Expression of functional ciliary neurotrophic factor receptors in immortalized gonadotrophin-releasing hormone-secreting neurones.

Ciliary neurotrophic factor (CNTF), a cytokine of the interleukin-6 superfamily, is known to exert pleiotropic actions, including regulation of food intake and permissive effects on reproduction, by facilitating the release of gonadotrophin-releasing hormone (GnRH) and gonadotrophins. CNTF activates membrane receptors (CNTF-Rs) composed of one ligand-specific binding subunit, defined CNTFR alpha, and two signal transducing subunits, termed leukaemia inhibitory factor receptor (LIFR) and gp130. However, it is not clear whether the effects of CNTF on GnRH release result from either a direct or an indirect action on GnRH-secreting hypothalamic neurones, or from a combination of these events. The hypothesis of a direct effect of CNTF was thus tested using the GT1-7 GnRH-secreting cell line. CNTF-R expression and CNTF-induced modulation of the Janus kinase (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway and of GnRH release were evaluated. GT1-7 cells were found to express CNTFR alpha, LIFR and gp130 genes, as shown by reverse transcription-polymerase chain reaction analysis, and the corresponding proteins, analysed by immunofluorescence and western blot. CNTFR alpha, LIFR and gp130 immunoreactive bands had an approximate size of 50, 190 and 130 kDa, respectively. Treatment of GT1-7 cells with 10(-12) M CNTF for 15-60 min resulted in a marked and transient increase of STAT3 phosphorylation via activation of JAK2. A 30-min exposure of GT1-7 cells to different CNTF concentrations increased the accumulation of GnRH into the culture medium, with a maximal effect at 10(-11) M. In conclusion, the present results provide new information about the regulation of the reproductive axis by CNTF, and suggest that it might operate at the hypothalamic level by directly influencing the activity of GnRH-secreting neurones, in addition to the possible indirect effects via interneurones proposed by previous studies.

Effect of central corticotropin releasing factor on hepatic circulation in rats: the role of the CRF2 receptor in the brain.

BACKGROUND: Corticotropin releasing factor (CRF) is distributed in the central nervous system and acts as a neurotransmitter to regulate gastric functions through vagal-muscarinic pathways. We have recently demonstrated that central CRF aggravates experimental acute liver injury in rats. In the present study, the central effect of CRF on hepatic circulation was investigated. METHODS: Hepatic surface perfusion was determined by laser Doppler flowmetry in urethane anaesthetised rats. Portal pressure and portal blood flow was simultaneously monitored. CRF (0.1-4 nmol), urocortin II (a selective CRF2 receptor agonist 2.5-100 pmol), or saline vehicle was injected intracisternally, and changes in hepatic circulation were observed for 120 minutes. We examined the effects of various pretreatments with K41498, a selective CRF2 receptor antagonist, atropine, 6-hydroxydopamine, hepatic plexus denervation, or hepatic branch vagotomy, respectively. RESULTS: Intracisternal injection of CRF (0.2-4 nmol) caused a dose dependent decrease in hepatic surface perfusion with a maximum response occurring 60 minutes post injection. Portal pressure was dose dependently elevated and portal blood flow was decreased by intracisternal CRF concurrently with the decrease in hepatic surface perfusion. These changes in hepatic circulation by intracisternal CRF were abolished by 6-hydroxydopamine and hepatic plexus denervation, but not by atropine or hepatic vagotomy. Urocortin II injected intracisternally decreased hepatic surface perfusion and elevated portal pressure at doses within the picomolar range. Intracisternal preadministration of K41498 inhibited the effect of central CRF on the hepatic circulation. CONCLUSION: These data suggest that CRF acts in the brain to decrease hepatic surface perfusion and elevate portal pressure through central CRF(2) receptor and sympathetic-noradrenergic pathways.

Food conversion is transiently affected during 4-week chronic administration of melanocortin agonist and antagonist in rats.

The central melanocortin system is involved in the regulation of food intake and body weight. In this study, we investigated the effect of a 4-week intracerebroventricular infusion of the melanocortin receptor agonist MT-II and the selective melanocortin-4 receptor antagonist HS024 on food intake and body weight homeostasis. The MT-II-treated rats ate less and lost considerably more weight than the control rats during the first week of treatment. During the second and third week, they gained weight and, by the end of the treatment period, the weight gain was similar to that of the control rats. The HS024 treatment caused hyperphagia and development of obesity during the entire period. Extensive accumulations of fat and a sixfold increase in leptin levels were observed in the HS024-treated rats, as compared with controls, after the 4-week period. Food conversion ratio, defined as body weight increase relative to weight of ingested food, was clearly increased in the HS024-treated rats, while it was lowered in the MT-II-treated rats compared with controls. The effect on food conversion ratio was transient, being greatest for both experimental groups during the first week and it was then attenuated to reach the level of controls at the end of the study. The results suggest that long-term injection of exogenous melanocortin receptor active substances may have an important transient effect on food conversion.

In vivo release of prolactin-releasing peptide in rat hypothalamus in association with luteinizing hormone and prolactin surges.

Prolactin (PRL)-releasing peptide (PrRP) is a novel hypothalamic peptide reported to be a potent and specific stimulator of PRL secretion. This author recently reported that PrRP might play a significant role in mediating the steroid-induced PRL surge in the rat. In order to examine the secretory profile of PrRP in the rat hypothalamus before and during the luteinizing hormone (LH) and PRL surges, this study employed the push-pull perfusion technique and determined the in vivo release of PrRP and also of gonadotropin-releasing hormone (GnRH) in ovariectomized rats primed with estradiol and progesterone. In the medial preoptic area (MPOA) where the GnRH neuronal perikarya exist, GnRH release was increased prior to the initiation of the LH surge, and PrRP also started rising even earlier than GnRH. In the median eminence-arcuate nucleus complex (ME-ARC), where GnRH neuronal fibers terminate, GnRH secretion started increasing before the commencement of the LH surge, but the release of PrRP did not change significantly. These results suggest that PrRP may play a role in mediating the steroid-induced LH surge by activating GnRH neurons in the MPOA. A possible involvement of PrRP in the PRL surge was not suggested from the present data. The lack of a significant alteration in PrRP release in the ME-ARC may argue against a direct hypophysiotropic action of the peptide.

Nitric oxide mediates leptin-induced preovulatory luteinizing hormone and prolactin surges in rats.

The objective of this study was to investigate whether nitric oxide (NO) plays a significant role in mediating the facilitatory action of leptin on the reproductive system. The covariate of reproductive function we used for evaluation was preovulatory surges of luteinizing hormone (LH) and prolactin (PRL), which were simulated by priming ovariectomized rats with estradiol and progesterone. A systemic treatment of normally-fed rats with an NO synthase inhibitor (N(W)-nitro-L-arginine methyl ester) significantly decreased the magnitude of both LH and PRL surges. Three-day-fasted rats did not show a significant surge of either LH or PRL. An intracerebroventricular administration of leptin to fasted rats led to a significant recovery of these hormonal surges, but a simultaneous administration of both the NO synthase inhibitor and leptin significantly abrogated the effects of leptin. This is the first report to demonstrate a significant intermediary role of NO in leptin-induced preovulatory LH and PRL surges in rats.

Ciliary neurotrophic factor, a gp130 cytokine, regulates preovulatory surges of luteinizing hormone and prolactin in the rat.

Ciliary neurotropic factor (CNTF) is a neuroregulatory cytokine belonging to the interleukin-6 type cytokine superfamily. Although a few studies have reported a facilitatory action of CNTF on the reproductive axis in rodents, information along this line is still very limited. In this study, we examined a possible role of CNTF in the generation of ovarian steroid-induced luteinizing hormone (LH) and prolactin (PRL) surges in the rat, a crucial physiological event in mammalian reproduction. Experiments were performed on both normally-fed and 3-day-fasted rats, ovariectomized and primed with estradiol and progesterone. Blood was collected every 30 min between 11:00 and 18:00 h, to measure LH and PRL. Drugs were given intracerebroventricularly at 11:00 h. Compared to control serum, undiluted as well as threefold dilutions of anti-CNTF serum caused partial but significant suppression of LH surges. Both concentrations of the antibody also delayed the onset of PRL surge to a comparable degree. Fasted rats did not exhibit significant surges of the hormones, while 0.3 and 1.0 nmol, but not 0.1 nmol, recombinant human CNTF led to a dose-dependent recovery of both LH and PRL surges. These results demonstrate for the first time a significant role of CNTF in the generation of preovulatory LH and PRL surges in the rat. CNTF may thus be another humoral signal linking nutrition and reproductive function.

Effect of central urocortin on carbon tetrachloride-induced acute liver injury in rats.

The effect of intracisternal injection of urocortin, an endogenous ligand for corticotropin-releasing factor (CRF) 2 receptor, on carbon tetrachloride (CCl4)-induced acute liver injury was investigated in rats. Intracisternal injection of urocortin dose-dependently enhanced elevation of serum alanine aminotransferase and aspartate aminotransferase levels induced by CCl4. Intracisternal urocortin also aggravated CCl4-induced histological changes of the liver. The aggravating effect of central urocortin on CCl4-induced acute liver injury was abolished by chemical sympathectomy, but not by vagotomy. These data demonstrate that urocortin acts in the brain to exacerbate acute liver injury through the sympathetic nervous system and suggest a possible involvement of the CRF2 receptor in the central CRF-induced exacerbation of acute liver injury in rats.

Marked uptake of technetium-99m pertechnetate by parathyroid adenoma.

We herewith report an unusual case of primary hyperparathyroidism whose parathyroid adenoma strongly accumulated technetium (Tc)-99m pertechnetate. A 41-year-old woman was referred to our department under the tentative diagnosis of primary hyperparathyroidism. Scintigraphy by thallium-201 chloride showed homogeneous uptake in the whole thyroid, whereas Tc-99m image revealed a strong local accumulation in the middle portion of the right thyroidal lobe. Neck exploration revealed a 12x8x5 mm tumor in the posterolateral region of the right thyroidal lobe, the pathology of which was parathyroid adenoma. In addition, a small nodule (8 mm in diameter) with pathological findings revealing follicular adenoma of the thyroid, was found within the medial portion of the right thyroidal lobe. Both lesions were removed by surgery, and a postoperative Tc-99m scintigraphy no longer demonstrated a significant uptake in the right thyroidal lobe. Since the thyroid adenoma was too small to be detected by any scintigraphic study and located much closer to the median line than the site of the marked accumulation of Tc-99m pertechnetate, it was considered very likely that the parathyroid adenoma concentrated Tc-99m. Search of literature revealed that there have been only thirteen cases of parathyroid tumor reported to date which significantly accumulated Tc-99m pertechnetate. The present patient represents another rare case of parathyroid adenoma showing sueh an unusual scintigraphic image.

Further evidence for a significant participation of the melanocortin 4 receptor in the preovulatory prolactin surge in the rat.

We previously reported that the melanocortin 4 receptor may play a significant role in mediating the preovulatory surges of luteinizing hormone and prolactin in the rat. In order to confirm this previous finding, in the present study we examined and compared the effects of intracerebroventricular administrations of 1.0 nmol of MT II (a non-selective melanocortin 3 and 4 receptor agonist) and 10 nmol of gamma(1)-melanocyte-stimulating hormone (a selective melanocortin 3 receptor agonist) on luteinizing hormone and prolactin surges in starved, gonadal steroid-primed ovariectomized female rats, which is a model deprived of inherent surges of the two hormones. MT II significantly recovered the surge of prolactin, but not of luteinizing hormone (although a tendency to increase was seen), and gamma(1)-melanocyte-stimulating hormone was without effect on both hormones. This study corroborated our previous report through a different and direct approach that the melanocortin 4 receptor, but not the melanocortin 3 receptor, plays a significant role in mediating the preovulatory prolactin surge in the rat.

Central regulation of hepatic function by neuropeptides.

In addition to classical neurotransmitters, such as acetylcholine and noradrenaline, neuropeptides have been recognized as new neurotransmitters and neuromodulators. Neuropeptides are widely distributed in the central nervous system as well as in peripheral nerves, and act as neurotransmitters to regulate various physiological functions. The digestive organs are no exception, and several neuropeptides in the central nervous system are shown to act in specific brain sites and control gastrointestinal functions, such as gastric acid secretion, and gastrointestinal motility, through the autonomic nervous system. Recently, a relationship between central neuropeptides and hepatic function through the autonomic nervous system has been revealed in animal models. Thyrotropin-releasing hormone acts in the medulla, in particular in the left dorsal vagal complex, to induce stimulation of hepatic blood flow and hepatic proliferation, and protect against experimental liver injury through vagal and cholinergic pathways. Corticotropin-releasing factor injected intracisternally elicits inhibition of the hepatic blood flow and exacerbates experimental liver injury through sympathetic and noradrenergic pathways. Neuropeptide Y acts in the left dorsal vagal complex, in particular in regard to the Y1 receptor subtype, to stimulate bile secretion. Other neuropeptides such as beta-endorphin and bombesin in the brain modulate hepatic proliferation and bile secretion. Through the use of neuropeptides, new knowledge of the central and peripheral mechanisms underlying brain regulation of hepatic function will be revealed. Further studies in regard to the physiological relevance of the central action of neuropeptides on specific brain sites should be performed to unravel the underlying pathways that mediate brain-liver interaction.

A significant participation of leukemia inhibitory factor in regulating the reproductive function in rats: a novel action of the pleiotropic cytokine.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine exhibiting diverse biological activities in various tissues and cell types. Accumulating evidence suggests a crucial role for LIF in regulating the hypothalamo-pituitary-adrenal axis, but information is limited regarding whether the cytokine also exerts a significant influence on other endocrine systems. In this study, we examined a possible involvement of LIF in the generation of ovarian steroid-induced luteinizing hormone (LH) and prolactin (PRL) surges in the rat. Experiments were performed on both normally fed and 3-day-fasted rats, which were ovariectomized and primed with estradiol and progesterone. From 11:00 to 18:00 h, blood was collected every 30 min to measure LH and PRL. All the following substances were given intracerebroventricularly at 11:00 h. Compared to control serum, undiluted and 5-times diluted preparations of anti-rat LIF serum caused a partial but significant suppression of LH surge to a similar extent. The two different concentrations of antibody also delayed the onset of PRL surge to a comparable degree. Fasted rats were devoid of significant surges of the hormones, while 1.0 and 3.0 nmol, but not 0.3 nmol, of recombinant murine LIF given to these animals led to a partial but significant recovery of both LH and PRL surges. This stimulatory potency of LIF on both hormones was already maximal at its 1.0-nmol dose. These results demonstrate for the first time a significant participation of LIF in the generation of LH and PRL surges in the rat. A novel action of the pleiotropic cytokine is reported herein.

Normalization of circulating leptin levels by fasting improves the reproductive function in obese OLETF female rats.

In order to examine a possible detrimental effect of hyperleptinemia on the reproductive system, we examined whether a decrease in circulating leptin levels by fasting affects the estradiol/progesterone-induced luteinizing hormone (LH) and prolactin (PRL) surges in genetically obese OLETF (Otsuka-Long-Evans-Tokushima-Fatty) rats. Experiments were performed on both normally fed and 3-day starved groups from ovariectomized OLETF rats and their controls LETO (Long-Evans-Tokushima-Otsuka). Starved LETO rats, whose leptin levels were less than 0.5 ng/ml, did not show a significant surge of either LH or PRL. Normally fed OLETF rats, whose leptin levels were 9.7 +/- 1.8 ng/ml, showed a significant but small surge for both LH and PRL. Interestingly, starved OLETF rats, whose leptin levels (4.1 +/- 0.7 ng/ml) were similar to those in normally fed LETO rats (3.3 +/- 0.4 ng/ml), had significantly greater surges of both hormones than normally fed OLETF group. This study demonstrates for the first time that the normalization of circulating leptin levels in female OLETF rats augments the steroid-induced LH and PRL surges, and also suggests a deleterious effect of hyperleptinemia on the reproductive axis.

A significant participation of orexin-A, a potent orexigenic peptide, in the preovulatory luteinizing hormone and prolactin surges in the rat.

Orexins are novel hypothalamic peptides which stimulate food intake. In view of the well-known tight connection between the nutritional state and the reproductive function, in this study we examined a possible role of orexin-A in the generation of ovarian steroid-induced luteinizing hormone (LH) and prolactin (PRL) surges in ovariectomized rats. Experiments were performed on both normally-fed and 3-day-fasted rats. Although fasting led to abolition of both LH and PRL surges, intracerebroventricular administration of orexin-A (0.3 and 3.0 nmol) resulted in a dose-dependent recovery of the hormonal surges. In addition, anti-orexin-A antisera given to normally-fed rats completely abrogated the surges of both hormones. These results demonstrate for the first time a significant participation of orexin-A in the preovulatory LH and PRL surges in the rat.

Agouti-related peptide prevents steroid-induced luteinizing hormone and prolactin surges in female rats.

We studied the effect of Agrp (agouti-related peptide) on LH (luteinizing hormone) and PRL (prolactin) surges in ovariectomized rats primed with estradiol and progesterone. The rats displayed characteristic LH and PRL surges that were completely abolished by starving. Injection of either 1 nmol or 3 nmol Agrp (83-132), a potent antagonist of the orexigenic MC3 and MC4 receptors, completely prevented both the LH and PRL surges. We also investigated the effects of either a single or double injection of anti-Agrp serum to fasted animals, which were without LH and PRL surges. A single injection of the antiserum was without effect, but the rats that received double injection of anti-Agrp serum partially reinstated both the LH and PRL surges. Although the onset of LH and PRL surges was significantly delayed in the double treated group, the highest levels of the surges for both hormones were statistically indistinguishable compared with the control group. These data give a clear indication that endogenous Agrp may be involved in LH and PRL surges during starvation, providing further evidence that the melanocortin system is important for these hormonal surges in female rats.

Involvement of prolactin-releasing peptide in the preovulatory luteinizing hormone and prolactin surges in the rat.

Prolactin (PRL)-releasing peptide (PrRP) is a novel hypothalamic peptide reported as a potent and specific stimulator of PRL secretion. In this study, we examined a possible role of PrRP in the ovarian steroid-induced PRL surge in the rat, simultaneously observing the change in luteinizing hormone (LH) surge. Experiments were performed on both normally-fed and three-day-fasted rats, which were ovariectomized and primed with estradiol and progesterone. From 11:00 to 18:00 h, blood was collected every 30 min to measure LH and PRL. All the following substances were given intracerebroventricularly at 11:00 h. Compared to control serum, anti-rat PrRP31 serum caused a significant reduction of the LH and PRL surges. The antiserum also delayed the onset of PRL surge. Fasted rats were devoid of significant surges of the hormones, while 3.0, but not 0.5 nmol of rat PrRP31 given to these animals produced a significant recovery of PRL surge. Although LH surge was not reinstated, basal LH secretion was transiently stimulated by 3.0 nmol of PrRP31. These results demonstrate for the first time a significant participation of PrRP in the preovulatory LH and PRL surges in the rat. Possible indirect pathways mediating this effect of PrRP were discussed, in view of the unique anatomical distribution of PrRP in the hypothalamus.

Evaluation of the role for prolactin-releasing peptide in prolactin secretion induced by ether stress and suckling in the rat: comparison with vasoactive intestinal peptide.

Prolactin (PRL)-releasing peptide (PrRP) is a recently discovered hypothalamic peptide possessing a specific stimulatory action on PRL secretion. In this study, we examined whether PrRP plays a role in mediating ether stress- and suckling-induced PRL secretion in rats through administering anti-PrRP antisera intracerebroventricularly. For comparison, we also tested the effect of anti-vasoactive intestinal peptide (VIP) antisera on the hormonal responses, since VIP is another candidate for a physiological PRL-releasing factor. The immunoneutralization of VIP, but not of PrRP, led to a significant suppression of PRL responses to both ether and suckling. These results suggest that PrRP may not play a significant role, or at least play a much weaker role than VIP, in mediating PRL release induced by ether stress and suckling in the rat.

A significant role of leukemia inhibitory factor in the brain and periphery in endotoxin stimulation of adrenocorticotropin secretion in the rat.

Leukemia inhibitory factor (LIF) is considered as another important cytokine regulating the activity of the hypothalamo-pituitary-adrenal axis. In this study, we examined the effects of intravenous (iv) and intracerebroventricular (icv) administrations of anti-LIF antibody on plasma adrenocorticotropin (ACTH) responses induced by intraperitoneal administration of lipopolysaccharide (LPS, 250 microg/kg) in male rats. Fifteen minutes before the LPS injection, anti-rat LIF antibody or control serum was given iv or icv. The antibody was administered at two different concentrations, i.e. undiluted and five-times diluted. Irrespective of the route of administration, the anti-LIF antibody partially but significantly suppressed ACTH responses to LPS, and its suppressive effect was similar between its two different concentrations. These results indicate that the anti-LIF antibody already exerted its maximal effects at its diluted preparation, and hence that the role of LIF in LPS-stimulated ACTH secretion is essentially partial. This is the first study to demonstrate in vivo that LIF in both the brain and general circulation plays a significant role in mediating endotoxin-stimulated ACTH secretion in the rat.

Downregulation of melanocortin receptors in brain areas involved in food intake and reward mechanisms in obese (OLETF) rats.

The melanocortin (MC)4 receptor is important for food intake and weight homeostasis as it mediates the orexigenic and anorexigenic effects of the MCs. OLETF (Otsuka-Long-Evans-Tokushima-Fatty) rats are a selective inbred strain of polygenic variant rats which overeat and develop obesity with elevated leptin levels. We investigated by autoradiography if the expression of MC receptors were altered in ovariectomized estradiol-replaced female OLETF rats compared to their controls (Long-Evans-Tokushima-Otsuka (LETO) rats). We found that OLETF rats show a reduction in total [125I]NDP-MSH MC receptor binding in the ventromedial hypothalamic nucleus, perhaps reflecting an increased release of MC peptides in this region. The levels of MC receptors in the arcuate nucleus and the paraventricular hypothalamic nucleus were not changed. Interestingly, the OLETF rats also showed reduced MC-receptor binding in areas such as the nucleus accumbens shell, and the ventral tegmental area, both of which are believed to be involved in reward systems. Similarities in the changes of MC receptor expression in obese animals and in animals treated with opiates may suggest a neurobiological link between food intake mediated through the MC receptors and reward.

Stimulation of prolactin secretion by chronic, but not acute, administration of leptin in the rat.

Leptin, the product of obese (ob) gene, has been reported to affect the secretion of all six anterior pituitary hormones, but data are especially scarce regarding the interplay between leptin and prolactin (PRL). Thus, in this study we examined and compared in vivo the effects of acute and chronic administrations of recombinant mouse leptin on PRL secretion in male rats. Normally-fed and 3-day-fasted rats received an intraperitoneal bolus injection of leptin [1.0 mg/kg body weight (BW)] or vehicle only. The leptin treatment was without effect on plasma PRL levels up to 5 h postadministration. Food deprivation for 3 days significantly decreased both PRL and leptin levels. This decrease in plasma PRL was prevented by a 3-day constant infusion of 75 microg/kg BW/day of leptin, which maintained plasma leptin levels similar to those of normally-fed rats. The administration of three times the higher dose of leptin (225 microg/kg BW/day) to fasted rats led to further increases in both PRL and leptin in the plasma. Thus, a dose-dependent stimulatory effect of chronic leptin treatment on PRL secretion was indicated. This study demonstrates that chronic, but not acute, administration of leptin stimulates PRL secretion in the rat.

Temporal profiles of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in the plasma and hypothalamic paraventricular nucleus after intravenous or intraperitoneal administration of lipopolysaccharide in the rat: estimation by push-pull perfusion.

Lipopolysaccharide (LPS) is known to stimulate the synthesis and secretion of various proinflammatory cytokines in both the peripheral immune cells and the brain. Yet, the relative contribution of peripheral and central cytokines to the LPS-induced activation of the hypothalamo-pituitary-adrenal axis is still poorly understood. In this study, utilizing the push-pull perfusion technique of the rat brain, we attempted to characterize in detail the temporal profiles of interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha after intravenous (i.v.) or intraperitoneal (i.p.) administration of LPS in both the general circulation and the hypothalamic paraventricular nucleus (PVN), which is the primary source of corticotropin releasing hormone (CRH). Temporal changes in plasma adrenocorticotropic hormone (ACTH) and CRH levels in the PVN were also monitored. We collected blood and perfusates every 30 min from 11:00 to 17:00 h. At 12:00 h, 1.0 or 2.5 mg/kg body weight of LPS was given via an i.v. or i.p. route, respectively. Peak ACTH response occurred 30 min after i.v. LPS and 1.5 h after ip LPS. Of the three cytokines measured in the plasma, TNF-alpha showed the fastest rise in synchrony with peak ACTH secretion after both i.v. and i.p. LPS. Although plasma IL-6 also showed a robust rise, its peak level occurred later than the ACTH peak. Elevation of plasma IL-1beta was the smallest among the three cytokines. CRH levels in the PVN reached their peaks 1 and 2.5 h after the ACTH peak following i.p. and i.v. LPS, respectively. Irrespective of the route of LPS administration, IL-6 and TNF-alpha levels in the PVN showed significant rises 1-2 h after the ACTH peak, but IL-1beta in the PVN did not significantly change during the entire period of observation. The results of the present study suggest that circulating TNF-alpha may play the most important role in triggering the early, peak phase of ACTH secretion after both i.v. and i.p. LPS. Although it is possible that brain TNF-alpha, IL-6, and circulating IL-6, may be involved in the later, protracted phase of ACTH secretion induced by LPS, IL-1beta in both the brain and peripheral circulation seems to play the smallest role in ACTH secretion. This is the first study to characterize the LPS-induced temporal changes in IL-1beta, IL-6, and TNF-alpha in both plasma and PVN simultaneously in conscious, freely moving rats.