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. Author manuscript; available in PMC: 2018 Nov 1.
Published in final edited form as: Alcohol Clin Exp Res. 2017 Sep 22;41(11):1886–1895. doi: 10.1111/acer.13484

CRF1 receptor-dependent increases in irritability-like behavior during abstinence from chronic intermittent ethanol vapor exposure

Adam Kimbrough 1, Giordano de Guglielmo 1, Jenni Kononoff 1, Marsida Kallupi 1, Eric P Zorrilla 1, Olivier George 1,*
PMCID: PMC5659873  NIHMSID: NIHMS901235  PMID: 28833238

Abstract

Background

In humans, emotional and physical signs of withdrawal from ethanol are commonly seen. Many of these symptoms, including anxiety-like and depression-like behavior, have been characterized in animal models of ethanol dependence. One issue with several current behavioral tests that measure withdrawal in animal models is they are often not repeatable within subjects over time. Additionally, irritability, one of the most common symptoms of ethanol withdrawal in humans, has not been well characterized in animal models. The corticotropin-releasing factor (CRF)-CRF1 receptor system has been suggested to be critical for the emergence of anxiety-like behavior in ethanol dependence, but the role of this system in irritability-like behavior has not been characterized.

Methods

The present study compared the effects of chronic intermittent ethanol vapor exposure (CIE)-induced ethanol dependence on irritability-like behavior in rats using the bottle-brush test during acute withdrawal and protracted abstinence. Rats were trained to self-administer ethanol in operant chambers and then either left in a nondependent state or made dependent via CIE. Naive, nondependent, and dependent rats were tested for irritability-like behavior in the bottle-brush test 8 h and 2 weeks into abstinence from ethanol. Separate cohorts of dependent and nondependent rats were used to examine the effect of the specific CRF1 receptor antagonist R121919 on irritability-like behavior.

Results

Dependent rats exhibited escalated ethanol intake compared with their own pre-CIE baseline and nondependent rats. At both time-points of abstinence, ethanol-dependent rats exhibited increased aggressive-like responses compared with naive and nondependent rats. R121919 reduced irritability-like behavior in both dependent and nondependent rats, but dependent rats were more sensitive to R121919.

Conclusions

Irritability-like behavior is a clinically relevant and reliable measure of negative emotional states that is partially mediated by activation of the CRF-CRF1 system and remains elevated during protracted abstinence in ethanol-dependent rats.

Keywords: CIE, addiction, alcohol use disorder, aggressive, corticotropin-releasing factor

Introduction

Alcoholism is a chronic relapsing disorder that is associated with compulsive drinking, the loss of control over intake, and the emergence of a negative emotional state during abstinence (Koob et al., 2004, Koob and Volkow, 2010). In humans, the emotional and physical signs of ethanol withdrawal include anxiety, irritability, mood swings, insomnia, tremors, convulsions, higher blood pressure, accelerated pulse, accelerated breathing, accelerated heart rate, dehydration, and delirium tremens (Schuckit et al., 1995, Finn and Crabbe, 1997). The emotional symptoms of ethanol dependence, including anxiety-like behavior and depression-like behavior, can be modeled in animals during ethanol dependence (Pleil et al., 2015; Marcinkiewcz et al., 2015; Thorsell et al., 2007; Varlinskaya et al., 2017; Kallupi et al., 2014; Gilpin et al., 2012; Gilpin et al., 2015; Heilig et al., 2010; Kliethermes et al., 2004; Pandey et al., 2015; Pandey et al., 2003; Valdez et al., 2004; Valdez et al., 2002b; Ehlers et al., 2013; Vetreno et al., 2016; Rylkova et al., 2009; McClintick and Grant, 2016; Egli et al., 2012; Buck et al., 2014). An important challenge for preclinical researchers is the fact that these tests are usually not repeatable within subjects over time, which prevents conducting reliable longitudinal studies. Moreover, increased irritability is a key negative emotional symptom that has been largely neglected.

Irritability is one of the most common withdrawal symptoms in humans (Lubman et al., 1983) and has been anecdotally noted during withdrawal in animal models but shown to be difficult to quantify experimentally (Frye and Ellis, 1977; Riihioja et al., 1997; Riihioja et al., 1999; Woldbye et al., 2002; Becker, 2000). Thus, it is critical to characterize irritability-like behavior during withdrawal and protracted abstinence from ethanol in dependent rats.

A commonly used method to study ethanol dependence and withdrawal is the chronic intermittent ethanol (CIE) vapor exposure model (Kissler et al., 2014; Gilpin et al., 2008b; Gilpin et al., 2008c; Staples et al., 2015; Leao et al., 2015; de Guglielmo et al., 2016; Kimbrough et al., 2017). Rats that are made dependent with CIE exhibit clinically relevant blood ethanol levels (BELs; 150–250 mg%), an increase in ethanol drinking when tested during early and protracted withdrawal, and compulsive-like ethanol drinking (e.g., responding despite adverse consequences; Kimbrough et al., 2017; Rogers et al., 1979; Roberts et al., 1996; O’Dell et al., 2004; Schulteis et al., 1995; Vendruscolo et al., 2012; Seif et al., 2013; Leao et al., 2015). Ethanol dependence that is induced by ethanol vapor results in withdrawal symptoms during both acute withdrawal (somatic and motivational) and protracted abstinence (motivational; (Macey et al., 1996; Sommer et al., 2008; Schulteis et al., 1995; Williams et al., 2012; Kallupi et al., 2014; Valdez et al., 2002a; Zhao et al., 2007; Vendruscolo and Roberts, 2014; de Guglielmo et al., 2017), but the effects of abstinence from CIE on irritability-like behavior during withdrawal and after protracted abstinence has not yet been reported.

Converging lines of evidence suggest that recruitment of the brain corticotropin-releasing factor (CRF)-CRF1 receptor system during withdrawal from CIE is critical for the emergence of anxiety-like behavior (Valdez et al., 2002b; Sabino et al., 2006; Finn et al., 2007; Gilpin et al., 2008b; Richardson et al., 2008b; Richardson et al., 2008a), but the role of the CRF-CRF1 system in irritability-like behavior during withdrawal remains to be demonstrated. In the present study, we hypothesized that (i) abstinence from ethanol after the escalation of ethanol drinking using the CIE model would increase irritability-like behavior in the bottle-brush test 8 h into withdrawal and after 2 weeks of protracted abstinence (Riittinen et al., 1986; Lagerspetz and Portin, 1968) and (ii) the CRF1 receptor antagonist R121919 would reduce irritability-like behavior in ethanol-dependent and nondependent rats, but more so with ethanol-dependent rats, consistent with the CIE-induced recruitment of CRF-CRF1 systems.

Materials and Methods

Animals

Adult male Wistar rats (Charles River), weighing 250–300 g at the beginning of the experiments, were used. The rats were group housed, two per cage, in a temperature-controlled (22°C) vivarium on a 12 h/12 h light/dark cycle (lights on at 10:00 PM) with ad libitum access to food and water. All of the procedures were conducted in strict adherence to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by The Scripps Research Institute Institutional Animal Care and Use Committee.

Operant ethanol self-administration

Three cohorts of rats were used in this study. The first cohort of rats (n = 24) was used to test irritability-like behavior in nondependent and dependent rats during acute (8 h) and protracted (2 weeks) abstinence. A second cohort of rats (n = 16) was used to test the effect of R121919 on irritability-like behavior in dependent rats. A third cohort of rats (n = 12) was used to test the effect of R121919 on irritability-like behavior in dependent rats. All of the cohorts self-administered 10% (w/v) ethanol during daily sessions in standard operant conditioning chambers (Med Associates) until stable responding was maintained as previously described (Kimbrough et al., 2017; de Guglielmo et al., 2016; Leao et al., 2015). The rats were first subjected to an overnight session in the operant chambers with access to one lever (front lever) that delivered water on a fixed-ratio 1 (FR1) schedule (i.e., each operant response was reinforced with 0.1 ml of the solution). Food was available ad libitum during this training period. After 1 day off, the rats were subjected to a 3 h session (FR1) for 1 day, a 2 h session (FR1) the next day, and a 1 h session (FR1) the next day, with one lever delivering ethanol (front lever, 0.1 ml). All of the subsequent sessions lasted 30 min, and two levers were available (front lever: ethanol; back lever: water) until stable levels of intake were reached. Upon completion of this procedure, the animals were allowed to self-administer a 10% (w/v) ethanol solution and water on an FR1 schedule of reinforcement. The animals in the first cohort were then divided into two groups (n = 12 dependent, n = 12 nondependent). The dependent group underwent the CIE protocol, and the nondependent group was left undisturbed in the vivarium. All of the rats in the second cohort underwent the CIE protocol to become ethanol-dependent. Rats in the third cohort underwent the same operant ethanol self-administration training but were not exposed to CIE.

Chronic intermittent ethanol vapor

The rats in the dependent groups were made ethanol-dependent by the CIE vapor procedure as previously described (O’Dell et al., 2004; Gilpin et al., 2008a). The rats underwent repeated daily cycles of 14 h vapor ON (BELs during vapor exposure ranged from 150 to 250 mg%) and 10 h vapor OFF, during which behavioral testing occurred (i.e., 6–8 h after the vapor was turned OFF), when brain ethanol levels and BELs are negligible (Gilpin et al., 2009). After 4 weeks of vapor exposure, the rats resumed operant self-administration sessions during withdrawal for 4 weeks to test for the escalation of ethanol intake before undergoing irritability-like behavior testing.

Blood ethanol measurements

Tail blood was collected and used to determine BELs using an oxygen-rate ethanol analyzer (Analox Instruments, Stourbridge, UK; Gilpin et al., 2009).

Irritability-like behavior

To test irritability-like behavior during ethanol withdrawal and protracted abstinence, we used the bottle-brush test, based on the methods of Riittinen et al. (1986) and Lagerspetz and Portin (1968) and modified slightly for rats. Irritability-like behavior was tested following the escalation of ethanol intake in the cohorts of dependent and nondependent rats and in age-matched ethanol-naive rats (n = 10). Testing occurred after 8 h of withdrawal or 2 weeks of protracted abstinence from CIE (in the dependent group). Irritability-like behavior was examined by measuring aggressive and defensive responses during the bottle-brush test.

Irritability-like behavior testing was performed in the middle half of the dark cycle under dark conditions with a red light for the researchers. The sessions were conducted in a randomized order for each animal. Testing consisted of 10 trials per rat in plastic cages (10.5 in × 19 in × 8 in; Ancare, Bellmore, NY, USA) with fresh bedding. During each trial, the rat started at the back of the cage. A bottle-brush was rotated toward the animal’s whiskers (from the front of the cage) by a treatment-naive experimenter. The brush was rotated around the whiskers of the rat for approximately 1 s. The brush was then rotated back to the front of the cage where it was allowed to hang vertically for approximately 2 s, during which behavioral responses were recorded. A 10-s intertrial interval was used. Three observers who were blind to treatment scored the behaviors in real time.

For each rat, separate sums of aggressive and defensive responses across all trials were determined for each observer. Aggressive and defensive response scores for each rat were then calculated by averaging the observers’ sums. This was then used to calculate a group mean and SEM.

The following were scored as aggressive responses: smelling the target, biting the target (during the initial phase of rotating the brush forward and back to the starting position), boxing the target, following the target, exploring the target (mounting the target or using paws or mouth to manipulate the brush without biting or boxing), and delayed biting (during the 2 s that the brush hung at the starting position). The following were scored as defensive responses: escaping from the target, digging/burying, jumping, climbing, vocalization, and grooming. Grooming and digging/burying were additionally recorded during the 10-s intertrial intervals. The aggressive and defensive responses that were measured were chosen based on the methods from Riittinen et al. (1986) and Lagerspetz and Portin (1968). Additionally, we selected these responses because they are easily distinguishable behaviors that can be separated into approach (aggressive) and avoidance (defensive) behaviors. Although smelling and exploration are not aggressive behaviors per se, they consistently occurred during a bout of aggressive behaviors (biting, boxing, and following) and thus were included in the list of aggressive behaviors.

Effects of CRF receptor antagonism on irritability-like behavior

The second cohort of rats that was made dependent on ethanol via CIE exposure was tested for irritability-like behavior 8 h into withdrawal. The third cohort of rats that was left nondependent was also tested for irritability-like behavior at the time of day they would normally have an operant self-administration of alcohol session. Thirty minutes before testing, the rats were injected i.p. with either vehicle (n = 8 dependent, n = 6 nondependent) or the selective CRF1 receptor antagonist R121919 (n = 8 dependent, n = 6 nondependent; 10 mg/kg; synthesized by Dr. Kenner Rice at the Drug Design and Synthesis Section, Chemical Biology Research Branch, National Institute on Drug Abuse, National Institutes of Health, Bethesda, MD, USA). R121919 was administered as a suspension in a vehicle that consisted of 5% dimethylsulfoxide, 5% Emulphor, and 90% distilled water. Following the injection of vehicle or R121919, the rats underwent the bottle-brush test as described above. The nondependent cohort was tested using a within-subjects design so that all 12 rats received both treatments. On one day, half of the rats received vehicle and half received R121919. They were then tested again 2 days later but received the opposite treatment.

Statistical analysis

The results are expressed as mean ± SEM. For the cohorts of dependent and nondependent rats, the last 3 days of ethanol intake before vapor exposure were averaged to obtain the pre-vapor baseline intake. The last 3 days of ethanol intake before bottle-brush testing were averaged to obtain the post-vapor (escalated) intake. The data were analyzed using repeated-measures analysis of variance (ANOVA), with group (nondependent and dependent) as the between-subjects factor and day (baseline intake and escalated intake) as the within-subjects factor. The data were also analyzed by week using repeated-measures ANOVA, with group (nondependent and dependent) as the between-subjects factor and week as the within-subjects factor. Behavioral data for the second cohort were analyzed using a one-way ANOVA to compare the average of the last 3 days of ethanol intake before vapor exposure (pre-vapor baseline intake) and the average of the last 3 days of ethanol intake before bottle-brush testing (post-vapor escalated intake). For the bottle-brush test, each time point (8 h of withdrawal and 2 weeks of protracted abstinence) was examined using a one-way ANOVA. The ANOVAs were followed by the Student-Newman-Keuls (SNK) post hoc test when appropriate. To evaluate the effects of R121919 on behavior in the bottle-brush test, t-tests were performed between the two groups. For the nondependent cohort that was tested with R121919, within-subjects t-tests were performed between the two treatments. Differences were considered significant at p < 0.05. All of the data were analyzed using Statistica 13 software (StatSoft, Palo Alto, CA, USA).

Results

Blood ethanol levels

Blood ethanol levels were measured during CIE. In dependent rats, BELs were maintained between 150 and 250 mg/100 ml in dependent rats. No differences in BELs were found between dependent cohorts (data not shown).

Operant ethanol self-administration during CIE exposure

For operant ethanol self-administration, the mixed factorial ANOVA, with group (nondependent and dependent) as the between-subjects factor and day (baseline intake and escalated intake) as the within-subjects factor, revealed a significant day × group interaction (F1,22 = 7.5, p < 0.05) and significant effects of day (F1,22 = 5.4, p < 0.05) and group (F1,22 = 4.1, p = 0.05). The SNK post hoc test revealed that both dependent and nondependent rats reached a stable baseline of responding for ethanol during training (36.8 ± 5.3 lever presses in dependent rats vs. 36.1 ± 4.7 lever presses in nondependent rats), with no significant difference between groups. After CIE exposure, dependent rats significantly escalated the number of lever presses for ethanol compared with nondependent rats (61.9 ± 7.7 lever presses in dependent rats vs. 34.0 ± 6.3 lever presses in nondependent rats; p < 0.05; Fig 1A) and compared with their own baseline responding (61.9 ± 7.7 lever presses in dependent CIE rats vs. 36.8 ± 5.3 lever presses in dependent baseline rats; p < 0.05; Fig. 1A).

Figure 1.

Figure 1

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Escalation of operant ethanol self-administration after CIE. Rats were trained to operantly self-administer ethanol until they reached a stable baseline of intake. Dependent rats were made dependent via chronic intermittent ethanol vapor. Dependent (n = 12) and nondependent (n = 12) rats were then tested daily for the escalation of operant ethanol self-administration. (A) Average of the last 3 days of pre-vapor (baseline) and post-vapor (escalation) prior to testing irritability-like behavior. Dependent rats significantly escalated their ethanol intake relative to baseline (white bar) after CIE (post vapor; black bar). Dependent rats also exhibited significantly higher ethanol intake after CIE compared with nondependent rats (post-vapor; black bar). Nondependent rats did not significantly escalate their ethanol intake relative to baseline (white bar). (B) Average ethanol intake per operant session during each week of CIE exposure. Dependent rats (black circles) significantly escalated their ethanol intake relative to their own baseline at weeks 2–4. Dependent rats significantly escalated their ethanol intake compared with nondependent rats (white circles) at weeks 3 and 4. *p < 0.05, dependent post-vapor each week vs. dependent baseline intake; #p < 0.05, dependent post-vapor each week vs. nondependent post-vapor each week.

When we analyzed the data according to weeks of ethanol self administration, the repeated-measures ANOVA, with group (nondependent and dependent) as the between-subjects factor and week as the within-subjects factor, revealed a significant week × group interaction (F4,88 = 3.9, p < 0.05) and a significant effect of week (F4,88 = 4.8, p < 0.005) but no effect of group (F1,22 = 3.6, p = 0.07). The SNK post hoc test revealed that dependent rats presented a significant increase in ethanol intake relative to their own baseline at weeks 3 and 4 of CIE exposure (Fig. 1B).

Irritability-like behavior at 8 h withdrawal and 2 weeks abstinence from CIE

When tested 8 h into withdrawal from ethanol vapor, there was a significant effect of group on aggressive responses (F2,31 = 11.8, p < 0.0005). The SNK post hoc test showed that ethanol-dependent rats had a significantly higher number of aggressive responses (26.3 ± 2.8) compared with both nondependent rats (14.4 ± 2.6) and naive rats (9.6 ± 1.6; Fig. 2A). No significant difference in defensive responses was found between groups (Fig. 2B).

Figure 2.

Figure 2

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Irritability-like behavior in ethanol-dependent rats 8 h into withdrawal from ethanol vapor. Rats underwent the bottle-brush test to assess aggressive and defensive responses. (A) Aggressive responses 8 h into withdrawal. Dependent rats (n = 12; black bar) exhibited a significant increase in the number of aggressive responses over the course of 10 trials compared with nondependent rats (n = 12; gray bar) and naive rats (n = 10; white bar). (B) Defensive responses 8 h into withdrawal. No significant differences in the number of defensive responses were found between groups. (C) Individual aggressive behaviors. Dependent rats exhibited a significant increase in biting compared with naive rats and smelling, boxing, following, exploration, and delayed biting compared with naive and nondependent rats. *p < 0.05, dependent vs. naive; #p < 0.05, dependent vs. nondependent.

When analyzing individual aggressive behaviors during 8 h withdrawal, there was a significant effect of group on smelling (F2,31 = 11.9, p < 0.0005). The SNK post hoc test showed that ethanol-dependent rats had a significantly higher number of smelling responses (4.9 ± 0.5) compared with both nondependent rats (2.5 ± 0.7) and naive rats (1.4 ± 0.3; Fig. 2C).

There was a significant effect of group on biting (F2,31 = 3.4, p < 0.05). The SNK post hoc test showed that ethanol-dependent rats had a significantly higher number of biting responses (1.2 ± 0.5) compared with naive rats (0.2 ± 0.1; Fig. 2C).

There was a significant effect of group on boxing (F2,31 = 6.7, p < 0.005). The SNK post hoc test showed that ethanol-dependent rats had a significantly higher number of boxing responses (7.8 ± 0.6) compared with both nondependent rats (5.5 ± 0.6) and naive rats (4.7 ± 0.7; Fig. 2C).

There was a significant effect of group on following (F2,31 = 7.8, p < 0.005). The SNK post hoc test showed that ethanol-dependent rats had a significantly higher number of following responses (5.3 ± 0.7) compared with both nondependent rats (3.5 ± 0.7) and naive rats (1.7 ± 0.5; Fig. 2C).

There was a significant effect of group on exploration (F2,31 = 11.2, p < 0.0005). The SNK post hoc test showed that ethanol-dependent rats had a significantly higher number of exploration responses (4.6 ± 0.6) compared with both nondependent rats (2.2 ± 0.5) and naive rats (1.1 ± 0.4; Fig. 2C).

There was a significant effect of group on delayed biting (F2,31 = 6.4, p < 0.005). The SNK post hoc test showed that ethanol-dependent rats had a significantly higher number of delayed biting responses (2.5 ± 0.6) compared with both nondependent rats (0.6 ± 0.3) and naive rats (0.5 ± 0.4; Fig. 2C).

When tested 2 weeks into protracted abstinence from ethanol vapor, there was a significant effect of group on aggressive responses (F2,31 = 6.7, p < 0.005). The SNK post hoc test showed that ethanol-dependent rats had a significantly higher number of aggressive responses (27.2 ± 2.7) compared with both nondependent rats (18 ± 2.0) and naive rats (14.8 ± 2.7; Fig. 3A). No significant difference in defensive responses was found between groups (Fig. 3B).

Figure 3.

Figure 3

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Irritability-like behavior following 2 weeks of protracted abstinence from ethanol vapor in dependent rats. Rats underwent the bottle-brush test to assess aggressive and defensive responses. (A) Aggressive responses after 2 weeks of protracted abstinence. Dependent rats (n = 12; black bar) exhibited a significant increase in the number of aggressive responses over the course of 10 trials compared with nondependent rats (n = 12; gray bar) and naive rats (n = 10; white bar). (B) Defensive responses after 2 weeks of protracted abstinence. No significant differences in the number of defensive responses were found between groups. (C) Individual aggressive behaviors. Dependent rats exhibited a significant increase in smelling compared with naive rats and exploration and delayed biting compared with naive and nondependent rats. *p < 0.05, dependent vs. naive; #p < 0.05, dependent vs. nondependent.

When analyzing individual aggressive behaviors during 2 weeks protracted abstinence from ethanol, there was a significant effect of group on smelling (F2,31 = 7.9, p < 0.005). The SNK post hoc test showed that ethanol-dependent rats had a significantly higher number of smelling responses (7.5 ± 0.5) compared and naive rats (3.7 ± 0.9). Nondependent rats also had a significantly higher number of smelling responses (6.3 ± 0.6) compared with naive rats (Fig. 3C).

There was a significant effect of group on exploration (F2,31 = 11.9, p < 0.0005). The SNK post hoc test showed that ethanol-dependent rats had a significantly higher number of exploration responses (5.2 ± 0.8) compared with both nondependent rats (2.7 ± 0.5) and naive rats (1.2 ± 0.4; Fig. 3C).

There was also a significant effect of group on delayed biting (F2,31 = 14.8, p < 0.00005). The SNK post hoc test showed that ethanol-dependent rats had a significantly higher number of delayed biting responses (3.5 ± 0.5) compared with both nondependent rats (0.6 ± 0.3) and naive rats (1.2 ± 0.5; Fig. 3C).

Effects of CRF receptor antagonism on irritability-like behavior in dependent rats

Operant ethanol self-administration in the vehicle group (baseline: 14.8 ± 4.2 lever presses; escalated: 46.8 ± 8.3 lever presses) and R121919 group (baseline: 15.8 ± 4.5 lever presses; escalated: 47.5 ± 7.5 lever presses) did not differ at either baseline or during escalation. The data from both groups were combined, and further analyses were performed to compare baseline vs. escalated intake in all dependent rats using a one-way repeated-measures ANOVA, with day (baseline intake and escalated intake) as the within-subjects factor. There was a significant effect of day on responding for ethanol (F1,15 = 22.1, p < 0.0005). The SNK post hoc test revealed that ethanol-dependent rats presented significantly higher ethanol intake during escalation compared with their own baseline (15.3 ± 3.0 lever presses at baseline vs. 47.1 ± 5.4 lever presses during escalation; Fig. 4A).

Figure 4.

Figure 4

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Escalation of ethanol intake and irritability-like behavior in ethanol-dependent rats (n = 16) 8 h into withdrawal from ethanol vapor. Rats were exposed to CIE to escalate ethanol intake relative to baseline. Escalated rats were then treated with vehicle (n = 8) or the CRF1 receptor antagonist R121919 (n = 8; 10 mg/kg) 30 min prior to testing. The rats then underwent the bottle-brush test to assess aggressive and defensive responses. (A) Average of the last 3 days pre-vapor (baseline) and post-vapor (escalation) prior to testing irritability-like behavior. Dependent rats significantly escalated their ethanol intake relative to baseline (white bar) after CIE (post-vapor; black bar). (B) Aggressive responses. R121919-treated rats (black bar) exhibited a significant reduction of the number of aggressive responses over the course of 10 trials compared with vehicle-treated rats (white bar). (C) Defensive responses. No significant differences in the number of defensive responses were found between groups. (D) Individual aggressive behaviors. R121919 significantly reduced smelling, boxing, and following in ethanol-dependent rats. *p < 0.05, R121919 vs. vehicle.

When ethanol-dependent rats were tested for irritability-like behavior in the bottle-brush test during acute withdrawal (after an injection of vehicle or R121919), there was a significant effect of group (vehicle vs. 10 mg/kg R121919; t14 = 2.2, p < 0.05). R121919 significantly decreased aggressive responses (8.3 ± 2.7) compared with vehicle-treated rats (18.2 ± 3.5; Fig. 4B). No significant difference in defensive responses was found between groups (Fig. 4C).

When analyzing individual aggressive behaviors in ethanol-dependent rats after R121919 treatment, there was a significant effect of group on smelling (vehicle vs. 10 mg/kg R121919; t14 = 2.7, p < 0.05). R121919 significantly decreased smelling responses (1.0 ± 0.4) compared with vehicle-treated rats (3.9 ± 1.0; Fig. 4D).

There was a significant effect of group on boxing (vehicle vs. 10 mg/kg R121919; t14 = 2.2, p < 0.05). R121919 significantly decreased boxing responses (4.8 ± 0.8) compared with vehicle-treated rats (7.2 ± 0.7; Fig. 4D).

There was a significant effect of group on following (vehicle vs. 10 mg/kg R121919; t14 = 2.7, p < 0.05). R121919 significantly decreased following responses (1.1 ± 0.6) compared with vehicle-treated rats (3.9 ± 0.9; Fig. 4D).

Effects of CRF receptor antagonism on irritability-like behavior in nondependent rats

When nondependent rats were tested for irritability-like behavior in the bottle-brush test after the injection of vehicle or R121919, there was a significant effect of group (vehicle vs. 10 mg/kg R121919; t11 = 2.1, p < 0.05). R121919 significantly decreased aggressive responses (6.2 ± 1.5) compared with vehicle-treated rats (9.4 ± 2.4; Fig. 5A). No significant difference in defensive responses was found between groups (Fig. 5B).

Figure 5.

Figure 5

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Within-subjects (n = 12) irritability-like behavior in nondependent rats that were treated with vehicle or the CRF1 receptor antagonist R121919 (10 mg/kg) 30 min prior to testing. The rats then underwent the bottle-brush test to assess aggressive and defensive responses. (A) Aggressive responses. R121919-treated rats (black bar) exhibited a significant reduction of the number of aggressive responses over the course of 10 trials compared with vehicle-treated rats (white bar). (B) Defensive responses. No significant differences in the number of defensive responses were found between groups. (C) Individual aggressive behaviors. R121919 significantly reduced smelling in nondependent rats. *p < 0.05, R121919 vs. vehicle.

When analyzing individual aggressive behaviors in nondependent rats after R121919 treatment, there was a significant effect of group on smelling (vehicle vs. 10 mg/kg R121919; t11 = 2.2, p < 0.05). R121919 significantly decreased smelling responses (1.2 ± 0.4) compared with vehicle-treated rats (1.8 ± 0.4; Fig. 5C).

Discussion

The present study found that rats that were made dependent on ethanol via CIE exhibited an increase in irritability-like behavior in the bottle-brush test 8 h into withdrawal and 2 weeks into protracted abstinence. At 8 h into withdrawal, all of the aggressive behaviors increased in dependent rats. At 2 weeks of protracted abstinence, only smelling, exploration, and delayed biting remained elevated in dependent rats, whereas the other behaviors returned to levels that were similar to the other groups. The specific CRF1 receptor antagonist R121919 at a dose that is known to block central CRF1 receptors decreased irritability-like behavior in both nondependent and dependent rats. However, when examining changes in individual aggressive behaviors in response to R121919, dependent rats showed reductions of more forms of aggressive behavior (smelling, boxing, and following) than nondependent rats (smelling only; Fig 4D, 5C), and this was also reflected by the greater reduction of their composite aggression score vs. their respective vehicle controls (9.9 in dependent vs. 3.2 in nondependent).

Similar to other studies (Gilpin et al., 2008c; Gilpin et al., 2008b; Leao et al., 2015; Williams et al., 2012; O’Dell et al., 2004; Walker et al., 2008), rats that were made dependent on ethanol via CIE significantly escalated their ethanol intake. Interestingly, we found that ethanol-dependent rats exhibited an increase in irritability-like behavior in the bottle-brush test 8 h into withdrawal and 2 weeks into protracted abstinence from ethanol vapor compared with both nondependent and naive rats. R121919 reduced irritability-like behavior 8 h into withdrawal in dependent rats. This effect was observed only for aggressive responses and not for defensive responses and to a greater degree in dependent vs. nondependent rats, indicating a behaviorally specific effect.

Aggressive irritability-like behavior has not been previously tested to measure negative emotional states during ethanol withdrawal in the rat CIE model. Animal models of irritability have been difficult to characterize, and reports are often limited to anecdotes or have used less specific or quantitative assessments, such as reactivity to handling (Frye and Ellis, 1977; Riihioja et al., 1997; Riihioja et al., 1999; Woldbye et al., 2002; Becker, 2000). An increase in irritability-like behavior 8 h into withdrawal from ethanol is consistent with the time course of other withdrawal symptoms.

Somatic withdrawal symptoms have been well characterized during withdrawal from ethanol vapor. Somatic withdrawal symptoms, such as tremors, abnormal gait, and tail stiffness, are often observed during acute withdrawal from ethanol vapor. These changes can be seen as early as 2 h into withdrawal and last up to 72 h (Macey et al., 1996; de Guglielmo et al., 2016; de Guglielmo et al., 2015). The motivational symptoms of withdrawal have been found to last much longer (> 2 weeks) into protracted abstinence. Increases in anxiety-like behavior in the elevated plus maze and depressive-like behavior (e.g., immobility) in the forced swim test have been reported in ethanol-dependent rats and mice during acute withdrawal from ethanol vapor and after protracted abstinence (Kallupi et al., 2014; Valdez et al., 2002a; Zhao et al., 2007; Williams et al., 2012; Marcinkiewcz et al., 2015; Shibasaki et al., 2012; Walker et al., 2010). Impairments in brain reward function, reflected by elevations of intracranial self-stimulation (ICSS) thresholds, have also been observed during acute withdrawal from ethanol vapor in dependent rats (Schulteis et al., 1995). Perhaps the closest measures of irritability that have been reported during withdrawal include 22 kHz ultrasonic vocalizations and measures of reactivity to touch or handling (including vocalizations; Frye and Ellis, 1977; Riihioja et al., 1997; Riihioja et al., 1999; Woldbye et al., 2002; Becker, 2000; Akunne and Soliman, 1988). Ultrasonic vocalizations are thought to represent distress in animals (Becker, 2000) and greatly increase during withdrawal from ethanol vapor and an ethanol liquid diet (Williams et al., 2012; Knapp et al., 1998; Buck et al., 2014). Neither of these measures, however, recapitulates the specific increase in aggressive responses that were observed in the present study, which might more specifically model the quick-to-anger, grouchiness, and hostility that is seen during ethanol withdrawal in humans.

In the present study, we observed an increase in irritability-like behavior after 2 weeks of protracted abstinence, which is consistent with motivational measures of ethanol withdrawal that have been shown to persist into protracted abstinence (2–8 weeks) from ethanol vapor (Roberts et al., 2000). Rats that underwent 4–6 weeks of abstinence from ethanol exhibited an increase in anxiety-like behavior and a decrease in the time spent on the open arms in the elevated plus maze (Valdez et al., 2002a; Zhao et al., 2007). However, an important advantage of using the bottle-brush test over the elevated plus maze test is that the former can be repeated in the same individuals. Our results revealed a consistent increase in irritability-like behavior during both acute and protracted abstinence in the same subjects.

Altogether, the present results suggest that irritability-like behavior may be a clinically relevant measure of a negative emotional state that may model the anxiety, irritability, and mood disturbances that can persist long after detoxification in ethanol-dependent humans (Brady et al., 2002; Martinotti et al., 2007; Miyata et al., 2008; Haney, 2005). Irritable aggression may be a prognostically relevant motivational withdrawal symptom that predicts an increase in craving (Chiang et al., 2002) and more relapse episodes in ethanol-dependent males during abstinence (Baars et al., 2013).

CRF-CRF1 receptor signaling has been shown to be critically involved in ethanol withdrawal-related behaviors (Zorrilla et al., 2013). CRF blockade with various receptor antagonists has been shown to reduce ethanol intake in dependent rats (Heilig and Koob, 2007; Funk et al., 2007; Valdez et al., 2002a; Funk et al., 2006) and anxiety-like behavior in dependent rats (Heilig and Koob, 2007; Baldwin et al., 1991, Rassnick et al., 1993, Valdez et al., 2003). Irritability-like behavior also appears to be inhibited by CRF1 receptor blockade, suggesting that irritability- and anxiety-like behavior may share common neurocircuitry. Combining the bottle-brush test with other reliable tests may help identify unique vs. overlapping neural substrates and the pathophysiological relevance of different motivational symptoms of withdrawal.

In summary, the present study found increases in irritable aggression-like behavior in ethanol-dependent rats during acute withdrawal and protracted abstinence. CRF1 antagonism reduced irritability-like behaviors in ethanol-dependent and nondependent rats, but dependent rats were more sensitive to the blockade of CRF1 receptors. The bottle-brush test may represent a clinically relevant, reliable, and reproducible method to investigate the neurobiological mechanisms that underlie the emergence of negative emotional states during abstinence and may facilitate drug discovery by providing a high-throughput within-subjects paradigm for the preclinical screening of novel medications for the treatment of symptoms of alcohol use disorders.

Acknowledgments

This work was supported by National Institutes of Health grants AA006420, AA020608, AA022977, and AA007456 and the Pearson Center for Alcoholism and Addiction Research. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The authors acknowledge the expert technical assistance of Maury Cole and editorial assistance of Michael Arends.

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