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Upregulation of Voluntary Alcohol Intake, Behavioral Sensitivity to Stress, and Amygdala Crhr1 Expression Following a History of Dependence

      Background

      A history of alcohol dependence recruits increased voluntary alcohol intake and sensitivity to stress. Corticotropin-releasing hormone (CRH) has been implicated in this transition, but underlying molecular mechanisms remain unclear.

      Methods

      A postdependent state was induced using intermittent alcohol exposure. Experiments were carried out following ≥3 weeks of recovery to eliminate contributions of acute withdrawal. Voluntary alcohol consumption was assessed in a two-bottle, free choice procedure. Behavioral sensitivity to stress was examined using fear suppression of behavior in a punished drinking (Vogel) conflict test. Effects of forced swim stress on voluntary alcohol intake were examined as a function of exposure history. Expression of Crh, Crhr1, and Crhr2 transcripts was analyzed by in situ hybridization histochemistry.

      Results

      Alcohol drinking was upregulated long-term following a history of dependence. Fear suppression of behavior was selectively potentiated in postdependent animals. This persisted 3 months after alcohol exposure and was reversed by the selective CRH-R1 antagonist 3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-8-(1-ethylpropyl)-2,6-dimethyl-imidazo[1,2-b]pyridazine (MTIP) (10 mg/kg). Forced swim stress increased alcohol intake in postdependent animals but not in control animals. Behavioral changes were paralleled by an upregulation of Crhr1 transcript expression within basolateral (BLA) and medial (MeA) amygdala and Crh messenger RNA (mRNA) in central amygdala (CeA). In contrast, Crhr2 expression was down in the BLA.

      Conclusions

      Neuroadaptations encompassing amygdala CRH signaling contribute to the behavioral phenotype of postdependent animals.

      Key Words

      Alcoholism develops over years and requires prolonged periods of brain exposure to intoxicating levels of alcohol. Over the course of this process, recruitment of elevated anxiety, low mood, and increased sensitivity to stress, here collectively labeled negative affect, has been postulated as being critical for the transition from a nondependent to a dependent state (
      • Breese G.R.
      • Overstreet D.H.
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      Conceptual framework for the etiology of alcoholism: A “ kindling”/stress hypothesis.
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      Pharmacological treatment of alcohol dependence: Target symptoms and target mechanisms.
      ,
      • Valdez G.R.
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      Allostasis and dysregulation of corticotropin-releasing factor and neuropeptide Y systems: Implications for the development of alcoholism.
      ). Clinically, negative affect is most prominent during acute alcohol withdrawal but persists into protracted abstinence (
      • Hershon H.I.
      Alcohol withdrawal symptoms and drinking behavior.
      ), as shown, for example, by potentiated startle responses (
      • Krystal J.H.
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      • Morgan C.A.
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      Evidence of acoustic startle hyperreflexia in recently detoxified early onset male alcoholics: Modulation by yohimbine and m-chlorophenylpiperazine (mCPP).
      ) and increased frequency of panic attacks (
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      • Nutt D.J.
      • Dwyer B.A.
      • Linnoila M.
      Alcoholism and panic disorder: Is the comorbidity more than coincidence?.
      ). Environmental stressors constitute a major category of stimuli capable of triggering relapse in humans and experimental animals (
      • Brownell K.D.
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      Understanding and preventing relapse.
      ;
      • Shaham Y.
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      • de Wit H.
      • Stewart J.
      The reinstatement model of drug relapse: History, methodology and major findings.
      ). Thus, increased sensitivity to stress in the postdependent state is likely to contribute to maintaining alcohol dependence. Identifying its neural substrates, therefore, is critical to developing novel alcoholism treatments.
      A history of alcohol dependence has been modeled in laboratory rats using prolonged exposure to alcohol vapor, which triggers long-lasting neural and behavioral plasticity that appear relevant for modeling human alcoholism. This type of manipulation produces persistently increased alcohol intake in genetically nonselected rats (
      • Rimondini R.
      • Arlinde C.
      • Sommer W.
      • Heilig M.
      Long-lasting increase in voluntary ethanol consumption and transcriptional regulation in the rat brain after intermittent exposure to alcohol.
      ;
      • Roberts A.J.
      • Heyser C.J.
      • Cole M.
      • Griffin P.
      • Koob G.F.
      Excessive ethanol drinking following a history of dependence: Animal model of allostasis.
      ). Exposure to repeated cycles of intoxication and withdrawal, which mimics the course of the clinical condition, is most effective for inducing increased alcohol drinking (
      • O’Dell L.E.
      • Roberts A.J.
      • Smith R.T.
      • Koob G.F.
      Enhanced alcohol self-administration after intermittent versus continuous alcohol vapor exposure.
      ;
      • Rimondini R.
      • Arlinde C.
      • Sommer W.
      • Heilig M.
      Long-lasting increase in voluntary ethanol consumption and transcriptional regulation in the rat brain after intermittent exposure to alcohol.
      ). Similar to the human condition, a minimum duration of dependence is required for lasting upregulation of alcohol preference (
      • Rimondini R.
      • Sommer W.
      • Heilig M.
      A temporal threshold for induction of persistent alcohol preference: Behavioral evidence in a rat model of intermittent intoxication.
      ). Elevated alcohol intake in postdependent rats is sensitive to the clinically effective compound, acamprosate, while alcohol intake of nondependent rats is unaffected by the same treatment (
      • Egli M.
      Can experimental paradigms and animal models be used to discover clinically effective medications for alcoholism?.
      ;
      • Heyser C.J.
      • Schulteis G.
      • Durbin P.
      • Koob G.F.
      Chronic acamprosate eliminates the alcohol deprivation effect while having limited effects on baseline responding for ethanol in rats.
      ;
      • Rimondini R.
      • Arlinde C.
      • Sommer W.
      • Heilig M.
      Long-lasting increase in voluntary ethanol consumption and transcriptional regulation in the rat brain after intermittent exposure to alcohol.
      ;
      • Spanagel R.
      • Zieglgansberger W.
      Anti-craving compounds for ethanol: New pharmacological tools to study addictive processes.
      ). Furthermore, the postdependent state is characterized by a persistently upregulated behavioral sensitivity to stress (
      • Valdez G.R.
      • Roberts A.J.
      • Chan K.
      • Davis H.
      • Brennan M.
      • Zorrilla E.P.
      • et al.
      Increased ethanol self-administration and anxiety-like behavior during acute ethanol withdrawal and protracted abstinence: Regulation by corticotropin-releasing factor.
      ,
      • Valdez G.R.
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      • Roberts A.J.
      • Koob G.F.
      Antagonism of corticotropin-releasing factor attenuates the enhanced responsiveness to stress observed during protracted ethanol abstinence.
      ). Together, these findings indicate that neuroadaptive processes induced by a prolonged exposure to cycles of intoxication and withdrawal parallel those in human alcoholism and might be able to shed light on underlying neural mechanisms.
      Corticotropin-releasing hormone (CRH) mediates behavioral stress responses through extrahypothalamic mechanisms. These actions of CRH are primarily mediated through the CRH-R1 receptor subtype, which has been proposed as an attractive target for medication development in anxiety, depression, and addiction (
      • Heinrichs S.C.
      • Koob G.F.
      Corticotropin-releasing factor in brain: A role in activation, arousal, and affect regulation.
      ,
      • Holsboer F.
      Corticotropin-releasing hormone modulators and depression.
      ,
      • Reul J.M.
      • Holsboer F.
      Corticotropin-releasing factor receptors 1 and 2 in anxiety and depression.
      ;
      • Sarnyai Z.
      • Shaham Y.
      • Heinrichs S.C.
      The role of corticotropin-releasing factor in drug addiction.
      ). Brain regions of particular importance for drug reward, including the medial prefrontal cortex (mPFC), the nucleus accumbens (NAcc), the bed nucleus of the stria terminalis (BNST), and several nuclei within the amygdala, including central (CeA), medial (MeA), and basolateral amygdala (BLA), are rich in CRH receptors, specifically of the R1 subtype (
      • Potter E.
      • Sutton S.
      • Donaldson C.
      • Chen R.
      • Perrin M.
      • Lewis K.
      • et al.
      Distribution of corticotropin-releasing factor receptor mRNA expression in the rat brain and pituitary.
      ;
      • Van P.K.
      • Viau V.
      • Bittencourt J.C.
      • Chan R.K.
      • Li H.Y.
      • Arias C.
      • et al.
      Distribution of mRNAs encoding CRF receptors in brain and pituitary of rat and mouse.
      ). Most of the CRH neurons targeting these regions originate from cortical interneurons or CeA (
      • Swanson L.W.
      • Sawchenko P.E.
      • Rivier J.
      • Vale W.W.
      Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: An immunohistochemical study.
      ), a structure that mediates fear and anxiety (
      • Davis M.
      • Walker D.L.
      • Lee Y.
      Amygdala and bed nucleus of the stria terminalis: Differential roles in fear and anxiety measured with the acoustic startle reflex.
      ;
      • LeDoux J.E.
      • Iwata J.
      • Cicchetti P.
      • Reis D.J.
      Different projections of the central amygdaloid nucleus mediate autonomic and behavioral correlates of conditioned fear.
      ;
      • Möller C.
      • Wiklund L.
      • Sommer W.
      • Thorsell A.
      • Heilig M.
      Decreased experimental anxiety and voluntary ethanol consumption in rats following central but not basolateral amygdala lesions.
      ). Anxious responding during acute alcohol withdrawal is attenuated by CRH antagonists, administered either systemically or directly into the CeA (
      • Baldwin H.A.
      • Rassnick S.
      • Rivier J.
      • Koob G.F.
      • Britton T.K.
      CRF antagonist reverses the “anxiogenic” response to ethanol withdrawal in the rat.
      ;
      • Funk C.K.
      • Zorrilla E.P.
      • Lee M.J.
      • Rice K.C.
      • Koob G.F.
      Corticotropin-releasing factor 1 antagonists selectively reduce ethanol self-administration in ethanol-dependent rats.
      ;
      • Knapp D.J.
      • Overstreet D.H.
      • Moy S.S.
      • Breese G.R.
      SB242084, flumazenil, and CRA1000 block ethanol withdrawal-induced anxiety in rats.
      ;
      • Overstreet D.H.
      • Knapp D.J.
      • Breese G.R.
      Modulation of multiple ethanol withdrawal-induced anxiety-like behavior by CRF and CRF1 receptors.
      ;
      • Rassnick S.
      • Heinrichs S.C.
      • Britton K.T.
      • Koob G.F.
      Microinjection of a corticotropin-releasing factor antagonist into the central nucleus of the amygdala reverses anxiogenic-like effects of ethanol withdrawal.
      ;
      • Valdez G.R.
      • Roberts A.J.
      • Chan K.
      • Davis H.
      • Brennan M.
      • Zorrilla E.P.
      • et al.
      Increased ethanol self-administration and anxiety-like behavior during acute ethanol withdrawal and protracted abstinence: Regulation by corticotropin-releasing factor.
      ). Furthermore, CRH antagonists block both elevated alcohol self-administration and potentiated anxiety-like responses to stressors seen during protracted abstinence following a history of dependence (
      • Breese G.R.
      • Overstreet D.H.
      • Knapp D.J.
      • Navarro M.
      Prior multiple ethanol withdrawals enhance stress-induced anxiety-like behavior: Inhibition by CRF1- and benzodiazepine-receptor antagonists and a 5-HT1a-receptor agonist.
      ;
      • Valdez G.R.
      • Roberts A.J.
      • Chan K.
      • Davis H.
      • Brennan M.
      • Zorrilla E.P.
      • et al.
      Increased ethanol self-administration and anxiety-like behavior during acute ethanol withdrawal and protracted abstinence: Regulation by corticotropin-releasing factor.
      ,
      • Valdez G.R.
      • Zorrilla E.P.
      • Roberts A.J.
      • Koob G.F.
      Antagonism of corticotropin-releasing factor attenuates the enhanced responsiveness to stress observed during protracted ethanol abstinence.
      ).
      Recruitment of central CRH signaling thus underlies two key features of the postdependent state, namely, long-term increased voluntary consumption of alcohol and persistently upregulated behavioral sensitivity to stress. The molecular mechanisms for these changes are largely unknown. Recently, we reported that in the genetically selected Marchigian-Sardinian alcohol preferring (msP) rat line (
      • Ciccocioppo R.
      • Economidou D.
      • Cippitelli A.
      • Cucculelli M.
      • Ubaldi M.
      • Soverchia L.
      • et al.
      Genetically selected Marchigian Sardinian alcohol-preferring (msP) rats: An animal model to study the neurobiology of alcoholism.
      ), high alcohol preference has cosegregated with increased behavioral sensitivity to stress, creating a phenocopy of the postdependent phenotype. A screen for differential gene expression identified an innate upregulation of the Crhr1 transcript in several brain regions of the msP line. Administration of the selective CRH-R1 antagonist, antalarmin, demonstrated a causal role of upregulated CRH-R1 receptors in the behavioral phenotype of msP rats (
      • Hansson A.C.
      • Cippitelli A.
      • Sommer W.H.
      • Fedeli A.
      • Bjork K.
      • Soverchia L.
      • et al.
      Variation at the rat Crhr1 locus and sensitivity to relapse into alcohol seeking induced by environmental stress.
      ).
      Here, we examined alcohol drinking, stress sensitivity, and expression of Crh and its receptors Crhr1 and Crhr2. Our hypothesis was that, similar to what we have found in msP rats, an upregulation of the Crhr1 transcript might be present in genetically nonselected rats following a history of dependence and that this upregulation would contribute to their behavioral phenotype.

      Methods and Materials

      Animals

      Male Wistar rats (Mollegard, Denmark), weighing 225 g to 250 g at outset of experiments, were housed four per cage at 20°C to 22°C, 45% to 55% controlled humidity, and reverse 12:12 hour light/dark cycle (lights off at 11:00 am) and tested during the dark phase. All procedures followed the European Commission Council Directive for Care and Use of Laboratory Animals (ethics permit S84/98, Stockholm South).

      Overall Design

      Animals were removed from alcohol or sham exposure after 4 or 7 weeks. After recovering for a period of 3 weeks, animals from each group were randomized to one of the three following experiments:
      • 1
        Voluntary Alcohol Drinking (7-week exposed: n = 10 vs. n = 8; 4-week exposed: n = 7 vs. n = 8, exposed and sham, respectively). After initial assessment of drinking, all 7-week animals continued voluntary alcohol drinking on the 24-hour access two-bottle free choice for 29 days, until assessed for effects of forced swim stress.
      • 2
        Stress Sensitivity in the Vogel Conflict Test (7-week only: n = 20 for exposed and sham, respectively). This experiment was carried out directly after the 3-week recovery period. Subjects were then kept for an additional 10 weeks, after which postdependent and control subjects were randomized to pretreatment with vehicle or CRH-R1 antagonist and retested (n = 10 per group).
      • 3
        Analysis of Gene Expression. Gene expression was assessed by in situ hybridization. Animals were sacrificed directly after the 3-week recovery period (n = 7 for both exposed and sham).

      Alcohol Vapor Exposure

      Exposure was as described (
      • Rimondini R.
      • Arlinde C.
      • Sommer W.
      • Heilig M.
      Long-lasting increase in voluntary ethanol consumption and transcriptional regulation in the rat brain after intermittent exposure to alcohol.
      ) in glass/steel chambers (1 × 1 × 1 m). High-performance liquid chromatography (HPLC) pumps (Knauer, Berlin, Germany) delivered alcohol into electrically heated stainless steel coils (60°C) connected to an airflow of 18 L per minute. Alcohol concentration was adjusted by changing pump flow and monitored via a spectrometer (Wilks, South Norwalk, Connecticut). Exposure was for 17 hours during each 24-hour period (on 4:00 pm; off 9:00 am). Rats were allowed to habituate to the chambers for 1 week, then exposed to low alcohol concentration for 1 week, and finally exposed to alcohol vapor yielding blood alcohol concentrations as shown in Table 1. Control animals were kept in identical chambers with normal airflow. Weekly, rats were weighed and blood was collected from the lateral tail vein, serum extracted, and assayed for ethanol using an nicotinamide adenine dinucleotide phosphate dehydrogenase/spectrophotometric assay kit (Sigma Aldrich Inc., St. Louis, Missouri) according to the manufacturer’s instructions.
      Table 1Blood Alcohol Concentrations Resulting from the Alcohol Vapor Exposure Used to Induce Dependence
      Exposure WeekBAC (mg/dL; mean ± SEM)Range
      1194.4 ± 29.290–340
      2313.9 ± 15.0227–377
      3237.3 ± 22.7181–339
      4204.4 ± 14.6152–293
      5309.8 ± 21.6239–426
      6388.1 ± 38.6257–574
      7303.0 ± 15.5273–416
      BAC, blood alcohol concentration.

      Alcohol Consumption and Its Modulation by Stress

      Alcohol consumption was measured as 24-hour access two-bottle free choice between 6% alcohol (wt/vol) in .2% saccharin solution, or vehicle, .2% saccharin solution only, as described (
      • Rimondini R.
      • Arlinde C.
      • Sommer W.
      • Heilig M.
      Long-lasting increase in voluntary ethanol consumption and transcriptional regulation in the rat brain after intermittent exposure to alcohol.
      ). One week was used to fade in alcohol, and consumption was measured over the following 2 weeks.
      To assess effects of stress on voluntary alcohol consumption, two-bottle free-choice drinking was continued for 29 days. Baseline data were obtained over a 3-day block on days 30 to 32 after initiation of drinking. The forced swim stress was carried out daily over a 3-day block on days 33 to 35 as described (
      • Vengeliene V.
      • Siegmund S.
      • Singer M.V.
      • Sinclair J.D.
      • Li T.K.
      • Spanagel R.
      A comparative study on alcohol-preferring rat lines: Effects of deprivation and stress phases on voluntary alcohol intake.
      ). Briefly, around 3:00 pm on each test day, animals were removed from their home cage and placed for 10 min in a plastic cylinder (45 × 20 cm) filled up to 35 cm with 19°C water. After completion of the forced swim, subjects were returned to their home cages. Following completion of the stress block, poststress drinking measures were obtained over a final 3-day block (days 36 to 38).

      Punished Drinking Test

      A modified Vogel drinking test was used as described (
      • Sommer W.
      • Möller C.
      • Wiklund L.
      • Thorsell A.
      • Rimondini R.
      • Nissbrandt H.
      • et al.
      Local 5,7-dihydroxytryptamine lesions of rat amygdala: Release in punished drinking, unaffected plus-maze behavior and ethanol consumption.
      ), and punished licks during the 8-min conflict interval were recorded. Unpunished licks during the preceding 4-min control interval were also recorded, as a control for possible effects on thirst or motor performance.
      To assess whether potentiated fear suppression of behavior in the conflict test is mediated by CRH-R1 activation, a novel imidazopyridazine CRH-R1 antagonist, 3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-8-(1-ethylpropyl)-2,6-dimethyl-imidazo[1,2-b]pyridazine (MTIP) (Lilly Research Laboratories, Indianapolis, Indiana) was used. The MTIP binds CRH-R1 receptors with nanomolar affinity, with no detectable activity at the CRH-R2 receptor or other common drug targets, and is highly brain penetrant. A 10 mg/kg was chosen because median effective dose (ED50) has been determined to approximately 1.5 mg, while 10 mg/kg produces a more than 90% blockade of several stress-induced behaviors (
      • Gehlert D.R.
      • Cippitelli A.
      • Thorsell A.
      • Le D.A.
      • Hipskind P.A.
      • Hamdouchi C.
      • et al.
      3-(4-chloro-2-morpholin-4-yl-thiazol-5-yl)-8-(1-ethylpropyl)-2,6-dimethyl-imidazo[1,2-b]pyridazine: A novel brain-penetrant, orally available corticotropin-releasing factor receptor 1 antagonist with efficacy in animal models of alcoholism.
      ). Vehicle (10% Tween [Sigma Aldrich Inc.] 80 in distilled water) or MTIP in vehicle were administered intraperitoneally (IP) 30 min prior to testing.

      In Situ Hybridization

      Procedures were performed as described previously (
      • Hansson A.C.
      • Sommer W.
      • Rimondini R.
      • Andbjer B.
      • Stromberg I.
      • Fuxe K.
      c-fos reduces corticosterone-mediated effects on neurotrophic factor expression in the rat hippocampal CA1 region.
      ,
      • Hansson A.C.
      • Cippitelli A.
      • Sommer W.H.
      • Fedeli A.
      • Bjork K.
      • Soverchia L.
      • et al.
      Variation at the rat Crhr1 locus and sensitivity to relapse into alcohol seeking induced by environmental stress.
      ). Rats were decapitated in the inactive phase (11:00 pm to 3:00 am), and brains were removed, snap frozen in −40°C isopentane, and stored at −70°C until use. The 10-μm brain sections were taken at Bregma levels 1) +2.5 to +1.7 mm, 2) −.3 to −.4 mm, 3) −1.7 to 2.0 mm, and 4) −2.3 to −3.3 mm (
      • Paxinos G.
      • Watson C.
      The Rat Brain in Stereotaxic Coordinates.
      ). Following hybridization with riboprobes for Crh, Crhr1, or Crhr2 messenger RNA (mRNA), sections were exposed to Fuji BAS-5000 Phosphorimager plates (Fujifilm, Tokyo, Japan). Digital images were analyzed using AIS Image Analysis Software (Imaging Research Inc., St. Catharines, Ontario, Canada). Regions of interest were chosen based on available functional data with microinjections of CRH receptor ligands within the amygdala and BNST. Values were converted to nCi/g using carbon 14 (14C) standards.

      Statistical Analysis

      Daily alcohol intake was averaged over 2 weeks. The 4-week and 7-week sham-exposed control groups did not differ from each other and were, therefore, pooled. Following one-way analysis of variance (ANOVA), each of the exposed groups was compared against the pooled control group by Dunnett’s post hoc test. In the swim-stress experiment, daily intake was averaged over each of the three blocks (baseline, stress, poststress), each of which was 3 days in duration. Drinking data were analyzed using two-way ANOVA, with history of dependence as between-subjects and the three drinking blocks as a within-subjects factor. Post hoc comparison was performed using the Newman-Keuls test.
      Punished licks violated assumptions of homogenous variances and were rank-transformed prior to analysis. In the initial experiment, a one-way ANOVA was carried out, with history of dependence as between-subjects factor. In the pharmacological experiment, a two-way analysis was carried out, with history of dependence and treatment (MTIP vs. vehicle) as factors. To assess whether any fear suppression by the conflict remained following MTIP treatment, unpunished and punished licks were both recalculated as rates (licks/min) to be directly comparable, and equality was tested using the confidence interval method. In each experiment, unpunished licks were separately analyzed as a control for nonspecific effects on thirst or motor performance.

      Gene Expression

      Data were compared by region-wise one-way ANOVAs, followed by the reverse Holm-Bonferroni correction (
      • Holm S.
      A simple sequentially rejective multiple test procedure.
      ,
      • Dow G.S.
      Effect of sample size and P-value filtering techniques on the detection of transcriptional changes induced in rat neuroblastoma (NG108) cells by mefloquine.
      ).

      Results

      Increased Alcohol Consumption Following a History of Dependence and Selective Increase of Alcohol Intake by Stress in Postdependent Rats

      Average daily intake of 7-week and 4-week exposed animals was compared with that of their pooled control animals, since the latter did not differ. A more than twofold increase was observed in 7-week but not 4-week exposed animals when compared with control animals [3.8 ± .35 and 1.12 ± .42, respectively, vs. 1.44 ± .28; F(2,30) = 17.5, p < .001, Dunnett’s post hoc test p < .001, 7-week exposed vs. control animals; Figure 1]. Vehicle intake was unaffected (data not shown).
      Figure thumbnail gr1
      Figure 1Long-lasting increases in voluntary alcohol consumption in rats with a history of dependence. Two-bottle, free-choice, continuous access alcohol in .2% saccharin versus .2% saccharin only was assessed after a 3-week resting period that followed the last exposure cycle. One week was allowed to gradually increase the alcohol concentration to 6% (wt/vol), and testing was over the following 2 weeks. Left panel: Consumption over the 2-week period. Right panel: Average daily consumption over the same period. There was a highly significant increase in alcohol consumption in the 7-week exposed group compared with their sham-exposed control group. Values are expressed as mean daily consumption ± SEM of 6% (vol/vol) alcohol consumption. ***p < .001. For detailed statistics, see Results.
      In the stress experiment, animals with a history of dependence continued to consume higher amounts of alcohol [main effect of exposure history: F(1,16) = 12.7, p = .003]. There was a significant overall effect of stress exposure [main effect: F(2,32) = 9.0, p = .0008], but this affected the two groups differentially, as shown by a significant interaction term [F(2,32) = 4.1, p = .027]. Post hoc analysis revealed that within the postdependent group, drinking both during the stress and the poststress block was significantly higher than during the prestress baseline block (p < .001). In contrast, within the sham-exposed control group, drinking during the stress and the poststress block was indistinguishable from that measured during the prestress baseline block (Figure 2). Vehicle intake did not differ between groups and was unaffected by stress exposure (data not shown).
      Figure thumbnail gr2
      Figure 2Increase in voluntary alcohol consumption in response to forced swim stress in animals with a history of dependence but not in nondependent animals. Following 7 weeks of intermittent alcohol vapor exposure and tested for voluntary alcohol drinking for 2 weeks (), subjects were tested over three blocks (prestress baseline, stress, poststress), each of 3-day duration. During the stress block, animals were subjected to daily forced swim stress. **p < .01, ***p < .001 versus prestress baseline value.

      Potentiated Fear Suppression of Behavior Following a History of Dependence and Its Reversal by the Selective CRH-R1 Antagonist MTIP

      After 3-week recovery, fear suppression followed a history of dependence, as shown by markedly lower rates of punished responding observed in 7-week exposed animals compared with their corresponding control group [F(1,36) = 14.3, p = .0006; Figure 3]. Unpunished responding did not differ between the groups.
      Figure thumbnail gr3
      Figure 3Long-term upregulated behavioral sensitivity to stress in rats with a history of dependence, induced by a 7-week exposure to daily cycles of intoxication and withdrawal in alcohol vapor chambers. To eliminate effects of acute withdrawal, fear-induced suppression of behavior in the punished drinking test was assessed after a 3-week resting period that followed the last exposure cycle. Punished licks recorded over an 8-min conflict period are given (mean ± SEM). Conflict testing was preceded by a 4-min period of unpunished drinking to control for potential nonspecific effects on thirst or motor performance. ***p < .001. For detailed statistics, see Results.
      Potentiation of fear suppression by a history of dependence persisted when tested 13 weeks after recovery [main effect of exposure history: F(1,35) = 5.4, p = .025]. At this time, pretreatment with the CRH-R1 antagonist, MTIP, had a robust anticonflict effect [main effect: F(1,35) = 13.2, p = .0009]. The MTIP eliminated the difference in conflict behavior related to exposure history. In fact, in both animals with and without a history of dependence, MTIP fully eliminated any fear suppression by conflict (equality between punished and unpunished lick rates within a 10% indifference interval: p < .05; Figure 4).
      Figure thumbnail gr4
      Figure 4Persistent increased behavioral sensitivity to stress following a history of dependence as measured in the conflict test, when animals were retested 13 weeks after completion of alcohol exposure (main effect of history of dependence: p = .025). The selective, brain penetrant CRH-R1 antagonist MTIP (10mg/kg) produced a robust overall anticonflict effect (main treatment effect: p < .001). The MTIP eliminated the increased sensitivity to stress in the postdependent group and, in fact, eliminated any fear suppression of behavior by conflict, as shown by punished lick rates becoming virtually identical to unpunished rates. Data are licks/min, mean ± SEM; equality of punished and unpunished lick rates within an indifference interval of 10%: p < .05 for both animals without a history of dependence and postdependent subjects. CRH, corticotropin-releasing hormone; MTIP, 3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-8-(1-ethylpropyl)-2,6-dimethyl-imidazo[1,2-b]pyridazine.

      Long-Lasting Increase of Crhr1 and Crh Gene Expression in the Amygdala Following a History of Dependence

      Postdependent animals had robustly elevated levels of Crh1 transcript in BLA [F(1,12) = 11.3, p < .01] and MeA [F(1,12) = 25.6, p < .001] but not in CeA or BNST (Figure 5C). The Crhr1 transcript was not reliably detected in the hypothalamic paraventricular nucleus (PVN).
      Figure thumbnail gr5
      Figure 5(A) Distribution of Crh transcript, encoding the CRH precursor, and the Crhr1 and Crhr2 transcripts, encoding the respective receptor subtype. Representative sections from the amygdala of rats without a history of dependence are shown at Bregma −2.5 mm. CeA: central; MeA: medial; and BLA: basolateral amygdala. Scale bar is 1 mm. Quantification of expression levels (nCi/g, mean ± SEM) for the respective transcript in postdependent rats versus rats without a history of dependence is shown in panels (B) through (D). (B) Crh expression was upregulated within CeA, which was the only amygdala region where measurable levels of this transcript were present. (C) Crhr1 message was robustly upregulated within BLA and MeA but not in CeA or BNST. (D) Expression of Crhr2 transcript was unaffected within the extended amygdala, with the exception of BLA, where a moderate decrease was seen. For all panels, *p < .05, **p < .01, ***p < .001 corrected for multiple tests. For detailed statistics, see Results. BLA, basolateral amygdala; BNST, bed nucleus of stria terminalis; CeA, central amygdala; CRH, corticotropin-releasing hormone; MeA, medial amygdala.
      Expression of the Crhr2 transcript was less affected in the extrahypothalamic regions studied, with the exception of a moderate decrease in the BLA [F(1,11) = 6.7, p = .025; Figure 5D]. Postdependent animals did not differ from control animals in Crhr2 expression within the PVN (22.5 ± 1.43 vs. 23.3 ± 1.17 nCi/g, mean ± SEM, respectively).
      Expression of the Crh transcript was elevated in CeA of postdependent animals [F(1,12) = 7.1, p = .02], while no difference was seen within the BNST (Figure 5B). Postdependent animals did not differ from control animals within the PVN (103.5 ± 3.5 vs. 107.4 ± 4.7).

      Discussion

      We report elevated voluntary alcohol consumption and potentiated stress sensitivity during protracted abstinence following a history of alcohol dependence. Postdependent animals were selectively sensitive to upregulation of alcohol consumption by a stressor. Long-term upregulation of the transcript encoding the CRH-R1 receptor was found within MeA and BLA of postdependent animals, while CRH-R1 antagonism eliminated the increased behavioral sensitivity to stress in the postdependent state.
      Our drinking data replicate prior reports (
      • O’Dell L.E.
      • Roberts A.J.
      • Smith R.T.
      • Koob G.F.
      Enhanced alcohol self-administration after intermittent versus continuous alcohol vapor exposure.
      ;
      • Rimondini R.
      • Arlinde C.
      • Sommer W.
      • Heilig M.
      Long-lasting increase in voluntary ethanol consumption and transcriptional regulation in the rat brain after intermittent exposure to alcohol.
      ;
      • Rimondini R.
      • Sommer W.
      • Heilig M.
      A temporal threshold for induction of persistent alcohol preference: Behavioral evidence in a rat model of intermittent intoxication.
      ;
      • Roberts A.J.
      • Heyser C.J.
      • Cole M.
      • Griffin P.
      • Koob G.F.
      Excessive ethanol drinking following a history of dependence: Animal model of allostasis.
      ). In the present study, alcohol intake more than doubled in animals with a history of dependence. This increase was seen after a 3-week recovery following completion of alcohol exposure and persisted for more than an additional month. It is therefore related to long-term neuroadaptations rather than acute withdrawal. A previously reported temporal threshold was replicated, so that a 7-week exposure upregulated alcohol intake, while a 4-week exposure did not. We have independently found that increased consumption in postdependent animals is not due to altered alcohol metabolism (Sommer et al., in preparation).
      Our conflict data show a long-lasting decrease in punished water drinking. This effect is not due to altered thirst, as shown by the unaffected rates of unpunished licks. Furthermore, it is also unlikely to be caused by altered nociception, since we have independently established that a history of dependence does not affect pain thresholds in the hot plate test (Heilig et al., unpublished data). Central administration of CRH has previously been shown to produce potentiated fear suppression in the Geller-Seifter test, while both prototypical benzodiazepine anxiolytics and intracerebroventricular administration of the nonselective CRH receptor antagonist α-helical-CRH9-41 reversed this potentiation (
      • Britton K.T.
      • Morgan J.
      • Rivier J.
      • Vale W.
      • Koob G.F.
      Chlordiazepoxide attenuates response suppression induced by corticotropin-releasing factor in the conflict test.
      ,
      • Britton K.T.
      • Lee G.
      • Vale W.
      • Rivier J.
      • Koob G.F.
      Corticotropin releasing factor (CRF) receptor antagonist blocks activating and ’anxiogenic’ actions of CRF in the rat.
      ). More recently, potent anticonflict effects were shown in the classical Vogel test with the nonpeptide CRH-R1 antagonists antalarmin and SSR125543A (
      • Griebel G.
      • Simiand J.
      • Steinberg R.
      • Jung M.
      • Gully D.
      • Roger P.
      • et al.
      4-(2-Chloro-4-methoxy-5-methylphenyl)-N-[(1S)-2-cyclopropyl-1-(3-fluoro-4-methylphenyl)ethyl]5-methyl-N-(2-propynyl)-1, 3-thiazol-2-amine hydrochloride (SSR125543A), a potent and selective corticotrophin-releasing factor(1) receptor antagonist II. Characterization in rodent models of stress-related disorders.
      ). The potentiated suppression of punished drinking observed in the present study is therefore likely to reflect a specific, CRH-mediated increase in behavioral sensitivity to stress. This finding is in line with prior reports that have demonstrated increased behavioral stress responses in postdependent rats (
      • Breese G.R.
      • Overstreet D.H.
      • Knapp D.J.
      • Navarro M.
      Prior multiple ethanol withdrawals enhance stress-induced anxiety-like behavior: Inhibition by CRF1- and benzodiazepine-receptor antagonists and a 5-HT1a-receptor agonist.
      ;
      • Overstreet D.H.
      • Knapp D.J.
      • Breese G.R.
      Accentuated decrease in social interaction in rats subjected to repeated ethanol withdrawals.
      ;
      • Rasmussen D.D.
      • Mitton D.R.
      • Green J.
      • Puchalski S.
      Chronic daily ethanol and withdrawal: 2 Behavioral changes during prolonged abstinence.
      ;
      • Valdez G.R.
      • Roberts A.J.
      • Chan K.
      • Davis H.
      • Brennan M.
      • Zorrilla E.P.
      • et al.
      Increased ethanol self-administration and anxiety-like behavior during acute ethanol withdrawal and protracted abstinence: Regulation by corticotropin-releasing factor.
      ,
      • Valdez G.R.
      • Zorrilla E.P.
      • Roberts A.J.
      • Koob G.F.
      Antagonism of corticotropin-releasing factor attenuates the enhanced responsiveness to stress observed during protracted ethanol abstinence.
      ). A history of multiple withdrawals, paralleling the clinical course of alcoholism, seems most effective in inducing anxiety-like behavior. For instance, rats subjected to three cycles of withdrawal from an alcohol diet, but not those exposed to a single withdrawal, showed reduced social interaction during early abstinence (
      • Overstreet D.H.
      • Knapp D.J.
      • Breese G.R.
      Accentuated decrease in social interaction in rats subjected to repeated ethanol withdrawals.
      ). Although this acute withdrawal effect subsided within 48 hours, a remaining recruitment of negative affect systems was demonstrated by the observation that following a history of three withdrawal episodes, a single future withdrawal from reexposure to chronic ethanol in control animals was anxiogenic. Interestingly, in this model, administration of a CRH antagonist during the first two cycles of withdrawal blocked the increase in anxiety behavior (
      • Knapp D.J.
      • Overstreet D.H.
      • Moy S.S.
      • Breese G.R.
      SB242084, flumazenil, and CRA1000 block ethanol withdrawal-induced anxiety in rats.
      ;
      • Overstreet D.H.
      • Knapp D.J.
      • Breese G.R.
      Modulation of multiple ethanol withdrawal-induced anxiety-like behavior by CRF and CRF1 receptors.
      ). Elevated anxiety has also been reported on the elevated plus-maze 4 weeks after a liquid alcohol diet (
      • Rasmussen D.D.
      • Mitton D.R.
      • Green J.
      • Puchalski S.
      Chronic daily ethanol and withdrawal: 2 Behavioral changes during prolonged abstinence.
      ) or alcohol vapor exposure (
      • Valdez G.R.
      • Roberts A.J.
      • Chan K.
      • Davis H.
      • Brennan M.
      • Zorrilla E.P.
      • et al.
      Increased ethanol self-administration and anxiety-like behavior during acute ethanol withdrawal and protracted abstinence: Regulation by corticotropin-releasing factor.
      ). In the latter case, no overt phenotype was seen in postdependent rats, but when testing was preceded by a restraint stress challenge, an exaggerated anxiogenic response was seen (
      • Valdez G.R.
      • Zorrilla E.P.
      • Roberts A.J.
      • Koob G.F.
      Antagonism of corticotropin-releasing factor attenuates the enhanced responsiveness to stress observed during protracted ethanol abstinence.
      ). Our present conflict data are similar in nature, as the conflict model itself is a stressor sufficient to recruit CeA activity and, in fact, relies on an activation of cell bodies in this structure for fear suppression of behavior (
      • Möller C.
      • Wiklund L.
      • Sommer W.
      • Thorsell A.
      • Heilig M.
      Decreased experimental anxiety and voluntary ethanol consumption in rats following central but not basolateral amygdala lesions.
      ). The fact that a stress-sensitive phenotype persists for more than 3 months after completion of alcohol exposure has not been reported previously, and the duration of this state is remarkable. It may be noted that all anxiety-like behaviors discussed here are unconditioned. In contrast, impaired associative fear learning has recently been reported in young human binge drinkers, presumably as a result of repeated cycles of intoxication and withdrawal (
      • Stephens D.N.
      • Ripley T.L.
      • Borlikova G.
      • Schubert M.
      • Albrecht D.
      • Hogarth L.
      • et al.
      Repeated ethanol exposure and withdrawal impairs human fear conditioning and depresses long-term potentiation in rat amygdala and hippocampus.
      ). This report proposed a “saturation” mechanism, whereby synaptic plasticity induced by a history of dependence reduces capacity for future learning, while allowing unconditioned stimuli access to neuronal pathways underlying conditioned fear.
      Elevated alcohol consumption and increased sensitivity to stress have previously been reported in postdependent rats, but little data exist to link these phenomena. A transient increase in voluntary alcohol intake has been reported in nonselected Wistar rats in response to the forced swim stress used here, but these animals had been consuming alcohol for 4 months and had experienced alcohol deprivation during the course of this history, features that set the scene for neuroadaptive changes (
      • Vengeliene V.
      • Siegmund S.
      • Singer M.V.
      • Sinclair J.D.
      • Li T.K.
      • Spanagel R.
      A comparative study on alcohol-preferring rat lines: Effects of deprivation and stress phases on voluntary alcohol intake.
      ). In the present study, nondependent Wistar rats did not increase their alcohol intake in response to repeated forced swim stress. In contrast, postdependent animals started out with elevated alcohol intake and showed a highly significant further increase in response to the stressor. Thus, the increased sensitivity to stress in the postdependent state translates into elevated motivation to consume alcohol in response to external stressors.
      Our data provide a putative molecular mechanism for the postdependent behavioral phenotype. Recently, we reported an innate upregulation of Crhr1 expression in the amygdala of msP rats that causes a behavioral phenotype similar to that seen in postdependent rats. This work also demonstrated that elevated Crhr1 transcript within the amygdala is strongly correlated with increased binding density (
      • Hansson A.C.
      • Cippitelli A.
      • Sommer W.H.
      • Fedeli A.
      • Bjork K.
      • Soverchia L.
      • et al.
      Variation at the rat Crhr1 locus and sensitivity to relapse into alcohol seeking induced by environmental stress.
      ). Here, we found that the postdependent phenotype is accompanied by a very similar upregulation of Crhr1 receptors within the BLA and MeA. Elimination of fear suppression in the conflict test by the CRH-R1 antagonist MTIP strongly suggests that, similar to what we found in the msP line, the upregulated Crhr1 expression in the postdependent state is causally related to the behavioral phenotype. This conclusion is further supported by our recent findings, in a separate study, that MTIP fully and dose-dependently blocks the increase in alcohol intake found in the postdependent state (Gehlert et al., 2007). These data in are in agreement with previous studies, which have demonstrated that both stress responses and elevated voluntary alcohol consumption in the postdependent state are blocked by CRH antagonism or specifically blockade of CRH-R1 receptors (
      • Breese G.R.
      • Overstreet D.H.
      • Knapp D.J.
      • Navarro M.
      Prior multiple ethanol withdrawals enhance stress-induced anxiety-like behavior: Inhibition by CRF1- and benzodiazepine-receptor antagonists and a 5-HT1a-receptor agonist.
      ;
      • Valdez G.R.
      • Roberts A.J.
      • Chan K.
      • Davis H.
      • Brennan M.
      • Zorrilla E.P.
      • et al.
      Increased ethanol self-administration and anxiety-like behavior during acute ethanol withdrawal and protracted abstinence: Regulation by corticotropin-releasing factor.
      ,
      • Valdez G.R.
      • Zorrilla E.P.
      • Roberts A.J.
      • Koob G.F.
      Antagonism of corticotropin-releasing factor attenuates the enhanced responsiveness to stress observed during protracted ethanol abstinence.
      ). Our findings are also in line with a recent study that showed a recruitment of local amygdala CRH circuits but not those in BNST or NAcc following a history of dependence (
      • Funk C.K.
      • O’Dell L.E.
      • Crawford E.F.
      • Koob G.F.
      Corticotropin-releasing factor within the central nucleus of the amygdala mediates enhanced ethanol self-administration in withdrawn, ethanol-dependent rats.
      ). Collectively, these findings suggest that a long-lasting upregulation of CRH-R1 receptors within the amygdala constitutes a shared neurobiological substrate underlying the behavioral phenotype of msP and postdependent rats. It remains to be determined to what extent CRH-R1 receptors in CeA, MeA, or BLA are involved.
      In the postdependent subjects, the upregulated Crhr1 expression was accompanied by an increase of Crh mRNA in the CeA. This finding confirms and expands on a prior report of increased amygdala CRH immunoreactivity in postdependent rats 6 weeks after termination of a liquid alcohol diet (
      • Zorrilla E.P.
      • Valdez G.R.
      • Weiss F.
      Changes in levels of regional CRF-like-immunoreactivity and plasma corticosterone during protracted drug withdrawal in dependent rats.
      ). Elevated tissue peptide content can result either from increased synthesis or decreased release. The observation of increased peptide levels in the previous report, together with elevated transcript in the present study, jointly establish that CRH synthesis is increased in CeA in the postdependent state. Thus, a presynaptic and a postsynaptic signaling component may act in concert to recruit the CRH system in postdependent animals. Finally, the Crhr2 transcript, although less affected, was oppositely regulated to Crhr1 within the BLA, in which a decrease was seen. This is in line with prior data showing that activation of CRH-R2 receptors is also capable of reversing the increased sensitivity to stress and elevated alcohol self-administration during the postdependent state (
      • Valdez G.R.
      • Sabino V.
      • Koob G.F.
      Increased anxiety-like behavior and ethanol self-administration in dependent rats: Reversal via corticotropin-releasing factor-2 receptor activation.
      ).
      Our expression analysis suggests that the dysregulation of CRH systems found during the postdependent state is relatively restricted to extrahypothalamic systems, while the PVN and control of the hypothalamic-pituitary-adrenal (HPA) axis seem unaffected. A limitation of our study is that we were not able to analyze pituitary expression of CRH receptor transcripts. However, our findings are in agreement with prior reports that stable, long-term changes in basal serum corticosterone levels are not found in postdependent rats (
      • Rimondini R.
      • Arlinde C.
      • Sommer W.
      • Heilig M.
      Long-lasting increase in voluntary ethanol consumption and transcriptional regulation in the rat brain after intermittent exposure to alcohol.
      ;
      • Zorrilla E.P.
      • Valdez G.R.
      • Weiss F.
      Changes in levels of regional CRF-like-immunoreactivity and plasma corticosterone during protracted drug withdrawal in dependent rats.
      ). The precise mechanism by which a parallel increase in CRH and CRH-R1 expression occurs within the amygdala in the postdependent state is unclear. The increased vulnerability of this structure may result from its unique organization, characterized by close proximity or intertwining of CRH synthesis and target sites (
      • Swanson L.W.
      • Petrovich G.D.
      What is the amygdala?.
      ). Also, CRH in CeA mimics actions of alcohol to potentiate local gamma-aminobutyric acid (GABA) transmission through a CRH-R1 mediated mechanism, possibly mediating an autoregulatory loop through recurrent collaterals (
      • Nie Z.
      • Schweitzer P.
      • Roberts A.J.
      • Madamba S.G.
      • Moore S.D.
      • Siggins G.R.
      Ethanol augments GABAergic transmission in the central amygdala via CRF1 receptors.
      ), while GABA responses to alcohol in CeA are upregulated in the postdependent state (
      • Nie Z.
      • Schweitzer P.
      • Roberts A.J.
      • Madamba S.G.
      • Moore S.D.
      • Siggins G.R.
      Ethanol augments GABAergic transmission in the central amygdala via CRF1 receptors.
      ;
      • Roberto M.
      • Madamba S.G.
      • Stouffer D.G.
      • Parsons L.H.
      • Siggins G.R.
      Increased GABA release in the central amygdala of ethanol-dependent rats.
      ). Intermittent exposure to alcohol intoxication and withdrawal may be particularly effective in driving an allostatic shift of the amygdala CRH system to a higher functional set point (
      • Valdez G.R.
      • Koob G.F.
      Allostasis and dysregulation of corticotropin-releasing factor and neuropeptide Y systems: Implications for the development of alcoholism.
      ), since Crhr1 gene expression regulation is expected to be on a slower scale than Crh itself. Ultimately, mechanisms regulating transcription must be recruited. An intriguing possibility is that these might be similar to those suggested in the phenomenon recently labeled “incubation of craving,” which refers to increased propensity for relapse-like behavior over time and which has been shown to rely on signaling through the extracellular signal-regulated kinase (ERK)-pathway, known to be capable of regulating transcription (
      • Grimm J.W.
      • Hope B.T.
      • Wise R.A.
      • Shaham Y.
      Neuroadaptation Incubation of cocaine craving after withdrawal.
      ;
      • Lu L.
      • Hope B.T.
      • Dempsey J.
      • Liu S.Y.
      • Bossert J.M.
      • Shaham Y.
      Central amygdala ERK signaling pathway is critical to incubation of cocaine craving.
      ).
      In summary, a recruitment of intra-amygdala but not hypothalamic CRH systems seems to be driving the postdependent phenotype. An increased stress sensitivity in this state may not be overt but is pronounced following a stress challenge. Postdependent upregulation of the CRH system gives rise to excessive rates of alcohol self-administration. Together, these data provide compelling evidence that a blockade of hyperactive signaling at CRH-R1 receptors in the postdependent state inhibit heavy drinking and reduced relapse risk.
      This research was supported by intramural National Institute on Alcohol Abuse and Alcoholism (NIAAA) funding, funding from the Swedish Medical Research Council, and the Karolinska Institute. This research has, in part, been carried out under a standard U.S. Government collaborative research and development agreement (CRADA) between the NIAAA and Eli Lilly Research Laboratories. Authors so listed are employees of Eli Lilly and Co. Remaining authors have no competing financial interest.

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