Corticotropin-releasing factor within the central nucleus of the amygdala mediates enhanced ethanol self-administration in withdrawn, ethanol-dependent rats

Abstract

Alcohol dependence is characterized by excessive consumption, loss of control over intake, and the presence of a withdrawal syndrome, including both motivational and physical symptoms. The motivational symptoms, including anxiety, have been hypothesized to be important factors eliciting excessive drinking during abstinence. Previous work has shown that ethanol-dependent rats also display enhanced anxiety-like behaviors and enhanced ethanol self-administration during withdrawal, likely resulting from dysregulation of brain corticotropin-releasing factor (CRF) stress systems. The present study was designed to explore the brain sites within the extended amygdala [central nucleus of the amygdala (CeA), lateral bed nucleus of the stria terminalis (BNST), and nucleus accumbens shell (NAcSh)] that mediate the increased ethanol self-administration observed during withdrawal. Ethanol-dependent animals showed an increase in ethanol self-administration after acute withdrawal relative to nondependent controls. The CRF antagonist D-Phe-CRF(12-41) ([D-Phe(12),Nle(21,38),C alpha MeLeu(37)]-rCRF(12-41)) was administered into the CeA, lateral BNST, or NAcSh of acute-withdrawn dependent and nondependent rats. Administered into the CeA, the antagonist reduced ethanol self-administration in dependent animals, with no effect in nondependent animals. Administration of D-Phe-CRF(12-41) into the lateral BNST and NAcSh was without effect on ethanol self-administration in dependent and nondependent animals. At the same time point of withdrawal, there was a decrease in CRF immunoreactivity within the CeA, suggesting an increased extracellular release of CRF during withdrawal. There was no change in CRF immunoreactivity in the BNST or NAcSh. These results indicate that CRF, specifically within the CeA, plays a role in mediating excessive ethanol consumption in ethanol-dependent animals.

Figure 1.

Effects of d-Phe-CRF_12–41 administered intra-CeA on ethanol and water self-administration in ethanol-dependent and nondependent rats. Ethanol dependence was induced by intermittent exposure to ethanol vapors for 4 weeks, and animals were subsequently tested for ethanol and water self-administration after 2 h of acute withdrawal. Withdrawn, ethanol-dependent animals displayed a significant increase in ethanol lever pressing compared with nondependent animals. d-Phe-CRF_12–41 significantly decreased ethanol self-administration in withdrawn, dependent but not nondependent animals when administered directly into the central nucleus of the amygdala. Neither ethanol vapor exposure nor d-Phe-CRF_12–41 alters water responding. *p < 0.0001 compared with same drug dose in nondependent animals. ^#p < 0.0001 compared with vehicle treatment in dependent animals. Error bars indicate SEM.

Figure 2.

Effects of d-Phe-CRF_12–41 administered intra-lateral BNST on ethanol and water self-administration in ethanol-dependent and nondependent rats. Ethanol dependence was induced by intermittent exposure to ethanol vapors for 4 weeks, and animals were subsequently tested for ethanol and water self-administration after 2 h of acute withdrawal. Withdrawn, ethanol-dependent animals displayed a significant increase in ethanol lever pressing compared with nondependent animals. d-Phe-CRF_12–41 was without effect in dependent and nondependent animals (on ethanol and water responding) when administered directly into the lateral bed nucleus of the stria terminalis. *p < 0.0001 compared with same drug dose in nondependent animals. Error bars indicate SEM.

Figure 3.

Effects of d-Phe-CRF_12–41 administered intra-NAcSh on ethanol and water self-administration in ethanol-dependent and nondependent rats. Ethanol dependence was induced by intermittent exposure to ethanol vapors for 4 weeks, and animals were subsequently tested for ethanol and water self-administration after 2 h of acute withdrawal. Withdrawn, ethanol-dependent animals displayed a significant increase in ethanol lever pressing compared with nondependent animals. d-Phe-CRF_12–41 was without effect in dependent and nondependent animals (on ethanol and water responding) when administered directly into the shell of the nucleus accumbens. *p < 0.0001 compared with same drug dose in nondependent animals. Error bars indicate SEM.

Figure 4.

Representative cannula site verification micrographs. At the completion of the experiments, brains were sectioned at 60 μm intervals, mounted, and stained with cresyl violet. Injection sites were verified under a light microscope. Only animals with correct, bilateral cannula placement were used for statistical analysis. Figures display representative slides from the CeA, lateral BNST, and NAcSh. Numbers in diagrams represent distance from bregma, based on the rat brain atlas by Paxinos and Watson (1998).

Figure 5.

CRF immunoreactivity in the CeA. Ethanol dependence was induced by intermittent exposure to ethanol vapors for 4 weeks, and animals were killed after 2 h of withdrawal. Brains were removed and CRF expression was measured in the central nucleus of the amygdala using immunohistochemical techniques. The data are expressed as the relative density of CRF immunoreactivity (n = 3 animals). A, CRF immunoreactivity was measured from five serial sections throughout the CeA. From bregma, these sections were −2.12, −2.30, −2.56, −2.80, and −3.14 mm. There was a significant decrease in CRF immunoreactivity in the CeA at bregma −2.80 and −3.14 mm in withdrawn, dependent animals relative to control. B, Representative micrographs (20×) from the CeA (bregma −2.80 mm) demonstrating CRF staining within this brain region. *p < 0.01 compared with nondependent animals. Error bars indicate SEM.

Figure 6.

CRF immunoreactivity in the lateral BNST. Ethanol dependence was induced by intermittent exposure to ethanol vapors for 4 weeks, and animals were killed after 2 h of withdrawal. Brains were removed and CRF expression was measured in the lateral BNST using immunohistochemical techniques. The data are expressed as the relative density of CRF immunoreactivity (n = 3 animals). A, CRF immunoreactivity was measured from five serial sections throughout the lateral BNST. From bregma, these sections were −0.20, −0.26, −0.30, −0.40, and −0.80 mm. Although there was a trend toward a decrease in dependent animals, there was no significant change in CRF immunoreactivity in the BNST in dependent animals compared with control. B, Representative micrographs (20×) from the lateral BNST (bregma −0.30 mm) demonstrating CRF staining within this brain region. Error bars indicate SEM.