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. Author manuscript; available in PMC: 2020 Apr 1.
Published in final edited form as: Neuropharmacology. 2019 Jan 16;148:210–219. doi: 10.1016/j.neuropharm.2019.01.017

Effect of chronic alcohol vapor exposure on reinstatement of alcohol seeking induced by U50,488.

Douglas Funk 1, Kathleen Coen 1, Sahar Tamadon 1, AD Lê 1,2,3
PMCID: PMC6424618  NIHMSID: NIHMS1519116  PMID: 30659838

Abstract

Alcohol dependence and stress are associated with relapse to alcohol during abstinence, but the underlying mechanisms are poorly understood. Kappa opioid receptors (KOR) are involved in alcohol reward and in the effects of stress. Previously, in non-dependent rats, we showed that KOR in the bed nucleus of the stria terminalis (BNST) mediate reinstatement of alcohol seeking induced by the selective KOR agonist U50,488. Here, we determine the effects of chronic, intermittent exposure to alcohol vapor on U50,488-induced reinstatement of alcohol seeking. We also study brain mechanisms involved using the neuronal activity marker Fos and phosphorylated p38 MAPK (p-p38), an intracellular messenger implicated in the effects of KOR stimulation. We trained male Long-Evans rats to self-administer alcohol (12% w/v) and exposed them to alcohol vapor (14 h vapor/10 h air) daily for 24 d or to the control condition, extinguished alcohol-reinforced responding and determined the dose response for U50,488-induced reinstatement. We then determined the effects of vapor exposure on U50,488-induced Fos and p-p38 expression. Vapor-exposed rats were more sensitive to U50,488-induced reinstatement. U50,488 increased Fos expression in brain areas involved in stress-induced relapse, and Fos activation in the ventral BNST was greater in vapor exposed rats. Vapor exposed rats had increased basal p-p38 expression in the dorsal BNST, LC and NTS. Our findings suggest that changes in the neuronal responses to KOR stimulation in the ventral BNST may be involved in the increased sensitivity to U50,488 accompanying dependence.

Keywords: Kappa opioid, Stress, Alcohol dependence, Fos, p38 MAPK

1. Introduction

Stressful life events are associated with increased alcohol intake and in abstinent individuals and higher rates of relapse (Sinha and Li, 2007). The kappa opioid receptor (KOR) is involved in stress-induced relapse to alcohol and other drugs. KOR and its endogenous ligand, dynorphin are distributed in brain areas involved in stress responses and stress-induced relapse including the bed nucleus of the stria terminalis (BNST), amygdala, hypothalamus and raphe nuclei (Morley et al., 1982; Nabeshima et al., 1992; Crowley et al., 2016). Selective KOR agonists such as U50,488 increase stress-related symptoms including dysphoria, anxiety-like behavior and adrenocortical activation while KOR blockade has anxiolytic-or antidepressant-like effects (Mague et al., 2003; Land et al., 2008; Van’t Veer et al., 2012; Le et al., 2018). Data from studies using the reinstatement procedure parallel this, in showing that KOR agonists induce reinstatement, while KOR antagonists block stress-induced reinstatement of drug and alcohol seeking (Beardsley et al., 2010; Jackson et al., 2012; Funk et al., 2014; Grella et al., 2014; Le et al., 2018). We recently demonstrated that BNST KORs are involved in U50,488-induced reinstatement of alcohol seeking in non-dependent rats (Le et al., 2018). U50,488-induced reinstatement was correlated with BNST Fos expression, antagonism of BNST KOR blocked U50,488-induced reinstatement, and intra-BNST U50,488 injections reinstated alcohol seeking. These findings are consistent with the presence of dynorphin and KOR in the BNST and the important role demonstrated for the BNST in drug seeking (Erb et al., 2001; Vranjkovic et al., 2014).

Chronic alcohol exposure is associated with changes in dynorphin and KOR levels and KOR sensitivity (D’Addario et al., 2013; Chavkin and Koob, 2016; Karkhanis et al., 2016; Rose et al., 2016). Systemic, intra-nucleus accumbens, BNST or amygdala blockade of KOR reduces increased alcohol intake observed early in withdrawal from chronic, intermittent exposure to alcohol vapor, without affecting intake in non-dependent rats (Walker and Koob, 2008; Nealey et al., 2011; Walker et al., 2011; Kissler and Walker, 2016; Erikson et al., 2018). The persistent increase in anxiety-like behavior produced by chronic alcohol exposure in rats (Marcinkiewcz et al., 2015) is also reduced by KOR blockade (Gillett et al., 2013). Together, these data suggest that changes in KOR may underlie the effects of alcohol dependence on measures of anxiety and on increased motivation to seek alcohol during early and protracted withdrawal. Stress- and cue-induced relapse to alcohol is also enhanced during protracted withdrawal after intermittent exposure to alcohol vapor or intragastric injections of alcohol (Liu and Weiss, 2002; de Guglielmo et al., 2015; Uhrig et al., 2017). The function of KOR in enhanced stress-induced alcohol seeking, during early or protracted withdrawal after alcohol dependence induction is not known.

KOR activation affects adenylate cyclase and ion channel conductance typical of other Gi/o-coupled receptors, while sustained KOR activation recruits the p38 mitogen-activated protein kinase pathway (p38) (Lemos et al., 2011). p38 is involved in the behavioral effects of KOR activation, including aversive effects of stress, stress-induced cocaine seeking and effects on extinction processes (Bruchas et al., 2007; Bruchas et al., 2008; Land et al., 2009; Schindler et al., 2012; Mannangatti et al., 2015; Heinsbroek et al., 2018). However, with the exception of work showing that p38 inhibitors reduce alcohol self-administration (Agoglia et al., 2015), the role of p38 in alcohol seeking is not known.

In the present study, we will examine the effects of chronic intermittent exposure to alcohol vapor on reinstatement induced by U50,488 during protracted withdrawal, and the underlying brain mechanisms. We hypothesize that chronic intermittent exposure to alcohol vapor will potentiate U50,488-induced reinstatement of alcohol seeking during protracted withdrawal. Given previous reports that KOR are involved in dependence-induced increases in alcohol taking, and our recent findings of the association of U50,488-induced reinstatement of alcohol seeking with BNST activatio2n, we predict that enhanced U50,488-induce reinstatement produced by alcohol vapor exposure will be accompanied by increased expression of the brain activity marker, Fos and the KOR activity marker p-p38 MAPK in the BNST.

2. Materials and methods

2.1. Subjects

We used 34 male Long-Evans rats from Charles River (Kingston, NY) weighing 225–250 g at the start of the experiments. We housed the rats individually under a 12:12 reversed light-dark cycle (light on from 7:00 p.m. to 7:00 a.m.; 21±1°C). Rats were fed 5 fo od pellets (25 g) 2 h after experimental sessions. All experimental testing was conducted Monday to Friday. Experimental procedures followed the NIH “Principles of laboratory animal care” (Eighth edition, 2011) and were approved by the local animal care committee. We excluded 5 rats, 4 due to low alcohol intake (<0.4 g/kg) and 1 due to illness.

2.2. Drugs

We prepared alcohol solution by diluting 95% alcohol in tap water. We obtained U50,488 hydrochloride from Vibrant Pharma (Brantford, ON) and dissolved it in distilled water. We based the U50,488 doses (free-base) and pre-treatment times on published studies (Funk et al., 2014; Grella et al., 2014; Kissler and Walker, 2016; Le et al., 2018).

2.3. Apparatus

2.3.1. Operant chambers

We used alcohol self-administration chambers (30×21×21 cm) equipped with two levers. Responding on one lever (active lever) activated an infusion pump (Razel Scientific), while responding on the other (inactive lever) was recorded, but did not activate the pump. Pump activation delivered 0.19 ml of 12% (w/v) alcohol into a drinking receptacle between the levers and initiated a 5-s timeout, during which the houselight was turned off and a compound tone + light cue was turned on. Houselight illumination signaled the start of the session.

2.3.2. Alcohol vapor chambers

Rats in the alcohol vapor group were placed sealed chambers (La Jolla Alcohol Research) and exposed to alcohol vapor introduced into the chambers by air pumped continuously over heated 95% alcohol (Russo et al., 2018). These rats received daily exposures (14 h alcohol vapor; 10 h air) in the chambers for 4 d/week (Tuesday-Friday) and were placed in their home cages on the weekend, for 6 weeks. Rats in the control group remained in their home cages.

2.4. Alcohol self-administration training and extinction of alcohol-reinforced responding

We trained rats to drink alcohol using a limited access procedure with access to alcohol for 30 min/day in ascending concentrations (3% w/v for 4 d, 6% for 5 d, 12% for 10 d) in drinking tubes. We then trained the rats to self-administer alcohol (12% w/v) in 1-h daily sessions on a fixed ratio-1 (FR-1) 5-s timeout reinforcement schedule for 4 d, FR-2 for 5 d and then FR-3 for 14 d, until they demonstrated 3 days of stable alcohol self-administration (variability<20%). Rats received limited access or operant sessions Monday-Friday and remained in their home cages on weekends.

The procedures we used during the extinction sessions were the same as during self-administration, except no alcohol was delivered. We began reinstatement testing after 10 extinction sessions, when rats reached the extinction criterion (<12 active presses/1 h). During the last 3 sessions prior to U50,488 dose response testing, we gave the rats vehicle injections (distilled water, s.c.) to habituate them to the injection procedure.

2.5. Reinstatement testing

We injected each rat with vehicle or one of the doses of U50,488 (1.25, 2.5, 5 mg/kg s.c.) and 30 min later placed them in the chambers for a 60-min reinstatement test. We gave the doses in counterbalanced order with at least two days of drug-free extinction sessions between tests when rats were at the extinction criteria.

2.6. Brain collection and Fos or p-p38 immunohistochemistry

At the end of the 60 min reinstatement test (90 min after vehicle or U50,488 injections), we deeply anesthetized the rats with sodium pentobarbital (100 mg/kg, i.p.), and perfused them transcardially with 100 ml of phosphate-buffered saline (pH 7.4) followed by 300 ml of chilled 4% paraformaldehyde in phosphate buffer. Fos protein expression is maximal (Kovacs et al., 2003), and significant increases in p-p38 in response to U50,488 have been demonstrated (Robles et al., 2014) at this sacrifice time. We removed their brains, stored them in buffered 4% paraformaldehyde for 1 h, and transferred them to 30% sucrose in phosphate buffer for 48 h, after which we froze them in dry ice and stored them at −70°C. We cut the brains at 40 microns on a cryostat and collected the sections into 0.1% Tween 20/PBS, and stored them in cryoprotectant (30% glycerol/ 30% ethylene glycol in phosphate buffer) at −20°C. We collected brain sections from dorsal and ventral mPFC, OFC, NAc core and shell, dorsal and ventral BNST, CeA, BLA, LH, DRN, MRN, VTA, LC and NTS, based on a brain atlas (Paxinos and Watson, 2005).

We made all immunohistochemical solutions in TBS-T (Tris-buffered saline with 0.2% Triton X-100). We rinsed the sections 3 times, heated them to 80°C in 10 mM Na citrate (pH 9) antibody retrieval solution for 30 min (Jiao et al., 1999), rinsed them 3 times, incubated them in 0.12% H2O2 in for 30 min to reduce background, rinsed 3 times, and blocked them in 3% normal goat serum (NGS) for 1 h. We then incubated the sections with rabbit anti-Fos (1:5,000, #2250, Lot # 9, Ref. 08/2017; Cell Signaling) or rabbit anti-p-p38 (1:500, #9211, Lot 24, Ref. 03/2018 Cell Signaling) with 3% NGS at room temperature overnight on a shaker, rinsed them 3 times and incubated them with biotinylated anti-rabbit secondary antibody for 1 hour (1:200, Vector Labs, BA1000) with 1 % normal goat serum. We then treated the sections with avidin/biotin horseradish peroxidase (Vectastain Elite, Lot # ZD0224) for 1 h, 3 rinses, and then 0.5% DAB solution (with 0.035% H2O2 and 0.032% NiCl2, pH=7.8), until the desired staining intensity was achieved (~ 4 min). We terminated the reaction with 3 rinses, mounted the sections on gelatin coated slides, dried them overnight, and then dehydrated and coverslipped them. Guided by a brain atlas (Paxinos and Watson, 2005), we digitized images of the brain sites with a light microscope and counted in a blind manner the numbers of Fos- or p-p38 positive neurons in each image, using particle analysis software (ImageJ, NIH). We calculated the mean counts per brain area per mm2 across images within each rat, and used these mean counts in the statistical analyses.

2.7. Statistical analyses

In Exp. 1a, we analyzed the effect of U50,488 on active and inactive lever-presses during the reinstatement test with mixed ANOVA using the within-subjects factors of U50,488 dose (0, 1.25, 2.5, 5 mg/kg) and between subjects factor of Vapor condition (air, alcohol vapor). In Exp. 1b, we analyzed the effects of U50,488 on active and inactive lever pressing during the reinstatement tests using 2 way ANOVA with the between-subjects factors of Vapor condition and U50,488 dose (0, 2.5 mg/kg). The lever press data from Exp. 1b were not normally distributed, so we conducted the analysis on square root transformed active and inactive lever presses. We analyzed the Fos and p-p38 expression data in each brain region using between subjects ANOVA with the factors of Vapor condition and U50,488 dose. We followed significant effects (p<0.05) from the ANOVAs using Bonferroni post-hoc tests (control vs. treatment).

2.8. Experimental procedures

2.8.1. Exp. 1a: Effects of alcohol vapor exposure on U50,488-induced reinstatement of alcohol seeking

The goal of Exp. 1a was to determine the effects of alcohol vapor exposure on U50,488-induced reinstatement of alcohol seeking during protracted withdrawal. After training for alcohol self-administration, we exposed rats to alcohol vapor (14h alcohol vapor/10 h clean air) for 4 d/week (Tuesday-Friday) for 6 weeks or to the control condition (home cage). The vapor-exposed rats were returned to their home cages Friday afternoon and received a 1 h alcohol self-administration session the following Monday. The rats in the control condition were not exposed to alcohol vapor but received the alcohol self-administration sessions together with the vapor-exposed rats. We assessed blood alcohol levels (BALs) in vapor-exposed rats in serum samples collected 7 h into 4th day of the 6th week of vapor and their alcohol content was measured enzymatically (Analox Instruments). The alcohol vapor exposed rats had a mean BAL of 228.26 ± 13.76 mg/dL.

Three days after the final alcohol vapor exposure we gave the rats 4 daily 1 h alcohol self-administration sessions, and then 10 daily 1 h extinction sessions until they reached the extinction criterion. We determined the effects of the prior exposure to alcohol vapor on U50,488-induced reinstatement by injecting rats with vehicle or one of the doses of U50,488 and 30 min later placing them in the chambers for a 1 h reinstatement test. We gave the U50,488 doses in counterbalanced order with at least two days between tests. The dose response for U50,488-induced reinstatement was conducted 26–35 d after the last exposure to alcohol vapor.

2.8.2. Exp. 1b: Effects alcohol vapor exposure on Fos and p-p38 expression associated with U50,488-induced reinstatement of alcohol seeking

The goal of Exp. 1b was to determine the effects of alcohol vapor exposure on U50,488-induced regional Fos and p-p38 expression in brain areas involved in stress-induced reinstatement of alcohol seeking during protracted withdrawal. After we completed the U50,488 dose-response testing in Exp. 1a, we tested the effects of U50,488 (2.5 mg/kg, most effective dose in vapor-exposed rats) on reinstatement of alcohol seeking in the control and vapor-exposed rats, and on Fos and p-p38 expression in individual brain regions. Rats were counterbalanced to vehicle and U50,488 groups based on their response to 2.5 mg/kg U50,488 in Exp. 1a. We injected the rats with vehicle or U50,488, and 30 min later placed them in the self-administration chambers for a 1 h reinstatement test. At the end of the test, we anaesthetized the rats with sodium pentobarbital, perfused them, and processed their brains for Fos or p-p38 immunohistochemistry. The rats were tested and perfused in Exp. 1b 40 d after the final alcohol vapor exposure.

We assessed the dose-response curve of U50,488-induced reinstatement 26–35 d and on U50,488-induced reinstatement, Fos and p-p38 expression 40 d after termination of vapor exposure. Increased reinstatement of alcohol seeking is observed 3 weeks after termination of vapor exposure (Liu and Weiss, 2002; de Guglielmo et al., 2015), while increased anxiety persists for at least 6 weeks (Knapp et al., 1998; Gillett et al., 2013; Marcinkiewcz et al., 2015). In addition, potential confounding effects of physical withdrawal symptoms are absent during these times in protracted withdrawal.

3. Results

3.1. Experiment 1a

3.1.1. Self-administration and extinction

In Exp. 1a we determined the effects of alcohol vapor exposure on the dose-response of U50,488 on reinstatement of alcohol seeking (see Fig. 1A for experimental timeline). As in our previous studies with similar training conditions (Le et al., 2018), rats demonstrated reliable alcohol self-administration (~0.7 g/kg at the end of training), and comparable extinction of the alcohol-reinforced responding (Figure 1B–C). Alcohol self-administration beginning 3 d after termination of vapor exposure (Fig, 1C) and extinction responding (1D) did not differ between alcohol vapor exposed and control rats (ps >0.05).

Figure 1. Alcohol vapor exposure is associated with increased sensitivity to the effects of U50,488 on reinstatement of alcohol seeking in Exp. 1a.

Figure 1.

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(A) Timeline of the experiment. (B) Self-administration training prior to vapor exposure: Mean±SEM number of active and inactive lever presses (left) and alcohol rewards and g/kg intake (right) during the 23 training sessions of all rats prior to vapor exposure. (C) Self-administration after vapor exposure: Mean±SEM number of active and inactive lever presses (left) and alcohol rewards and g/kg intake (right) during the 4 training sessions conducted at FR3 beginning on the third day after the final vapor exposure or the control condition. (D) Extinction: Mean±SEM number of non-reinforced presses on the previously active lever and on the inactive lever during the 10 extinction days in the control and vapor-exposed rats. (E) Reinstatement: Mean±SEM number of non-reinforced presses on the previously active lever (left) and on the inactive lever (right) during the 1-h reinstatement tests in control and vapor-exposed rats. * Different from vehicle (0 dose), + different from control rats, p<0.05, n=29. FR, fixed ratio, s.c., subcutaneous.

3.1.2. Reinstatement

Fig 1E shows that systemic injections of U50,488 increased active lever pressing during the reinstatement tests (F3,81=8.19, p=0.00) that was significantly enhanced in rats exposed to alcohol vapor, which was reflected in a significant U50,488 dose × Vapor condition interaction (F3,81=2.95, p=0.037).

In control rats, post hoc tests showed that the 5 mg/kg dose of U50,488 elicited a trend towards higher active lever pressing compared to vehicle-treated rats (p=0.054). In vapor-exposed rats, the 1.25 and 2.5 mg/kg doses of U50,488 induced significantly higher active lever pressing than vehicle (ps=0.00), while the 5 mg/kg dose caused a trend towards higher pressing (p=0.051) (Figure 1D, left). The analysis of the inactive lever data showed a significant effect of U50,488 dose (F3,81=2.78, p=0.046) as inactive lever responding tended to be higher when the rats were administered U50,488 (Figure 1D, right). The data in Exp. 1a show that rats previously exposed to alcohol vapor show a potentiated response to U50,488 compared to controls.

3.2. Experiment 1b

Exp. 1b was done to determine if changes in the expression of the neuronal activity marker Fos and p-p38, a marker for activation of the p38 signaling pathway elicited by KOR activation accompany the enhanced U50,488-induced reinstatement seen in rats previously exposed to alcohol vapor (see Fig. 1A for experimental timeline). The dose of U50,488 used was 2.5 mg/kg, at which we showed a significant potentiation of reinstatement of alcohol seeking in alcohol vapor-exposed rats compared to controls in Exp. 1a.

3.2.1. Reinstatement

We observed reinstatement of alcohol seeking after systemic injections of 2.5 mg/kg of U50,488, as indicated by a significant effect of U50,488 dose (F1,25=5.74, p=0.024)(Figure 2A) in the analysis of active lever press data. Post hoc tests showed that vapor-exposed rats treated with U50,488 had higher active lever pressing than those treated with vehicle (p=0.028). Inactive lever pressing was not affected by U50,488 or prior exposure to alcohol vapor (ps>0.05).

Figure 2. U50 488-induced reinstatement of alcohol seeking and effects on regional Fos expression in control and alcohol vapor-exposed rats in Exp. 1b.

Figure 2.

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(A) Reinstatement: Mean±SEM number of non-reinforced presses on the previously active lever (left) and on the inactive lever (right) during the 1-h reinstatement tests. (B) Fos expression: Mean±SEM number of Fos-labeled neurons in the dorsal and ventral mPFC, OFC, NAc core and shell, dorsal and ventral BNST CeA, BLA, LH, DRN, MRN, VTA, LC and NTS of control and vapor-exposed rats injected with vehicle or U50,488 (2.5 mg/kg, s.c.). * Different from vehicle (0 dose), # significant main effect of U50,488 dose ps<0.05, n=7–8/group.

3.2.2. Fos expression

After the reinstatement tests, injections of U50,488 (2.5 mg/kg) induced significant Fos expression, as reflected by a main effect of U50,488 dose, in all of the brain regions examined, the dorsal mPFC (F1,28=11.07, p=0.003), ventral mPFC (F1,28=7.25, p=0.012), OFC (F1,28=10.97, p=0.003), NAc core (F1,28=37.86, p=0.001), NAc shell (F1,28=30.1, p=0.001), dorsal BNST (F1,28=19.37, p=0.001), ventral BNST (F1,28=64.86, p=0.001), CeA (F1,28=27.01, p=0.001), BLA (F1,28=4.65, p=0.041), LH (F1,28=53.86, p=0.001), VTA (F1,27=8.49, p=0.008), DRN (F1,28=4.80, p=0.038), MRN (F1,28=7.67, p=0.010), LC (F1,27=47.12, p=0.001) and NTS (F1,28=11.88, p=0.002) (Fig. 2B).

The analysis of Fos expression in the ventral BNST showed a significant U50,488 dose × Vapor condition interaction (F1,28=5.93, p=0.02), but not in any other brain area. Post-hoc tests revealed that U50,488 increased Fos expression in the ventral BNST of both control (p=0.001) and vapor-exposed rats (p=0.001), and that ventral BNST Fos expression was higher in vapor exposed rats administered U50,488 compared to controls administered U50,488 (p=0.001). There was also a significant main effect of Vapor condition on Fos in the ventral BNST (F1,28=6.73, p=0.016), but not in any other brain area. Figure 4A shows representative brain sections illustrating Fos expression in the dorsal and ventral BNST in control and vapor exposed rats treated with vehicle or 2.5 mg/kg U50,488.

Figure 4. Photomicrographs of Fos and p-p38 expression in Exp.1b.

Figure 4.

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(A) Alcohol vapor exposure is associated with increased U50,488-induced Fos expression in the ventral, but not dorsal BNST. Representative photomicrographs of Fos expression in the dorsal BNST (top) and ventral BNST (bottom) from control and alcohol vapor exposed rats treated with vehicle or U50,488 (2.5 mg/kg, s.c.)(scale bar=300 μm). (B) Alcohol vapor exposure is associated with increased p-p38 expression in the dorsal BNST, LC and NTS. Representative photomicrographs of p-p38 expression in the dorsal BNST (top) (scale bar 300 μm), LC (middle) and NTS (bottom) from control and alcohol vapor-exposed rats treated with vehicle or U50,488 (scale bars=100 μm).

3.2.3. p-p38 expression

After the reinstatement tests, rats injected with U50,488 (2.5 mg/kg) had slightly but significantly higher p-p38 expression in the NAc core (U50,488 dose: F1,25=5.78, p=0.024) (Figure 3) compared to vehicle-treated rats, in both control and vapor-treated rats. There was a significant effect of Vapor condition on p-p38 expression in the dorsal BNST (F1,24=7.89, p=0.01), LC (F1,24=23.55, p=0.001) and NTS (F1,25=8.38, p=0.008), because rats previously exposed to alcohol vapor had higher levels of p-p38 in these regions compared to controls. There was no significant U50,488 dose × Vapor condition interaction in p-p38 expression in any brain area measured. Figure 4B shows representative brain sections illustrating p-p38 expression in the dorsal BNST, LC and NTS in control and vapor-exposed rats treated with vehicle or U50,488. Figure 5 shows the brain areas in which Fos and p-p38 immunoreactivity was sampled, drawn on plates from a brain atlas (Paxinos and Watson, 2005).

Figure 3. Regional p-p38 expression in control and alcohol vapor-exposed rats administered vehicle or U50,488 in Exp. 1b.

Figure 3.

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Mean±SEM number of p-p38-labeled neurons in the dorsal and ventral mPFC, OFC, NAc core and shell, dorsal and ventral BNST CeA, BLA, LH, VTA, DRN, MRN, VTA, LC and NTS of control and alcohol vapor-exposed rats injected with vehicle or U50,488 (2.5 mg/kg, s.c.). * Main effect of U50,488 dose. # Main effect of Vapor condition ps<0.05, n=6–8/group.

Figure 5. Sampling regions of the different brain areas analyzed in Exp. 1b.

Figure 5.

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(A-H) Distance from bregma in mm are indicated on each section (Paxinos and Watson, 2005). The rectangular microscope fields were aimed in the centre of the regions depicted. The size used to capture Fos images in all regions was 0.46 × 0.35 mm (100X). The same magnification and field size was used to capture p-p38 images in all regions except the LC and NTS. For p-p38 in the LC and NTS a field size of 0.23 × 0.17 mm (200X) was used.

The data from Exp. 1b shows that U50,488-induced reinstatement is associated with increased Fos expression in multiple brain areas. Prior exposure to alcohol vapor led to greater U50,488-induced Fos expression in the ventral BNST. The results with p-p38 expression suggest that a history of alcohol vapor exposure results in increases in p38 phosphorylation in dorsal BNST, LC and NTS.

4. Discussion

The 4 main findings of our study are: 1) prior chronic, intermittent exposure to alcohol vapor potentiated the effects of U50,488 on reinstatement, 2) this effect was associated with increased U50,488-induced Fos expression in the ventral BNST, 3) alcohol vapor exposure increased p-p38 expression in the dorsal BNST, LC and NTS and 4) these effects of alcohol vapor exposure on U50,488-induced reinstatement, U50,488 Fos expression and p-p38 expression were observed during protracted withdrawal, 25–40 days after vapor exposure. These results suggest that the enhanced U50,488-induced reinstatement in rats with a history of chronic alcohol vapor exposure is associated with enhanced neuronal responses in the ventral BNST. This is consistent with our previous findings showing that KOR in the BNST mediate U50,488-induced reinstatement of alcohol seeking, and with the literature on the function of the BNST in responses to stress and stress-induced reinstatement of drug seeking. Our present results also suggest that increased p38 MAPK activity in the dorsal BNST, LC and NTS may have a role in the effects of chronic exposure to alcohol vapor.

4.1. Alcohol vapor exposure and U50,488-induced reinstatement of alcohol seeking

We did not observe a significant effect of U50,488 in controls at the 1.25 or 2.5 mg/kg dose, or in either controls or vapor-exposed rats at the 5 mg/kg dose, although there was a trend in both cases (p=0.054 and 0.051 respectively). It is difficult to explain this pattern because in our previous studies in non-dependent rats, we reported a trend at 2.5 mg/kg and significant reinstatement at 5 mg/kg U50,488 (Funk et al., 2014), and in our most recent study, significant reinstatement at both 2.5 and 5 mg/kg (Le et al., 2018). In our study on nicotine-seeking, both the 2.5 and 5 mg/kg doses of U50,488 induced reinstatement, with a larger effect occurring at 2.5 mg (Grella et al., 2014). Other studies report an inverted U-shaped dose function for U50,488-induced changes in anxiety-like behavior and impulsivity (Privette and Terrian, 1995; Walker and Kissler, 2013), suggesting that at higher doses, KOR agonists may recruit other neuronal systems that have inhibitory effects on performance. In our study, the prior exposure of the rats to alcohol vapor may have potentiated these performance-inhibiting effects of the higher doses of U50,488, and contributed to the inverted U shaped dose response we observed.

4.2. Effects of prior exposure to alcohol vapor on U50,488-induced brain activation

Our present findings show that the enhancement of U50,488-induced Fos expression in rats previously exposed to alcohol vapor is site-specific, in that we observed potentiation only in the ventral BNST. The effects of alcohol dependence on stress-induced Fos have been previously examined, but only during acute withdrawal (1–10 h after termination of exposure) (Knapp et al., 1998; Retson et al., 2015). In these studies, withdrawal from alcohol was reported to augment stress-induced Fos expression throughout the brain, but the data are confounded by the strong Fos induction seen during the acute stage of alcohol withdrawal when physical withdrawal symptoms are present (Knapp et al., 1998; Marcinkiewcz et al., 2015; de Guglielmo et al., 2016). Our present work extends these data on stress-induced Fos expression to a time after withdrawal from alcohol when this confound is absent, 40 d after termination of vapor exposure.

4.3. Role of KOR in the BNST in enhanced reinstatement in vapor-exposed rats

KORs in the CeA, NAC and BNST are involved in increased alcohol self-administration during acute withdrawal from alcohol (Nealey et al., 2011; Kissler et al., 2014; Kissler and Walker, 2016; Erikson et al., 2018). We previously showed that in non-dependent rats, Fos expression in the dorsal and ventral BNST was correlated with U50,488-induced reinstatement of alcohol seeking, and that BNST injections of U50,488 induced reinstatement (Le et al., 2018). Here, we observed potentiation of Fos expression by U50,488 in the ventral BNST of rats exposed to alcohol vapor. Together, these results may suggest the involvement of ventral BNST KOR in the potentiating effects of vapor exposure on U50,488-induced reinstatement during protracted withdrawal. This is supported by data showing that increases in alcohol self-administration seen early in withdrawal are blocked by BNST KOR antagonism (Erikson et al., 2018), and that there are persistent increases in BNST neuron excitability in alcohol dependent rats (Marcinkiewcz et al., 2015). However, although our present data show a significant potentiation of U50,488-induced Fos expression in the ventral BNST, at a dose that had greater effects in vapor-exposed vs. control rats, they must be interpreted with caution, as it was shown with single U50,488 dose.

4.4. Effects of U50,488 and alcohol vapor exposure on p-p38 expression

We determined whether p-p38 expression was affected by U50,488 and alcohol vapor exposure, because the p38 MAPK pathway is implicated in the aversive effects of KOR agonists and in stress- and cocaine-induced reinstatement of conditioned place preference to cocaine (Bruchas et al., 2007; Land et al., 2009; Schindler et al., 2012; Mannangatti et al., 2015). U50,488 produced a small increase in p-p38 expression in the NAC core, but not in any other region, and did not interact with vapor exposure. We also found increases in basal expression of p-p38 in the dorsal BNST, LC and NTS in rats chronically exposed to alcohol vapor. It could be speculated that a long-lasting elevation in dynorphin release (Lindholm et al., 2000; Kuzmin et al., 2013), and therefore increased stimulation of KOR, were responsible for the increased p-p38 expression we observed in the rats exposed to alcohol vapor in our study. Alternatively, other effects alcohol vapor exposure, such as a vapor-induced increase in total p38 MAPK levels, could have contributed to the increased p-p38 expression we observed.

4.5. Mechanisms underlying effects of chronic exposure to alcohol vapor during protracted withdrawal

Our data indicate that the effects of chronic vapor exposure on U50,488-induced reinstatement and brain activation occur during protracted withdrawal, with enhanced U50,488-induced reinstatement occurring 25–40 d after the last vapor exposure and on Fos expression, 40 d after the last vapor exposure. In support of our present behavioral data, footshock-induced relapse was shown to be potentiated 19–21 d after withdrawal from alcohol vapor or intragastric alcohol (Liu and Weiss, 2002; de Guglielmo et al., 2015). Consistent with these data, increased anxiety-like behavior, a state associated with relapse liability, was observed up to 6 weeks after withdrawal (Knapp et al., 1998; Gillett et al., 2013; Marcinkiewcz et al., 2015). Our data adds to these findings on the long-lasting effects of chronic exposure to high doses of alcohol, in showing that enhanced reinstatement induced by a stressor, in the form of U50,488 can be seen up to 40 days after such exposure. Our findings of potentiated U50,488-induced Fos induction in the ventral BNST of rats previously exposed to alcohol vapor provide a neuronal correlate of these behavioral effects.

One candidate for a transmitter involved in the long-lasting effects of vapor exposure on U50,488-induced relapse is the stress-related neuropeptide, corticotropin releasing factor (CRF). Persistent changes in CRF and its receptors have been reported in alcohol dependent rats (Eisenhardt et al., 2015); this is relevant to our present data, as CRF is colocalized with dynorphin in brain areas involved in stress-induced relapse, and there are CRF receptor - KOR interactions in reinstatement (Marchant et al., 2007; Funk et al., 2014). Another is noradrenaline, a transmitter integral to the effects of stress. The BNST, especially the ventral region, is densely innervated by noradrenaline projections and antagonism of BNST adrenoceptors blocks stress responses and stress-induced reinstatement of drug seeking (Cecchi et al., 2002; Zheng and Rinaman, 2013; McReynolds et al., 2014; Vranjkovic et al., 2014).

4.6. Methodological and interpretational considerations

A limitation of our data is that intermittent alcohol vapor exposure did not increase alcohol self-administration. In previous studies demonstrating vapor-induced increases, rats were given self-administration sessions during acute withdrawal (6–12 h), and It is thought that rats increase alcohol intake because it can alleviate the aversive consequences of withdrawal (Vendruscolo and Roberts, 2014; Tunstall et al., 2017). In the present study, in contrast, we tested self-administration 3 d after alcohol vapor offset, when physical withdrawal symptoms have dissipated. Our findings are consistent with other studies that did not find increases in alcohol self-administration, when tested 3 or more d after withdrawal (Sidhpura et al., 2010; de Guglielmo et al., 2015). It is important to note, however, that even in the absence of increased alcohol self-administration, these two studies demonstrated long-lasting effects of chronic alcohol treatment on self-administration and reinstatement (Sidhpura et al., 2010; de Guglielmo et al., 2015), that are in line with our present findings of potentiated U50,488-induced reinstatement in vapor-exposed rats during protracted withdrawal.

We observed effects of alcohol vapor 26–40 days after termination of exposure, but we did not determine the time course of U50,488-induced reinstatement or Fos/p-p38 expression during withdrawal. Previous work has shown that effects of drug dependence may be expressed in a dynamic manner from the acute dependence phase to protracted withdrawal (Kuhar and Pilotte, 1996). In order to examine the persistence of the effects of alcohol vapor exposure, a full determination of the time course is necessary.

An interpretational issue is that we saw minimal U50,488-induced p-p38 expression, that achieved statistical significance only in the NAC core; if the p38 signaling pathway is involved in the effects U50,488 on reinstatement or Fos expression we would expect enhanced expression in the BNST or other regions. A possible explanation for this could be the time of brain collection after the U50,488 injections we employed, which we based on a study conducted in California mice showing that 2.5–10 mg/kg U50,488 increased p-p38 in the NAC shell at 90 min after systemic injection (Robles et al., 2014). However, an earlier study that conducted a time course on the effects of U50,488 on p-p38 expression found increases in the dorsal raphe 30 min after injection, but not at 2 h (Lemos et al., 2011), although the dose of U50,488 and species used were not specified. A related issue is that we determined the effects of U50,488 on p-p38 with a single dose, at a single time after vapor exposure. To fully understand the effects of U50,488 on regional p-p38 expression and its potential interaction with alcohol dependence, a dose response and time course study is critical.

The neuronal populations in some of the brain regions we examined, such as the NAC (Al-Hasani et al., 2015; Floresco et al., 2018) and BNST (Crestani et al., 2013) do not have homogeneous functions but rather comprise distinct subpopulations of neurons that encode in some cases opposite effects on behavior (e.g. reward vs. aversion). This may an especially important consideration in the case of U50,488, that has both aversive and appetitive properties (Land et al., 2008). U50,488 would be expected to affect all KOR-containing neurons regardless of the valence they encode. Therefore, the U50,488-induced Fos expression may not represent the specific neuronal populations that underlie reinstatement. Further studies employing double-labeling methods and selective inactivation procedures (e.g. Daun02 (Funk et al., 2016; Koya et al., 2016)) may help to identify the activated neurons involved in U50,488-induced reinstatement.

An important question is whether the alcohol vapor-induced enhancement of the effects of U50,488 on relapse and Fos was due to increased KOR sensitivity. Enhanced U50,488-induced inhibition of dopamine release from NAc tissue slices 72 h after alcohol, suggestive of increased KOR sensitivity have been reported (Rose et al., 2016). The BNST receives dopamine innervation (Park et al., 2012), and it could therefore be suggested that alcohol dependence affects this projection in a similar way, although the downstream mechanisms enhancing reinstatement are not known.

A recent study provides evidence of increased sensitivity of KOR in the BNST in alcohol dependent rats. Increases in BNST KOR mRNA in vapor-exposed rats self-administering alcohol were reported. This study also showed that withdrawal induced increases-in alcohol self-administration were blocked by intra-BNST administration of nor-BNI (Erikson et al., 2018). Taken together with our present findings of increased U50,488-induced reinstatement and Fos in the BNST of vapor-exposed rats and our previous work showing intra-BNST injections of U50,488 induce reinstatement, these findings support the idea that enhanced sensitivity of BNST KOR are related to the potentiation of U50,488-induced alcohol seeking by alcohol vapor exposure that we observed in the present study.

4.7. Concluding remarks

Our present results show that chronic intermittent exposure to alcohol vapor leads to enhanced U50,488-induced reinstatement of alcohol seeking that is accompanied by increased neuronal activation in the ventral BNST. These findings support our previous work on the important role for the BNST in U50,488-induced reinstatement in non-dependent rats, and extend them to rats chronically exposed to alcohol vapor. Our results suggest that changes in the response of the ventral BNST to U50,488 may participate in the vapor-induced enhancement of U50,488-induced reinstatement of alcohol seeking during protracted withdrawal.

Highlights.

  • exposure to alcohol vapor made rats more sensitive to U50,488-induced reinstatement of alcohol seeking

  • this increased reinstatement was accompanied by increased expression of the neuronal activity marker, Fos in the ventral BNST

  • alcohol vapor exposure increased basal expression of phosphorylated p38 MAPK in a subset of brain regions

Acknowledgements

We wish to thank Dr. Yavin Shaham for his helpful comments on this manuscript.

Funding

The study was supported by NIAAA-NIH funds awarded to ADL (R01-AA024341).

Footnotes

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Declarations of interest: none

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