The peripheral injection of alcohol stimulates the activity of the hypothalamic-pituitary-adrenal (HPA) axis, but the ready penetration of this drug in most bodily compartments has made it difficult to identify its specific sites of action. Here we determined whether alcohol can directly influence the corticotropes. We first determined whether alcohol acted within the brain to stimulate neurons in the paraventricular nucleus (PVN) of the hypothalamus, which synthesizes corticotropin-releasing factor (CRF) and vasopressin (VP). To test this hypothesis, we injected alcohol intracerebroventricularly (icv; 5 microl of 200-proof; 86 micromol) and compared these results with those obtained after its ip administration (3.0 g/kg). Although not causing neuronal damage and not leading to detectable levels of the drug in the general circulation, icv alcohol significantly up-regulated PVN CRF heteronuclear RNA levels and increased plasma ACTH levels, a change comparable to the one observed in the ip model. To determine whether alcohol stimulated the corticotropes independently of CRF and/or VP, we injected the drug ip or icv and measured changes in anterior pituitary proopiomelanocortin (POMC) transcripts and ACTH release in the presence or absence of endogenous CRF and/or VP. Intracerebroventricular and ip alcohol significantly increased POMC primary transcript levels, measured by ribonuclease protection assay, over a time-course that corresponded to ACTH release. Both the POMC and the ACTH responses were completely abolished by removal of CRF and VP. Collectively, these results indicate that alcohol-induced activation of the corticotropes does not represent a direct influence of the drug on the pituitary but requires CRF and VP.

We previously reported that in adult male rats, the intracerebroventricular (icv) injection of corticotropin-releasing factor (CRF) or the beta-adrenergic agonist isoproterenol (ISO) significantly inhibited the ability of human chorionic gonadotropin (hCG) to stimulate testosterone (T) secretion. The finding that this phenomenon also took place when LH release had been blocked with an LHRH antagonist suggested that icv CRF and ISO did not alter Leydig cell function by influencing the activity of pituitary gonadotrophs. We therefore proposed the existence of a neural pathway connecting the brain to the testes, whose activation by icv CRF or ISO interfered with T secretion. Based on the intratesticular injection of the transganglionic tracer pseudorabies virus, we recently identified the paraventricular nucleus (PVN) of the hypothalamus as a component of this neural link. The aim of the present work was to investigate the functional role of this brain area in mediating the ability of CRF and ISO to inhibit the ability of hCG to stimulate T secretion. We first demonstrated that local microinfusion of CRF or ISO directly into the PVN mimicked the effect of their icv injection, suggesting that the PVN does indeed represent a site of action of ISO and CRF in altering Leydig cell responsiveness to gonadotropin. In contrast, neither CRF nor ISO microinfusion into the central amygdala or the frontal cortex influenced hCG-stimulated T secretion. To further investigate the role of the PVN in ISO- and CRF-induced blunting of hCG stimulation of T, we determined the effect of icv CRF or ISO on testicular activity of rats with electrolytic lesions of the PVN. These lesions, which did not in themselves influence Leydig cell responsiveness to hCG, blocked the effect of both icv ISO and CRF on hCG-induced T release. Collectively, these results support the hypothesis that CRF- and ISO-induced activation of cells in the area of the PVN decreases the ability of gonadotropin to release T and suggests that this nucleus represents an important site of the proposed neural connection between the brain and the testes.

We previously reported the existence of a descending multisynaptic, pituitary-independent, neural pathway between the hypothalamus and the testes in the male rat. Stimulation of this pathway by the intracerebroventricular (icv) injection of IL-1beta or corticotropin-releasing factor blunts the testosterone (T) response to human chorionic gonadotropin (hCG). This response is mediated at least in part by catecholamine beta-adrenergic receptor activation. The present work was performed to further investigate the role of brain catecholamines and testicular blood flow in this pathway. The icv injection of 5 microl of 200 proof ethanol (EtOH; 86 micromol) did not result in detectable levels of the drug in the general circulation and did not induce neuronal damage, but rapidly blunted hCG-induced T release while not decreasing LH levels or altering testicular blood flow. EtOH significantly up-regulated transcripts of the immediate-early gene c-fos in the paraventricular nucleus (PVN) of the hypothalamus. Lesions of the PVN blocked the inhibitory effect of IL-1beta on T, but only partially interfered with the influence of EtOH. PVN catecholamine turnover significantly increased after icv injection of IL-1beta, but not EtOH. Brain catecholamine depletion due to the neurotoxin 6-hydroxydopamine did not alter the ability of hCG to induce T release, but significantly reversed the inhibitory effect of icv EtOH or IL-1beta on this response. Collectively, these results indicate that icv-injected IL-1beta or EtOH blunts hCG-induced T secretion through a catecholamine-mediated mechanism that does not depend on either peripherally mediated effects or pituitary LH, and that the PVN plays a role in these effects.

We previously reported anatomical and functional evidence for a direct, inhibitory neural pathway that regulates testosterone (T) secretion independently of the pituitary. This pathway is activated by the intracerebroventricular (icv) administration of agents that stimulate stress responses, such as IL-1beta, corticotropin-releasing factor (CRF), and norepinephrine (NE), which results in a blunted T response to the administration of human chorionic gonadotropin (hCG). Blunting of the T response is mediated by central beta-adrenergic receptor stimulation. CRF, but not ethanol (EtOH) or IL-1beta, acts directly on the paraventricular nucleus of the hypothalamus to activate the pathway. Here we explored the role played by brain areas hypothesized to be part of this pathway, such as neurons in the dorsal pons [including the locus coeruleus (LC) of the brainstem], where NE is produced. Microinfusion of EtOH or IL-1beta, but not CRF, into these neurons activated the pathway. Electrolytic lesions of this region significantly reversed the inhibitory effect of icv-administered EtOH on hCG-induced T release, while having no effect on the ability of IL-1beta or CRF to do so. However, the icv administration of IL-1beta, EtOH, or CRF, in doses that rapidly inhibit the T response to hCG, all caused a significant depletion of NE from the LC. Collectively, these results indicate that in addition to the paraventricular nucleus, the brainstem area containing the LC is part of a neural pathway that connects the brain to the testes independently of the pituitary. We also speculate that EtOH may stimulate this pathway through NE-dependent activation of the dorsal pons.

The study was designed to determine whether the ability of central oxytocin (OT) to stimulate gonadotropin-releasing hormone (GnRH) on the afternoon of proestrus (PE) in the cycling female rat is mediated at the level of GnRH terminals within the median eminence (ME), or at higher hypothalamic levels where GnRH cell bodies and axons are located. Determining the location of this OT effect in vivo has proven difficult. Therefore, an in vitro system utilizing ME or basal hypothalamic (BH) explants containing GnRH terminals, or GnRH neurons including the cell bodies, axons and terminals, respectively, were harvested from regular cycling female rats at 15:00 h on PE or diestrus (DI). The explants were allowed to preincubate in Krebs Ringer Bicarbonate Buffer containing glucose, ascorbic acid, calcium, and a metalloprotease inhibitor (KRBG) and enriched with 95% O₂/5% CO₂ at 37°C until a stable baseline release of GnRH was achieved (30 min). The 0.05 level of probability was used as the minimum criterion of significance in all experiments. The ability of OT (10^–15–10^–9M) to stimulate the release of GnRH was determined in both ME and BH explants on PE and DI. The results demonstrated a sensitive, dose-dependent ability of OT to stimulate GnRH release from PE BH explants which was observed only in PE. Furthermore, OT failed to significantly stimulate GnRH release from ME explants on either PE or DI. The data indicate that the PE BH explant paradigm can be used to examine the manner and mechanisms by which OT influences GnRH release on the afternoon of PE. Furthermore, the results indicate for the first time that the stimulatory action of OT by itself on preovulatory GnRH release in cycling female rats is not mediated at the level of the GnRH terminals within the ME, but requires neuronal interactions and mechanisms within the BH explants.