Modulation of multiple ethanol withdrawal-induced anxiety-like behavior by CRF and CRF1 receptors

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Abstract

Previous work demonstrated that rats subjected to multiple withdrawals from chronic ethanol exhibit a sensitization of anxiety-like behavior compared to animals withdrawn from treatment with an equal but continuous amount of ethanol. This study sought to examine whether corticotropin-releasing factor (CRF) could modulate this ethanol-withdrawal-induced anxiety-like behavior. Initially, rats were administered with CRF (1 μg) or vehicle intraventricularly on two occasions 5 days apart while on control diet (CD) followed by exposure to 7% ethanol diet (ED) for 5 days, with social interaction assessed 5 h into withdrawal. Social interaction was significantly reduced in the CRF-treated animals compared to vehicle-treated rats and vehicle- and CRF-treated rats maintained on CD, indicative that CRF given before ethanol exposure was capable of inducing an adaptive change that sensitized withdrawal-induced anxiety-like behavior. Next, the CRF1 receptor antagonist CRA1000 (3 mg/kg, systemically), the CRF2 receptor antagonist antisauvagine-30 (20 μg intraventricularly), or vehicle was injected 4 h after the ethanol was removed following the first and second cycles of chronic ethanol exposure and the effect on the multiple-withdrawal-induced anxiety-like behavior determined after the third withdrawal cycle. The CRF1 receptor antagonist blocked the reduced social interaction behavior, whereas the CRF2 receptor antagonist was without effect. Similar pretreatment with another CRF1 receptor antagonist CP-154,526 (10 mg/kg systemically) during the first and second withdrawals also counteracted anxiety-like behavior. These findings indicate that the CRF system and CRF1 receptors play key roles in the adaptive change responsible for the anxiety-like behavior induced by repeated withdrawals from chronic ethanol.

Introduction

Repeated ethanol exposures and withdrawals induce long-lasting adaptive changes in the brain that are reflected by behavioral consequences (e.g., Holter et al., 1998, Malcolm et al., 2000, McCown and Breese, 1990). In this respect, a recent investigation showed that anxiety-like behavior, as indexed by the social interaction test, increased in rats repeatedly withdrawn from exposure to ethanol (Overstreet et al., 2002). Rats exposed continuously for 15 days to a diet containing 4.5% ethanol exhibited a normal level of social interaction upon withdrawal, indicative that the ethanol alone was not responsible for the sensitization of the anxiety-like behavior associated with the repeated withdrawals (Overstreet et al., 2002).

Several investigators have reported alterations in the hypothalamo–pituitary–adrenal (HPA) axis after chronic ethanol treatment (e.g., Rasmussen et al., 2000, Rivier and Lee, 2001). Antagonists of corticotropin-releasing factor (CRF) have been reported to reduce anxiety-like behavior observed in ethanol-withdrawn rats Koob et al., 1998, Rassnick et al., 1993 and attenuate foot shock-induced reinstatement of ethanol-seeking behavior (Le et al., 2000). While CRF, by driving the HPA axis, could be a key factor in the adaptive changes associated with chronic ethanol, a recent study demonstrated that adrenalectomy does not modulate foot shock-induced reinstatement of ethanol-seeking behavior (Le et al., 2000). Based upon this background, it is hypothesized that CRF contributes to the sensitized anxiety-like behavior observed in rats repeatedly withdrawn from chronic ethanol diet (ED).

To examine the role of CRF in the multiple-withdrawal-induced sensitization, it was tested whether central administration of CRF would substitute for the initial two withdrawals at 6 and 11 days of the multiple withdrawal protocol to induce anxiety-like behavior. Subsequently, it was determined if selective antagonists for CRF1 and CRF2 receptors would prevent the anxiety-like behavior seen with repeated withdrawals. These studies will support the proposed hypothesis that CRF acting on CRF1 receptors contributes to the anxiety-like behavior observed during repeated ethanol withdrawals.

Section snippets

Animals

Male Sprague–Dawley rats (Charles-River, Raleigh) were purchased at 40 days of age (160–180 g). After giving 5 days to adapt to local conditions (22 °C, 50% humidity, 12:12-h light–dark cycle with lights on between 0900 and 2100 h), they were placed on a nutritionally complete diet used previously in our laboratory (e.g., Frye et al., 1983, Moy et al., 2000, Overstreet et al., 2002). Intakes of the liquid diet were recorded daily, and body weights were measured weekly. These experiments were

Effect of intraventricular CRF administration on withdrawal-induced anxiety-like behavior

Rats that were initially treated with a single dose of CRF intraventricularly exhibited lower social interaction behavior than the rats given artificial cerebrospinal fluid, confirming the anxiogenic properties of CRF [Fig. 1; F(3,45)=13.32, P<.001]. Interestingly, the control rats that were used as partners for the CRF- and vehicle-treated rats (PART-C and PART-V, respectively) spent as much time in social interaction as the vehicle-treated rats, although their partners differed greatly in the

Discussion

Previous studies have demonstrated that restraint stress applied at weekly intervals prior to 5 days of 4.5% ED resulted in sensitization of a withdrawal-induced reduction in social interaction behavior (Breese et al., 2003). Inasmuch as the present findings confirm that a single withdrawal from 7% ED does not induce anxiety-like behavior (Overstreet et al., 2002), we were able to examine whether CRF would substitute for multiple stresses to sensitize anxiety.

It was initially demonstrated that

Acknowledgments

This research was supported by funding from NIAAA (2 P60 AA-011605-06, AA-014073, AA-014284). We wish to thank Taisho Pharmaceutical (Saitama, Japan) for the generous supply of CRA1000 and Pfizer (Groton, CN) for CP-154,526. We thank Dhritiman Muckerjee, Qi Yu, and Mili Senapati for expert technical assistance.

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