The role of oestradiol in sexually dimorphic hypothalamic-pituitary-adrena axis responses to intracerebroventricular ethanol administration in the rat

Abstract

Systemic ethanol (EtOH) administration activates the hypothalamic-pituitary-adrenal (HPA) axis of rats in a sexually dimorphic manner. The present studies tested the role played by the central nervous system (CNS) in this phenomenon. To localise the effects of the drug to the brain, we utilised an experimental paradigm whereby a small, nontoxic amount of the drug was delivered via intracerebroventricular (i.c.v.) injection. EtoH administered i.c.v. rapidly diffuses throughout the cerebrospinal fluid and brain, and does not cause neuronal damage or have any long-term physiological or behavioural effects. Experimental groups included intact males, intact cycling females, and ovariectomised (OVX) animals with or without replacement oestradiol (E(2)). Intracerebroventricular EtOH-induced HPA hormonal activation was determined by measuring plasma adrenocorticotrophin (ACTH) levels. Activation of brain areas that both regulate HPA function and are responsive to gonadal hormones was determined using expression of the transcription factor c-fos (Fos) as a marker of neuronal activity. We observed sex- and oestrous cycle- dependent differences in HPA activation by EtOH as measured by both these parameters. ACTH secretion was highest in females in pro-oestrus or oestrus, just prior to or after the endogenous peak of E(2), as was Fos expression in the paraventricular nucleus of the hypothalamus (PVN) and the locus coreuleus (LC) of the brainstem. In OVX animals, E(2) replacement caused an increase in PVN and LC Fos expression in response to i.c.v. EtOH compared to OVX controls, but a decrease in ACTH secretion. Taken together, these results indicate that at the level of the CNS, EtOH stimulates HPA activity more robustly at times when the effects of E(2) are high, but that E(2) alone is not responsible for this effect. The data further suggest that the LC plays an important role in the circuitry, which appears to be different from that activated following the systemic administration of EtOH.

Figure 1

The icv administration of EtOH (5 μL of 100%) significantly stimulated ACTH secretion compared to vehicle in: A) intact males, B) intact females in estrus or diestrus, and C) intact females in proestrus or metestrus. Each time point represents mean ACTH ± SEM. ***, P < 0.001 versus vehicle; **, P < 0.01 versus vehicle; *, P < 0.05 versus vehicle; a, P < 0.05 versus the same time point in diestrus (Panel B), or metestrus (Panel C); N = 6–9 animals/group.

Figure 2

The icv administration of EtOH (5 μL of 100%) significantly increased Fos expression in the PVN compared to vehicles in all experimental animals females, with this effect being highest on proestrus (Panel A). Panel B shows representative 10X pictures of PVN Fos expression (black nuclei) in response to icv EtOH (5μL of 100%) or vehicle in intact males (two panels on left) and females in estrus (two panels on right). Bar height represents the mean ± SEM. **, P < 0.01 versus vehicle; ***, P < 0.001 versus vehicle; a, P < 0.05 versus metestrus/diestrus animals receiving EtOH. N = 8 (male) or 11–12 (female) animals per group and 4–6 PVN sections per animal.

Figure 3

The icv administration of EtOH (5 μL of 100%) stimulated Fos expression in the LC compared to vehicle in intact females but not in males. Panel A shows average icv EtOH vs vehicle Fos counts for males and females in metestrus/diestrus or proestrus/estrus. Panel B shows representative 10X pictures of Fos expression (black nuclei) in the LC in response to icv EtOH (5 μL of 100%) or vehicle in males (left two panels) and estrous females (right two panels). Bar height represents the mean ± SEM. **, P < 0.01 versus vehicle; a, P < 0.05 versus metestrus and diestrus/EtOH; N = 11–12 animals per group and 4–6 LC sections per animal.

Figure 4

The icv administration of EtOH (5 μL of 100%) stimulated Fos expression as compared to vehicle in the cingulate cortex of males and intact, cycling females. Bar height represents the mean ± SEM. **, P < 0.01 versus estrus/vehicle. N = 12 animals per group and 5–6 cingulate cortex sections per animal.

Figure 5

The icv administration of EtOH (5 μl of 100%) significantly stimulated ACTH secretion as compared to vehicle in OVX shams and OVX animals receiving replacement E2 injections (10 μg/day for 7 days or 50 μg every fourth day). Panel A shows a time course for ACTH secretion following icv EtOH or vehicle and Panel B shows cumulative ACTH secretion over 60 minutes. *, P < 0.05 versus vehicle; a, P < 0.01 versus EtOH/OVX + 10 μg and 50 μg E2; b, P < 0.05 versus EtOH/50 μg E2; N = 8 animals/group.

Figure 6

The icv administration of EtOH (5 μl of 100%) significantly stimulated Fos expression in the PVN of OVX animals with or without E2 (10 μg/day for 7 days) replacement (panel A) as compared to controls, and in the LC of OVX animals with E2 replacement (panel B). **, P < 0.01 versus vehicle; ***, P < 0.001 versus vehicle; a, P < 0.01 versus EtOH/OVX with or without E2 replacement. N = 6–8 animals per group and 4–6 PVN or LC sections per animal.