Alcohol self-administration acutely stimulates the hypothalamic-pituitary-adrenal axis, but alcohol dependence leads to a dampened neuroendocrine state

Abstract

Clinical studies link disruption of the neuroendocrine stress system with alcoholism, but remaining unknown is whether functional differences in the hypothalamic-pituitary-adrenal (HPA) axis precede alcohol abuse and dependence or result from chronic exposure to this drug. Using an operant self-administration animal model of alcohol dependence and serial blood sampling, we show that longterm exposure to alcohol causes significant impairment of HPA function in adult male Wistar rats. Acute alcohol (voluntary self-administration or experimenter-administered) stimulated the release of corticosterone and its upstream regulator, adrenocorticotropic hormone, but chronic exposure sufficient to produce dependence led to a dampened neuroendocrine state. HPA responses to alcohol were most robust in 'low-responding' non-dependent animals (averaging < 0.2 mg/kg/session), intermediate in nondependent animals (averaging approximately 0.4 mg/kg/session), and most blunted in dependent animals (averaging approximately 1.0 mg/kg/session) following several weeks of daily 30-min self-administration sessions, suggesting that neuroendocrine tolerance can be initiated prior to dependence and relates to the amount of alcohol consumed. Decreased expression of corticotropin-releasing factor (CRF) mRNA expression in the paraventricular nucleus of the hypothalamus and reduced sensitivity of the pituitary to CRF may contribute to, but do not completely explain, neuroendocrine tolerance. The present results, combined with previous studies, suggest that multiple adaptations to stress regulatory systems may be brought about by excessive drinking, including a compromised hormonal response and a sensitized brain stress response that together contribute to dependence.

Fig. 1

Design of the alcohol dependence model (A), blood alcohol levels (left graph), and physical withdrawal signs (right graph) following removal from chronic intermittent exposure to alcohol vapors or air control (B), and operant self-administration data summarizing enhanced responding for alcohol in dependent animals following dependence induction (C). Adult male Wistar rats were trained using a sweetened-solution fading paradigm to orally self-administer alcohol (10% w/v ethanol, alcohol) or water. Following maintenance on 10% w/v alcohol for 20–30 days, stable responders were evenly divided into two groups and exposed to intermittent alcohol vapors, 14 h on/10 h off (‘dependent’) or control air (‘non-dependent’). After 4 weeks of dependence induction, animals were tested at least twice before being assigned to Experiments 1–4. After removal from vapors, blood alcohol levels dropped rapidly, reaching undetectable levels within 4–6 h (*P < 0.001, compared with 0 h; **P < 0.001, blood alcohol levels compared with 2 h; B, left graph). Mild physical withdrawal began to rise 2 h into withdrawal, peaked from 6 to 8 h, and descended thereafter (*P < 0.002, compared with 0 h; ^#P < 0.01, compared with air control group; B, right graph). Operant data from all trained animals of the present study are shown in (C) (Experiments 1, 3 and 4, light cycle) and (D) (Experiment 2, dark cycle). Post-vapor testing was done when dependent animals were 6–8 h into withdrawal. Bars in (C) represent mean alcohol intake (g/kg) in dependent animals (black bars, n = 22) tested before (‘pre-vapor’) and after (‘post-vapor’) dependence induction compared with non-dependent animals (gray bars, n = 28) tested at these same times (data from Experiments 1, 3 and 4 combined). Bars in (D) represent mean alcohol intake (g/kg) in dependent animals (black bars, n = 5) tested before (‘pre-vapor’) and after (‘post-vapor’) dependence induction compared with non-dependent (gray bars, n = 7) and low-responding non-dependent (white bars, n = 4) controls tested at these same times (Experiment 2). Enhanced responding for alcohol was evident in dependent animals compared with non-dependent controls following chronic intermittent alcohol vapor. Post hoc analysis indicated that post-vapor responding for alcohol was higher in dependent animals (*P < 0.0001, compared with pre-vapor responding; ^#P < 0.001, compared with non-dependent controls during post-vapor testing; C and D). Low-responding animals had reduced alcohol intake compared with non-dependent and dependent animals pre-vapor (^##P < 0.01) and compared with dependent animals post-vapor (^##P < 0.01). To convert blood alcohol levels from g% (g/dL) to mM, divide values by 0.0046 (e.g. 0.200 g% is 43.5 mM). Data are expressed as mean ± SEM.

Fig. 2

Alcohol consumption (cumulative responses, A, and total g/kg intake, inset), blood alcohol levels (blood alcohol levels, g%; simple regression scatter plot of alcohol consumption and 30 min blood alcohol levels, B; blood alcohol levels over time, C), plasma adrenocorticotropic hormone (ACTH; pg/mL, D), and plasma corticosterone (cort; ng/mL, E) responses to self-administered alcohol (10% w/v) in non-dependent (gray bar/circles, n = 7) and dependent (black bar/circles, n = 11) animals 6–8 h into withdrawal from vapors. Gray shading indicates when the levers were available in the operant chambers. Dependent animals consumed twice as much alcohol as non-dependent animals (^#P = 0.01, A, inset), reflected in blood alcohol levels that were > 0.150 g% (32.6 mM) in this group (C). The amount of alcohol consumed was strongly correlated with blood alcohol levels immediately following the session (B). (C) Self-administration of alcohol significantly increased blood alcohol levels in both groups (*P < 0.05, compared with 0 min, C), but blood alcohol levels were significantly higher in dependent animals at all time-points except at 0 min, when blood alcohol levels were at baseline (^#P < 0.05, compared with non-dependent animals). Self-administration of alcohol elevated ACTH in both groups of animals (*P = 0.02, compared with 0 min, D), and the degree to which ACTH was increased from basal levels was positively correlated with blood alcohol levels (15 min blood alcohol levels and percent increase in ACTH from basal levels, r = 0.52, P = 0.02, no indicator shown). Despite the increase, ACTH remained lower at all time-points in dependent animals (^#P = 0.01, compared with non-dependent animals, D). Corticosterone was also lower in dependent animals at 0 min (^#P < 0.02, compared with non-dependent animals, E). Self-administration of alcohol elicited a slight increase in corticosterone at 15 min, and the degree to which corticosterone increased was positively correlated with blood alcohol levels (r = 0.58, P = 0.04, no indicator shown). To convert blood alcohol levels from g% (g/dL) to mM, divide values by 0.0046 (e.g. 0.150 g% is 32.6 mM); to convert ACTH_1–39 (pg/mL) to pM, divide values by 4.5410 (e.g. 50 pg/mL is 11.01 pM); to convert corticosterone (ng/mL) to nM, divide values by 0.3464 (e.g. 100 ng/mL is 288.7 nM). Data are expressed as mean ± SEM.

Fig. 3

Blood alcohol levels (g%, A), adrenocorticotropic hormone (ACTH; pg/mL, B) and corticosterone levels (ng/mL, C) before and after an alcohol challenge (1 g/kg, i.v.) in low-responding (white circles, n = 4), non-dependent (n = 7) and dependent (black circles, n = 5) animals 6–8 h into withdrawal from vapors (dark cycle). Alcohol was administered (gray arrow) immediately after the first sample was collected (basal, 0 min). Baseline (0 min) blood alcohol levels were undetectable, and alcohol elicited equivalent increases in blood alcohol levels in all groups (*P < 0.0001; compared with 0 min). No differences in HPA axis activity were evident under basal conditions, but alcohol elicited vastly different ACTH and corticosterone responses in the three groups. The most robust HPA responses were observed in low-responding animals, intermediate responses in non-dependent animals and nearly flattened responses in dependent animals. Post hoc analyses indicated significant peaks in ACTH and corticosterone at 15 min in low-responding and non-dependent animals, and corticosterone levels remained elevated at 45 min only in low-responding animals (*P < 0.008, compared with 0 min). Low-responding animals had significantly higher ACTH and corticosterone levels 15 and 30 min after the alcohol challenge (^#P < 0.01, compared with dependent animals; ^##P < 0.01, compared with dependent and non-dependent animals). To convert blood alcohol levels from g% (g/dL) to mM, divide values by 0.0046 (e.g. 0.200 g% is 43.5 mM); to convert ACTH_1–39 (pg/mL) to pM, divide values by 4.5410 (e.g. 500 pg/mL is 110.1 pM); to convert corticosterone (ng/mL) to nM, divide values by 0.3464 (e.g. 400 ng/mL is 1154.7 nM). Data are expressed as mean ± SEM.

Fig. 4

Adrenocorticotropic hormone (ACTH) responses following exogenously administered rat/human corticotropin-releasing factor (r/hCRF). A dose–response curve for r/hCRF was first conducted in alcohol-naive animals (n = 6 per dose: 0.1, 0.3, 1.0 and 3.0 μg/kg, i.v.), indicating that all doses increased ACTH (*P < 0.0001, A), with a peak at 15 min, and that the 0.3 μg/kg dose of CRF elicited a mid-range ACTH response. The 0.3 μg/kg dose of r/hCRF was then administered to non-dependent (gray circles, n = 5) and dependent (black circles, n = 4) rats 6–8 h into withdrawal from vapors (14.00–16.00 h), and blood samples were obtained 0 (basal, before injection), 15, 30 and 45 min later for measurement of plasma ACTH. Although basal ACTH was significantly lower in dependent animals (^#P = 0.0007, compared with non-dependent animals at 0 min, B), CRF elicited a rapid rise in ACTH and eliminated the hormonal difference between groups (*P < 0.0001, compared with 0 min, B). To convert ACTH_1–39 (pg/mL) to pM, divide values by 4.5410 (e.g. 200 pg/mL is 44.0 pM). Data are expressed as mean ± SEM.

Fig. 5

Photomicrographs showing corticotropin-releasing factor (CRF) mRNA signal in the paraventricular nucleus of the hypothalamus (PVN, A) and group means of transcript optical density (OD, arbitrary units of signal intensity corrected for background) in the parvocellular portion of the PVN (pPVN) in alcohol-naive (white bar, n = 6), non-dependent (gray bar, n = 10) and dependent (black bar, n = 13) animals 6–8 h into withdrawal from vapors (14.00–16.00 h, B). CRF mRNA was significantly decreased in the pPVN in dependent animals compared with alcohol-naive controls (*P = 0.01, B), but not compared with non-dependent animals. Groups did not differ in CRF mRNA levels in the magnocellular division of the PVN (mPVN, data not shown). Data are expressed as mean ± SEM.