Roles of the locus coeruleus and adrenergic receptors in brain-mediated hypothalamic-pituitary-adrenal axis responses to intracerebroventricular alcohol

Abstract

Background: Alcohol activates the hypothalamic-pituitary-adrenal (HPA) axis through its actions in both the periphery and the central nervous system (CNS). The studies presented here were designed to test the CNS-specific noradrenergic mechanisms by which alcohol stimulates HPA activity in the male rat.

Methods: We used an experimental paradigm in which a small, nontoxic amount (5 μl) of alcohol was slowly microinfused intracerebroventricularly (icv). Alcohol was administered icv to animals with lesions of the locus coeruleus (LC) or in animals pretreated with α- or β-adrenergic receptor antagonists. Hormonal HPA activation was determined by measuring secretion of the pituitary stress hormone adrenocorticotropin (ACTH). Neuronal activation was determined by quantification of the expression of the transcription factor c-fos (Fos).

Results: As expected, icv alcohol stimulated ACTH secretion from the pituitary and Fos expression in the paraventricular nucleus of the hypothalamus (PVN). Bilateral electrolytic LC lesions blocked the ability of icv alcohol to stimulate ACTH secretion. Pretreatment with icv propranolol increased basal ACTH secretion levels, but icv alcohol did not increase this effect. Propranolol also blunted icv alcohol-induced PVN Fos expression. A low dose of phenoxybenzamine, an α-adrenergic receptor antagonist, did not affect the ability of icv alcohol to stimulate ACTH release. However, a higher dose of the drug was able to block the ACTH response to icv alcohol. Despite this, phenoxybenzamine did not inhibit alcohol-induced Fos expression. Icv pretreatment with corynanthine, a selective α-1 adrenergic receptor antagonist, modestly raised basal ACTH levels and blocked the icv alcohol-induced secretion of this hormone.

Conclusions: These results indicate that the LC and norepinephrine play important roles in HPA activation caused by icv alcohol administration, but that the specific adrenergic receptor subtypes involved in this phenomenon still need to be identified.

Figure 1

A) Lesions of the brainstem area containing the locus coeruleus (LCx) blocked the ability of icv alcohol (EtOH, 5 μL of 100%) to stimulate ACTH secretion when compared to sham animal. B) Pictures of a representative LC lesion and the LC of a control animal. Bar height represents the mean ± SEM. **, P < 0.01 versus LCx/alcohol; N = 8–10 animals per treatment group.

Figure 2

A) The icv administration of alcohol (EtOH, 5 μL of 100%) significantly increased ACTH secretion compared to its vehicle. Pretreatment with a low dose of iv phenoxybenzamine (PBZ, 1 mg/kg in 0.2ml saline) did not affect the ability of icv alcohol to increase ACTH secretion. B) Pretreatment with a high dose of phenoxybenzamine (10 mg/kg in 0.2ml saline) completely inhibited the ability of icv alcohol to induce ACTH secretion. Bar height represents the mean ± SEM. ***, P < 0.001 versus vehicle/vehicle, phenoxybenzamine/vehicle and phenoxybenzamine/alcohol; N = 6–8 animals.

Figure 3

A) The icv administration of alcohol (EtOH, 5 μL of 100%) significantly increased ACTH secretion compared to its vehicle. Pretreatment with icv corynanthine (150 μg in 5 μL of saline) elevated basal ACTH levels, but icv alcohol did not add to this effect. Bar height represents the mean ± SEM. *, P < 0.05 versus vehicle/vehicle; **, P < 0.01 versus vehicle/vehicle; a, P < 0.05 versus corynanthine/alcohol, N = 6–8 animals.

Figure 4

A) The icv administration of alcohol (EtOH, 5μL of 100%) significantly increased ACTH secretion compared to its vehicle. Pretreatment with icv propranolol (5 μg in 5 μL saline) elevated basal ACTH levels, but icv alcohol did not add to this effect. B) Cumulative ACTH as a percent of the basal value showed that propranolol significantly inhibited alcohol-induced ACTH secretion as compared to basal levels. Cumulative values were derived by adding icv alcohol-stimulated ACTH levels at 15, 30, and 60 minutes after icv injection. Bar height represents the mean ± SEM. **, P < 0.01 versus vehicle/vehicle; ***, P < 0.001 versus cumulative propranolol/vehicle and propranolol/alcohol; N = 5–8 animals.

Figure 5

The icv administration alcohol (EtOH, 5 μL of 100%) significantly increased Fos expression in the paraventricular nucleus of the hypothalamus (PVN) compared to vehicle controls. A) The pretreatment icv propranolol (5μg in 5μl saline) elevated Fos expression in the PVN. Alcohol given icv did not add to this effect. B) Pretreatment with iv phenoxybenzamine (10 mg/kg in 0.2 ml saline) elevated Fos expression in the PVN, but did NOT affect the ability of icv alcohol to stimulate Fos expression in the PVN. C) Representative 10× pictures of Fos expression in the PVN in all treatment groups. Bar height represents the mean ± SEM. *, P < 0.05 versus vehicle/vehicle; **, P < 0.01 versus vehicle/vehicle; a, P < 0.05 versus vehicle/vehicle; b, P < 0.05 versus propranolol/alcohol. N = 5–7 animals and 3–4 PVN sections per animal.