} \mathrm{d}r \tag{22}$$

With the help of Equations (20) and (21), the closed form of *w* can be written as

$$\begin{aligned} w(r, z) &= w\_0 + \sum\_{n=1}^{\infty} w\_n\\ w(r, z) &= c\_1 \chi(r) + c\_2 \chi\_1(r) + \frac{\mathrm{d}p}{\mathrm{d}z} \chi\_2(r) \end{aligned} \tag{23}$$

Using the boundary conditions (13), the values of constants *c*1 and *c*2 can be written as

$$\begin{split} c\_{1} &= \frac{\chi\_{1}(r\_{1}) - \chi\_{1}(r\_{2})}{\chi(r\_{1})\ \chi\_{1}(r\_{2}) - \chi(r\_{2})\ \chi\_{1}(r\_{1})} - \frac{\mathrm{d}p}{\mathrm{d}z} \left[ \frac{\chi\_{2}(r\_{1})}{\chi(r\_{1})} \frac{\chi\_{1}(r\_{2}) - \chi\_{2}(r\_{2})}{\chi\_{1}(r\_{2}) - \chi(r\_{2})} \frac{\chi\_{1}(r\_{1})}{\chi\_{1}(r\_{1})} \right] \\ c\_{2} &= -\frac{1}{\chi\_{1}(r\_{2})} - \frac{\mathrm{d}p}{\mathrm{d}z} \frac{\chi\_{2}(r\_{2})}{\chi\_{1}(r\_{2})} - c\_{1} \frac{\chi(r\_{2})}{\chi\_{1}(r\_{2})} \end{split} \tag{24}$$

where these χ- *s* are defined in Appendix A.

The closed form solution (13) is represented in terms of integrals for any kind of general variable viscosity. These integrals can be computed for particular values of variable viscosity μ. Here, three cases of variable viscosity are taken into account, μ(*r*) = 1, *r*, and 1 *r* .