Corticotropin-Releasing Factor 1 Antagonists Selectively Reduce Ethanol Self-Administration in Ethanol-Dependent Rats

Methods and Materials

Results

Discussion

Excessive, uncontrolled drinking and the presence of a withdrawal syndrome following cessation of alcohol intake are two of the diagnostic criteria for dependence in humans (American Psychiatric Association 1994). Further, human alcoholics will report that the negative affect state, especially enhanced anxiety and stress, experienced during withdrawal is the main factor eliciting relapse and binge drinking during periods of abstinence (Hershon 1977). Some of these negative affect symptoms can persist months after chronic ethanol cessation (Begleiter and Porjesz 1979). Animal model systems for ethanol dependence have been developed which mimic excessive drinking and withdrawal (O’Dell et al 2004; Roberts et al 2000). Here, we have induced ethanol dependence in male Wistar rats by exposing the animals to intermittent ethanol vapors for 4 weeks. We report that three nonpeptide, small molecule CRF₁ antagonists, antalarmin, MJL-1-109-2, and R121919, significantly and selectively reduce ethanol self-administration in ethanol-dependent rats. These data indicate that the CRF₁ receptor subtype is important in mediating excessive ethanol intake in ethanol-dependent animals.

One potential issue with this study may be the specificity of the antagonists for CRF₁ receptors. Importantly, the portion of the CRF₁ receptor to which these antagonists bind is not contained in the CRF₂ or CRF-binding peptide (Zorrilla and Koob 2004). Further, these nonpeptide antagonists have all been used in multiple studies demonstrating their specificity and effectiveness. Antalarmin has a K_i value of 1.0 nm and antagonizes both central and peripheral actions of CRF in vivo at a dose of 20 mg/kg, similar to the dose we found effective here (Webster et al 1996). In rats, antalarmin, at doses of 10 or 20 mg/kg, has also been shown to inhibit fear conditioning (Deak et al 1999), inhibit CRF- and novelty-induced anxiety-like behaviors (Zorrilla et al 2002), inhibit the locomotor activating effects of CRF (Zorrilla et al 2002), and inhibit naloxone-precipitated place aversion in morphine-dependent animals (Stinus et al 2005). Importantly, at the doses used in this study, antalarmin does not have nonspecific effects, as it does at higher doses (Zorrilla et al 2002). Here, we show that antalarmin dose-dependently and significantly reduced ethanol self-administration in dependent animals. Antalarmin had no effect in nondependent animals.

The CRF₁ receptor antagonist R121919 also reduces anxiety-like behavior in animal models. In vitro studies have found that R121919 is highly selective for the CRF₁ receptor (Heinrichs et al 2002) and has no reported pharmacological interactions (Zorrilla and Koob 2004). In rats, R121919, at doses similar to those used in this study (20–30 mg/kg), inhibits stress-induced plasma adrenocorticotropic hormone levels, inhibits anxiogenic-like behaviors, and inhibits CRF-induced locomotor activity (Chen et al 2004; Heinrichs et al 2002; Keck et al 2001). The CRF₁ receptor antagonist R121919 has also been shown to be effective in reducing anxious and depressed symptoms in an open-label human clinical trial for treatment of depression (Kunzel et al 2003; Zobel et al 2000). Similar to antalarmin, we show here that R121919 dose-dependently and significantly reduced ethanol self-administration in dependent animals. Our data indicate a plateau in the level of reduction of ethanol responding in dependent animals, which may result from receptor occupancy of CRF₁ receptors by R121919. Heinrichs et al (2002) have previously reported receptor occupancy levels for R121919, demonstrating nearly 50% occupancy at 2.5 mg/kg and 100% occupancy at 20 mg/kg. Thus, R121919 may be occupying the majority of CRF₁ receptors at 10 mg/kg, producing a plateau effect at the highest dose (20 mg/kg). Alternatively, other receptors may also be involved in mediating the enhanced intake of ethanol in dependent animals, such as the CRF₂ receptor (discussed below). R121919 had no effect in nondependent rats.

The CRF₁ receptor antagonist MJL-1-109-2 has been the least studied drug of the three used here but is an analog of the well-studied compound DMP696 (He et al 2000; Jagoda et al 2003). However, MJL-1-109-2, similar to antalarmin and R121919, also is able to penetrate the blood brain barrier and has a K_i value of 1.9 nm (Jagoda et al 2003). Here, MJL-1-109-2 significantly attenuated ethanol self-administration in dependent animals. MJL-1-109-2 was without effect in nondependent animals. Furthermore, studies in our group have shown that MJL-1-109-2 does not reduce food intake at the doses used here (Sabino et al, unpublished data, 2005), further suggesting a behaviorally selective action of MJL-1-109-2 on excessive ethanol drinking and not consummatory behavior in general. The use of three CRF₁ specific antagonists strengthens our conclusion that the CRF₁ receptor subtype is important for mediating excessive drinking in ethanol-dependent animals.

CRF mediates the neuroendocrine (Rivier et al 1982; Vale et al 1981), autonomic (Vale et al 1983), and behavioral responses to stress and anxiety (Britton et al 1982; Koob and Heinrichs 1999; Sutton et al 1982). During the development of ethanol dependence, both hypothalamic as well as extrahypothalamic CRF systems become dysregulated. In rats, acute ethanol activates the HPA axis (Rivier et al 1984), while chronic exposure to ethanol, as well as ethanol withdrawal, leads to an attenuation of HPA axis activity (Dave et al 1986; Lee et al 2000; Rivier et al 1990; Zorrilla et al 2001). In contrast, extrahypothalamic CRF systems become hyperactive during withdrawal from chronic ethanol exposure. There is an increase in extracellular CRF release in both the central nucleus of the amygdala (CeA) (Merlo Pich et al 1995) and bed nucleus of the stria terminalis (BNST) (Olive et al 2002). A decrease in CRF-like immunoreactivity in the amygdala during acute withdrawal has also been reported, which likely reflects an increase in CRF release in these brain regions (Zorrilla et al 2001). Ethanol-dependent animals also display enhanced sensitivity to an intracerebroventricular (ICV) infusion of CRF, as measured by electrophysiology (Slawecki et al 1999) and locomotor activity (Ehlers and Chaplin 1987). The authors suggest that this increased sensitivity to CRF may be the result of an increase in CRF receptor expression. Thus, while CRF activity within the HPA axis appears to become blunted with ethanol dependence, extrahypothalmic CRF systems become hyperactive.

It was hypothesized that this dysregulation of brain CRF “stress” systems underlies both the enhanced anxiety-like behaviors as well as contributes to the enhanced ethanol self-administration associated with withdrawal. Ethanol-dependent rodents display enhanced anxiety-like behaviors during acute and protracted withdrawal as measured by the elevated plus maze test (Baldwin et al 1991; Valdez et al 2002b), enhanced acoustic startle (Macey et al 1996), social interaction test (Overstreet et al 2002), and light/dark box (Kliethermes et al 2004). In rats, the subtype nonselective, peptide CRF receptor antagonists α-helical CRF_9–41 and D-Phe-CRF_12–41 (intracerebroventricular administration) reduce ethanol withdrawal-induced anxiety-like behavior (Baldwin et al 1991; Valdez et al 2002b). However, α-helical CRF_9–41 was found to be ineffective in reducing other physical symptoms of withdrawal, such as body tremor and tail stiffness (Baldwin et al 1991). This same antagonist, α-helical CRF_9–41, also decreased ethanol withdrawal-induced anxiety-like behavior when injected directly into the CeA (Rassnick et al 1993). Using a model involving multiple ethanol withdrawals, Overstreet et al (2004) have also shown an enhancement of anxiety-like behaviors in ethanol-dependent rats, an effect reversed by systemic administration of CRF₁ specific antagonists. Thus, CRF₁ receptors appear to mediate the enhanced anxiety associated with withdrawal.

Ethanol-dependent animals also display enhanced ethanol self-administration during acute and protracted periods of withdrawal (O’Dell et al 2004; Roberts et al 2000; Valdez et al 2002b). We suggest that dependent animals self-administer excessive amounts of ethanol during withdrawal to alleviate the CRF-mediated increase in negative affect, such as enhanced anxiety or hyperarousal. Indeed, data from a study by Olive et al (2002) suggest that animals will self-administer ethanol during periods of withdrawal to attenuate the increased CRF release in the extended amygdala. Further, the enhanced ethanol self-administration during withdrawal can be reversed by intracerebroventricular administration of subtype nonselective, peptide CRF receptor antagonists (Valdez et al 2002b) and, as we show here, systemic administration of small molecule CRF₁ antagonists. The subtype nonselective, peptide CRF receptor antagonist D-Phe-CRF_12–41 reduces ethanol self-administration during acute withdrawal (2 hours) as well as protracted withdrawal (3 to 5 weeks) in dependent rats (Valdez et al 2002b). However, the antagonist had no effect in nondependent animals. Here, we have found that three CRF₁ specific antagonists reduce ethanol self-administration in dependent rats during acute withdrawal, suggesting that the ICV effects of D-Phe-CRF_12–41 (Valdez et al 2002b, 2003b) are likely mediated by a CRF₁ receptor mechanism. It will be important for future studies to also address the effectiveness of these CRF₁ receptor antagonists in reducing ethanol self-administration during periods of protracted abstinence as well.

The data presented in this study support a role for CRF₁ receptors in mediating excessive ethanol drinking during withdrawal in dependent animals. CRF₁ receptors are located throughout the brain, primarily in the pituitary gland, cerebral cortex, cerebellum, hippocampus, hindbrain, and olfactory bulb (Van Pett et al 2000). CRF₁ receptors are also expressed in areas of the extended amygdala, such as the BNST and central and basolateral nuclei of the amygdala (Chen et al 2000; Van Pett et al 2000), regions which also have increased CRF release during ethanol withdrawal (Merlo Pich et al 1995; Olive et al 2002). Several lines of evidence indicate that the CRF₁ receptor plays an important role in mediating anxiety-like behavior. CRF₁ receptor deficient mice display reduced basal (Muller et al 2003; Smith et al 1998; Timpl et al 1998) and ethanol withdrawal-induced anxiety-like behavior (Timpl et al 1998). CRF₁ antisense oligodeoxynucleotides reduce anxiety-like behavior in rats when injected intracerebroventricularly (Heinrichs et al 1997; Liebsch et al 1999) or directly into the CeA (Liebsch et al 1995). Further, multiple studies have found that selective CRF₁ receptor antagonists inhibit anxiety-like behaviors in animals (Zorrilla and Koob 2004). Although not directly addressed in this study, the important role of CRF₁ receptors in mediating anxiety-like behaviors provides strong support for the hypothesis that CRF₁ antagonists reduce excessive drinking during withdrawal by reducing withdrawal-associated anxiety. In addition, studies from our lab have shown that ethanol-dependent rats display heightened anxiety-like behaviors following a 2-hour withdrawal period, which were reversed by IVC administration of subtype nonselective CRF receptor antagonists (Valdez et al 2002b), further suggesting that CRF₁ receptors reduce ethanol intake by reducing heightened anxiety-like behaviors.

The specific brain regions and pathways involved in mediating CRF₁ antagonist-induced decreases in ethanol self-administration in dependent animals remain unknown. Several studies suggest that regions of the extended amygdala may be involved in mediating CRF-induced anxiety (Merlo Pich et al 1995; Muller et al 2003; Olive et al 2002; Rassnick et al 1993), and the same regions may mediate excessive ethanol consumption during withdrawal as well. Specifically, the CeA, a region mediating anxiogenic-like behaviors (Liang et al 1992; Walker and Davis 1997), is involved in mediating both acute effects of ethanol as well as the anxiogenic effects of chronic ethanol exposure (Pandey et al 2003; Rassnick et al 1993; Roberto et al 2004; Yoshimoto et al 2000). Thus, this brain region may be involved in mediating the increased ethanol self-administration during withdrawal. The basolateral nucleus of the amygdala may also play a role, as CRF released from the CeA can activate basolateral CRF₁ neurons by volume transmission (Roozendaal et al 2002). Previous studies using in vivo and in vitro autoradiography have demonstrated that the CRF₁ antagonists used here bind to CRF₁ receptors within these amygdala structures (Heinrichs et al 2002; Jagoda et al 2003). Future studies will be important to determine which brains regions, primarily within the extended amygdala, are important in mediating excessive drinking during withdrawal.

None of the CRF₁ receptor antagonists altered ethanol self-administration in nondependent animals. However, one study (Lodge and Lawrence 2003a) has previously shown that antalarmin reduces volitional ethanol consumption in isolation-reared, Fawn-Hooded rats. Isolation rearing produces an anxious, high-alcohol preferring phenotype, similar to other alcohol-preferring strains of rats (Lodge and Lawrence 2003b). These alcohol-preferring animals have been shown to have dysregulated CRF systems (Ehlers et al 1992; Hwang et al 2004), dopaminergic systems (Lodge and Lawrence 2003b), and serotonin systems (Hensler et al 2004). Thus, alcohol-preferring animals, such as those used in the Lodge and Lawrence (2003a) study, likely do not represent a true ethanol-nondependent phenotype. These alcohol-preferring strains more closely resemble ethanol-dependent animals than nondependent animals in our model system.

The present study suggests an important role for CRF₁ receptors in mediating ethanol self-administration in dependent animals. These data do not rule out a potential role for CRF₂ receptors in modulating this response as well. Urocortin 3, a specific CRF₂ agonist, injected intracerebroventricularly, can reduce enhanced ethanol self-administration and anxiety-like behaviors associated with acute ethanol withdrawal in ethanol-dependent rats (Valdez et al 2003a). In rats, CRF₂ agonists produce anxiolytic-like behaviors (Valdez et al 2002a, 2003a), and CRF₂ deficient mice display enhanced anxiety-like behavior (Bale et al 2000; Kishimoto et al 2000). These data suggest that CRF₁ and CRF₂ receptors may have opposing roles in controlling ethanol withdrawal-induced anxiety and ethanol self-administration, consistent with the opposing electrophysiological actions of CRF₁ and CRF₂ ligands on glutamatergic transmission (Liu et al 2004).

We have studied the acute effects of CRF₁ antagonists on ethanol self-administration in ethanol-dependent versus nondependent animals. However, the chronic effects of these antagonists will also have important clinical implications. For example, some studies have shown alterations in the dopaminergic system following chronic exposure to antalarmin (Lawrence et al 2005). Several other studies have reported no dysregulation of the HPA axis following chronic administration of CRF₁ antagonists (Oshima et al 2003; Wong et al 1999). In future studies, we will more thoroughly examine different durations of dosing and dosing patterns of the antagonists used here.

These data have important clinical implications for the treatment of alcohol dependence in humans. Dysregulation of CRF stress systems may represent a long-lasting change within the brain resulting from chronic ethanol consumption. Upon cessation of ethanol use, stress systems become overactive, likely causing negative affect states, such as heightened anxiety and depression. Novel pharmacotherapies aimed at treating alcohol-induced anxiety and depression may prove to be successful in treating relapse and excessive drinking during withdrawal. Thus, the development of these CRF₁ antagonists, antalarmin, MJL-1-109-2, and R121919, as a novel class of pharmacotherapy may be promising for the treatment of alcoholism.