Seasonal glucocorticoid production correlates with a suite of small-magnitude environmental, demographic, and physiological effects in mandrills.

OBJECTIVES: The activation of the hypothalamic-pituitary-adrenal axis is a neuroendocrine response to external and internal changes that animals face on a predictable or unpredictable basis. Across species, variation in glucocorticoid production has been related to such changes. In this study, we investigated the predictable, seasonal sources of variation in the levels of fecal glucocorticoid metabolites (fGCM) in a large natural population of mandrills (Mandrillus sphinx) in Southern Gabon. MATERIALS AND METHODS: Using five years of regular behavioral monitoring and hormone analyses performed on 1,233 fecal samples collected on 99 individuals of both sexes and all ages and General Linear Mixed Models, we studied the three main seasonal predictors of fGCM concentrations: (i) weather conditions, (ii) number of adult males, and (iii) female reproductive status. These three predictors all vary seasonally in mandrills. RESULTS: We first showed an increase in fGCM concentrations during the short dry season while controlling for other factors. Pregnant females, which include the large majority of adult females at this time of the year, mainly drove this increase, although a combination of other small-magnitude, season-related effects linked to climatic events and demographic changes also partly explained this seasonal trend. Indeed, fGCM concentrations increased with both low temperatures (and low rainfall) and high numbers of adult males present in the group. These seasonal changes, while correlated, held true throughout the studied years and when restricting our analyses to a given season. Finally, we found that older mandrills showed on average higher fGCM concentrations than younger ones and that medium-ranked females exhibited the highest levels of fGCMs. DISCUSSION: The observed patterns suggest that plasticity in mandrills' metabolism in the form of glucocorticoid production allows them to adjust to predictable changes in climatic, demographic and physiological conditions by mobilizing and redirecting energetic resources toward appropriate, calibrated seasonal responses.

Continuous i.c.v. infusion of brain-derived neurotrophic factor modifies hypothalamic-pituitary-adrenal axis activity, locomotor activity and body temperature rhythms in adult male rats.

Brain-derived neurotrophic factor is a neurotrophin belonging to the nerve growth factor family, which is involved in the differentiation and survival of many types of neurons. It also participates in neuroprotection and neuronal plasticity in adult rats. Our previous studies showed that a single brain-derived neurotrophic factor injection modifies hypothalamic-pituitary-adrenal axis activity in adult male rats. To investigate the effect of chronic brain-derived neurotrophic factor administration on some physiological parameters, adult rats were implanted with osmotic micro-pumps to deliver brain-derived neurotrophic factor continuously for 14 days in the lateral ventricle (12 microg/day/rat). mRNA levels were evaluated by in situ hybridization analysis, peptide contents and plasma hormone concentrations by radioimmunoassay. Animals were also equipped with telemetric transmitters to study locomotor activity and temperature rhythms modifications, since hypothalamic-pituitary-adrenal axis is known to modulate these two parameters. Decreased body weight was used as a control of brain-derived neurotrophic factor access to hypothalamic areas as already documented. In the hypothalamus the continuous brain-derived neurotrophic factor treatment increases: (i) the mRNA steady state levels of corticotropin releasing hormone and arginin-vasopressin in the paraventricular nucleus, the supraoptic nucleus, and the suprachiasmatic nucleus; (ii) the surface of corticotropin releasing hormone and arginin-vasopressin mRNA signals in these nuclei as detected by in situ hybridization, and (iii) the corticotropin releasing hormone and arginin-vasopressin contents. The plasma concentrations of adrenocorticotropic hormone and corticosterone were decreased and increased, respectively. Finally, this treatment increased daily locomotor activity and temperature, and provoked some circadian perturbations. These results obtained after chronic brain-derived neurotrophic factor administration extend data on the brain-derived neurotrophic factor involvement in the hypothalamic-pituitary-adrenal axis regulation and illustrate its effects on the locomotor and temperature rhythms. They also allow demonstrating that the regulation of the hypothalamic-pituitary-adrenal axis by brain-derived neurotrophic factor differs according to the brain-derived neurotrophic factor administration mode, i.e. acute injection or chronic administration.

Different responses of plasma ACTH and corticosterone and of plasma interleukin-1 beta to single and recurrent endotoxin challenges.

In a parallel study in 10 individual rats, three time series of plasma concentrations of ACTH, corticosterone (CORT), and interleukin-1 beta (IL-1 beta) were measured before (time 0) and at intervals between 15 and 480 min following intra-arterial (i.a.) infusions of 25 microgram/kg lipopolysaccharide (LPS). All LPS injections were given at 9 AM. The first time series was performed on naive rats (day 1). A sequence of six daily injections (days 3-8) of the same dose of LPS followed. The post-LPS time course of the plasma ACTH, CORT and IL-1 beta levels were studies on days 3 (second injection) and 8 (seventh injection). The first LPS injection induced a rapid (30 min) eightfold rise in plasma ACTH and CORT, culminating in concentrations 30 times the baseline at 60 min (ACTH) and 15 times baseline at 120 min (CORT). Both hormones receded back to the initial basal level at 480 min. On the other hand, IL-1 beta increased slowly to peak at 13 times baseline 120 min before declining to minimal seven- to ninefold basal levels, 480 min and even 48 h post-LPS. During the second phase of the experiment starting 48 h after the initial LPS priming sequence, the ACTH and CORT responses to daily recurrent LPS injections again differed from those of IL-1 beta. The post-LPS time courses of the ACTH and CORT reaction displayed a typical pattern of a progressive attenuation studied at days 3 and 8. The peak amplitudes at days 3 and 8 were reduced to 60 and 10%, respectively, for ACTH, and to 85 and 45% for CORT of those observed at the first LPS test. The duration of the response (both) was also shortened from 480 min (first LPS test) to 300 min at days 3 and 8. The post-LPS patterns of the IL-1 beta responses were characterized, first by basal levels seven to nine times higher than the initial baseline values (day 1), and by a rapid suppression of the post-LPS response, with only a slight (30%) increase at day 3 and no increase at day 8. Thus, after both acute and recurrent LPS administration, ACTH/CORT and IL-1 beta reacted differently to the endotoxin challenge. The two LPS reactive systems were not correlated. This is inconsistent with the often proposed role of increased plasma IL-1 beta release as an intermediary factor in the LPS-induced recruitment of the corticotropic axis in general infections.

Circadian variations in the amplitude of corticotropin-releasing hormone 41 (CRH41) episodic release measured in vivo in male rats: correlations with diurnal fluctuations in hypothalamic and median eminence CRH41 contents.

The possible correlation between the circadian and episodic release of corticotropin-releasing hormone 41 (CRH41) in male rats was explored in a comparative study, including the measurement at 0700 hr and 1700 hr of (1) the quantitative parameters of the episodic release pattern of CRH41 into the push-pull-cannulated median eminence (ME); (2) CRH41 content measured by radioimmunoassay in the hypothalamus, and immunocytochemically in the ME; and (3) plasma adrenocorticotropic hormone (ACTH). The data showed that in early evening, the 3.4-fold rise in plasma ACTH coincided with a doubling of CRH41 content in the hypothalamus and in the ME, and of the CRH41 release from the perfused ME. The immunocytochemical data further indicated that the ME area labeled with CRH41 immunoreactivity, rather than the labeling intensity of CRH41-stained neurons, increased in the evening, which may point to an evening recruitment of additional CRH41-producing neurons as the origin of the evening increment in CRH41 and ACTH releases. Finally, the computerized analysis of the CRH41-releasing pattern with three different algorithms (Pulsar, Ultra, and the Santen and Bardin algorithm) showed for the first time that the evening rise in CRH41 output was associated with correlative increases of three parameters of the episodic pattern--peak amplitude (+55% to +80%), peak duration (+20%), and mean absolute peak values (+73%)--while the pulse frequency remained at the baseline level of 3 cycles.hr-1. The data suggest the occurrence of a connection between the circadian pacemaker and the machinery generating the episodic release of CRH41.

Involvement of Endogenous Brain-Derived Neurotrophic Factor in Hypothalamic-Pituitary-Adrenal Axis Activity.

Brain-derived neurotrophic factor (BDNF) appears to be highly involved in hypothalamic-pituitary-adrenal (HPA) axis regulation during adulthood, playing an important role in homeostasis maintenance. The present study aimed to determine the involvement of BDNF in HPA axis activity under basal and stress conditions via partial inhibition of this endogenous neurotrophin. Experiments were conducted in rats and mice with two complementary approaches: (i) BDNF knockdown with stereotaxic delivery of BDNF-specific small interfering RNA (siRNA) into the lateral ventricle of adult male rats and (ii) genetically induced knockdown (KD) of BDNF expression specifically in the central nervous system during the first ontogenesis in mice (KD mice). Delivery of siRNA in the rat brain decreased BDNF levels in the hippocampus (-31%) and hypothalamus (-35%) but not in the amygdala, frontal cortex and pituitary. In addition, siRNA induced no change of the basal HPA axis activity. BDNF siRNA rats exhibited decreased BDNF levels and concomitant altered adrenocortoctrophic hormone (ACTH) and corticosterone responses to restraint stress, suggesting the involvement of BDNF in the HPA axis adaptive response to stress. In KD mice, BDNF levels in the hippocampus and hypothalamus were decreased by 20% in heterozygous and by 60% in homozygous animals compared to wild-type littermates. Although, in heterozygous KD mice, no significant change was observed in the basal levels of plasma ACTH and corticosterone, both hormones were significantly increased in homozygous KD mice, demonstrating that robust cerebral BDNF inhibition (60%) is necessary to affect basal HPA axis activity. All of these results in both rats and mice demonstrate the involvement and importance of a robust endogenous pool of BDNF in basal HPA axis regulation and the pivotal function of de novo BDNF synthesis in the establishment of an adapted response to stress.

Osmotic stress increases brain-derived neurotrophic factor messenger RNA expression in the hypothalamic supraoptic nucleus with differential regulation of its transcripts. Relation to arginine-vasopressin content.

We have shown that osmotic stress increases brain-derived neurotrophic factor (BDNF) mRNA in the supraoptic nucleus and that this increase seems to be determined by the high expression of transcripts containing exon I. The paraventricular nucleus is another hypothalamic neuronal subset where BDNF mRNA is also sensitive to osmotic stress stimulation. In this nucleus, transcripts containing exon I were not modified but only those containing exon II. By contrast, transcripts containing exon III did not exhibit any variation in our experimental conditions. The presence of BDNF mRNA in both paraventricular and supraoptic hypothalamic nuclei was recently reported. These nuclei are extremely sensitive to osmotic stimuli and their neurons secrete oxytocin and arginine-vasopressin in the posterior pituitary gland. This study was thus designed to investigate the possible involvement of BDNF in the response of supraoptic nucleus to osmotic stress stimulus. Osmotic stress was induced by hypertonic saline injection (1.35% NaCl) administered to animals 3 h before analysis. We used non-isotopic in situ hybridization to study the expression of BDNF mRNA and its transcripts with antisense riboprobes on histological brain sections, including paraventricular and supraoptic nuclei from control and osmotic stress-stimulated animals. To investigate a possible correlation between the expression of BDNF mRNA and arginine-vasopressin, the peptide content was analyzed by immunohistochemistry in both paraventricular and supraoptic nuclei at two different times after hyperosmotic injection. The results showed that BDNF mRNA expression preceded the arginine-vasopressin increase. In addition, on serial adjacent histological sections of supraoptic nucleus (10 microm), both BDNF and arginine-vasopressin mRNAs were visualized by isotopic in situ hybridization and the images were overlaid, showing that almost all of the hybridization signals were overlapped. Taken together our results are in keeping with the hypothesis that activation of the different BDNF promoters seems to be region-specific. Besides, the temporal correlation between both BDNF mRNA expression and arginine-vasopressin content, as well as the morphological vicinity between their respective producing cells in the supraoptic nucleus, suggest an autocrine or paracrine action for this neurotrophin in the regulation of arginine-vasopressin secretion.

Immobilization stress rapidly modulates BDNF mRNA expression in the hypothalamus of adult male rats.

We demonstrated that short times (15 min) of immobilization stress application induced a very rapid increase in brain-derived neurotrophic factor (BDNF) mRNA expression in rat hypothalamus followed by a BDNF protein increase. The early change in total BDNF mRNA level seems to reflect increased expression of the BDNF transcript containing exon III, which was also rapidly (15 min) modified. The paraventricular and supraoptic nuclei, two hypothalamic nuclei closely related to the stress response and known to express BDNF mRNA, were analyzed by in situ hybridization following immobilization stress. In the parvocellular region of the paraventricular nucleus, BDNF mRNA levels increased very quickly as early as 15 min. In contrast, in the two other regions examined, the lateral and ventral magnocellular regions of the paraventricular nucleus, as well as in the supraoptic nucleus, signals above control were increased later, at 60 min. After stress application, plasma adrenocorticotropic hormone and corticosterone levels were strongly and significantly increased at 15 min. These studies demonstrated that immobilization stress challenge very rapidly enhanced BDNF mRNA levels as well as the protein, suggesting that BDNF may play a role in plasticity processes related to the stress response.

The octadecaneuropeptide-induced response of corticotropin-releasing hormone messenger RNA levels is mediated by GABA(A) receptors and modulated by endogenous steroids.

The involvement of endogenous benzodiazepine octadecaneuropeptide in the regulation of corticotropin-releasing hormone messenger RNA expression has been studied using in situ hybridization technique. Intracerebroventricular injection of octadecaneuropeptide (4 microg/kg) induced a 26% decrease in the corticotropin-releasing hormone messenger RNA expression in the hypothalamic paraventricular nucleus. Concomitant injection of octadecaneuropeptide and i.p. injection of the GABA(A) receptor agonist muscimol (4 mg/kg) potentiated the corticotropin-releasing hormone messenger RNA decrease ( - 34%). The depressing effect of octadecaneuropeptide on corticotropin-releasing hormone gene expression was totally reversed by pretreatment of the animals with the GABA(A) receptor antagonist picrotoxin (5 mg/kg; i.p.) or by pretreatment with the benzodiazepine receptor antagonist flumazenil (4 mg/kg; i.p.). To determine the reciprocal involvement of adrenal and sexual steroids in this regulation, animals are adrenalectomized and/or castrated. Adrenalectomy reversed the effect induced by octadecaneuropeptide, which increased corticotropin-releasing hormone messenger RNA expression (+21%), while castration did not modify the negative influence of octadecaneuropeptide. When rats were adrenalectomized and castrated, the adrenalectomy influence was predominant, since octadecaneuropeptide increased significantly the hybridization signal (+18%). The involvement of neurosteroids, especially reduced metabolites of progesterone was also investigated. The concomitant injection of octadecaneuropeptide and subcutaneous injection of the 5alpha-reductase inhibitor MK-906 (14 mg/kg) to adrenalectomized and castrated rats, reduced significantly by 60% the increase of corticotropin-releasing hormone messenger RNA expression induced by octadecaneuropeptide. These results indicate that in vivo the endogenous benzodiazepine octadecaneuropeptide, via an activation of the benzodiazepine sites of the GABA(A) receptor, negatively modulates corticotropin-releasing hormone neuronal activity and that this modulation can be negatively or positively influenced by central and peripheral steroids.

Differential involvement of adrenal and gonadal steroids in anterior and intermediate pituitary pro-opiomelanocortin mRNA expression induced by the endogenous benzodiazepine, octadecaneuropeptide, in adult male rats.

The involvement of the endogenous benzodiazepine, octadecaneuropeptide (ODN), in the regulation of proopiomelanocortin (POMC) mRNA expression at the pituitary level, and the influence of adrenal and gonadal steroids, have been studied using a quantitative in situ hybridization technique. I.c.v. injection of ODN (4 micrograms/kg) in sham-operated rats induced a 17 and 7% decrease in the POMC mRNA expression in anterior and intermediate pituitary lobes respectively. To determine the reciprocal involvement of adrenal and gonadal steroids in this regulation, animals were adrenalectomized and/or castrated. Adrenalectomy significantly increased POMC mRNA expression by 48% at the anterior pituitary level, but induced a 10% decrease of hybridization signal at the intermediate pituitary lobe (vs control sham-operated). Adrenal ablation reversed the effect induced by ODN and increased POMC mRNA expression at the anterior and intermediate pituitary levels by 60 and 10% respectively, compared with control sham-operated. By contrast, castration, which produced a decrease in POMC mRNA in the anterior pituitary and an increase in the intermediate lobe, did not modify the negative influence of ODN observed in sham-operated animals. When rats were adrenalectomized and castrated, the adrenalectomy influence was predominant at the anterior pituitary level, since ODN increased significantly the hybridization signal (+68% vs control sham-operated), while the castration influence was predominant at the intermediate pituitary level, since ODN induced an 11% decrease in POMC mRNA signal compared with control sham-operated. These studies indicate that, in vivo, the decrease in POMC mRNA expression in the anterior and intermediate pituitary induced by an endogenous benzodiazepine is differently modulated by adrenal and gonadal steroids, with a predominant influence of adrenal steroids at the anterior pituitary level and gonadal steroids at the intermediate pituitary level.

Age-related decrease in the hypothalamic CRH mRNA expression is reduced by dehydroepiandrosterone (DHEA) treatment in male and female rats.

Aging is frequently associated with changes in physiological and cognitive processes. Among these changes, it has been shown that aging has a profound impact on the hypothalamo-pituitary-adrenocortical (HPA) axis. Since there is controversy about the influence of aging on the HPA axis, the first objective of the present study was to clarify the effects of aging on the HPA axis using the genetic expression of hypothalamic corticotropin-releasing hormone (CRH). Changes in the CRH mRNA expression were quantified in the hypothalamic paraventricular nucleus (PVN) using in situ hybridization, in young (50 days) and old (18 months) rats of both sexes. The second objective was to determine the effects of dehydroepiandrosterone (DHEA) treatment on the age-related changes in CRH mRNA in both sexes. In aged animals of both sexes, CRH mRNA levels in PVN were significantly reduced by 20%. DHEA treatment increased the CRH mRNA expression in young rats and reversed the decrease in the CRH mRNA expression in old rats. Young males were more sensitive to DHEA than young females (+15% vs. +9%) whereas in old animals females showed more striking changes than males (+38% vs. +24%). These findings clearly demonstrate that in old apparently healthy rats of both sexes aging process induced a significant decrease in basal CRH mRNA levels and that DHEA treatment which exerts a positive influence on CRH gene expression completely reversed the decrease of the CRH mRNA levels associated with aging.

Concomitant changes in CRH mRNA levels in rat hippocampus and hypothalamus following immobilization stress.

By using in situ hybridization, we have demonstrated an increased expression of corticotropin-releasing hormone (CRH) mRNA in the hippocampus following immobilization stress (3 h) in rats. It paralleled that measured in the hypothalamic paraventricular nucleus (PVN). Pretreatment of control and stressed rats with MK-801 (a NMDA receptor antagonist) further increased CRH mRNA expression, in the two structures. The concomitant up-regulation of CRH mRNA expression in these structures suggests a common regulatory finality for a single molecule at two different loci.

Involvement of neurosteroids in the effect of the endogenous benzodiazepine receptor ligand octadecaneuropeptide (ODN) on gonadotropin-releasing hormone gene expression in rat brain.

We have recently demonstrated that different activators of the GABAA receptor complex including reduced progesterone metabolites and the endozepine octodecaneuropeptide (ODN) exert an inhibitory influence on GnRH gene expression. In order to investigate the possible involvement of neurosteroids, especially progesterone metabolites in the effect of ODN, we have evaluated in adrenalectomized and castrated male rats the influence of pretreatment with an inhibitor of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) trilostane (TRIL) and an inhibitor of 5 alpha-reductase MK-906 on GnRH mRNA levels in ODN-treated rats. TRIL completely prevented the inhibitory influence of ODN on GnRH mRNA. It was also found that the inhibitor of 3 beta-HSD as well as pregnenolone sulfate (PREG-S), which has been shown to be increased following TRIL treatment, could induce an increase in GnRH mRNA. MK-906 could also completely reverse the negative influence of ODN. When administered alone, this antagonist of 5 alpha-reductase induced an increase in GnRH mRNA. These results clearly indicate that the inhibition of two key enzymes for the synthesis of reduced progesterone metabolites can completely prevent the inhibitory influence of the endozepine ODN, suggesting that the effect of this endogenous ligand might be completely or partially mediated by an activation of the synthesis of active progesterone metabolites. On the other hand, it remains possible that, as a consequence of enzymatic inhibition, an increase in some precursors known as antagonists of GABAA may play a role in the prevention of the ODN effect by the two enzyme antagonists.

Effects of ageing and dehydroepiandrosterone administration on pro-opiomelanocortin mRNA expression in the anterior and intermediate lobes of the rat pituitary.

There is still controversy about the influence of ageing on the activity of the hypothalamo-pituitary-adrenocortical (HPA) axis in the rat. The first objective of the present study was to evaluate the influence of ageing on the activity of the HPA axis by measuring pituitary proopiomelanocortin (POMC, the precursor of ACTH and alphaH) mRNA levels in the anterior and intermediate lobes in young (50-55-day old) and aged (18-month-old) rats of both sexes. The second goal of the study was to evaluate the effect of 2.5 day administration of dehydroepiandrosterone (DHEA), a steroid precursor which has been shown to improve some ageing-associated deficits. In the young male anterior pituitary, DHEA induced a 17.5% increase in POMC mRNA levels. In aged males, anterior pituitary POMC mRNA levels were 22% lower than those detected in young animals. DHEA treatment produced a 26% increase, then completely restoring mRNA levels when compared to those found in young vehicle-treated males. In the young female, DHEA did not induce any changes in anterior pituitary POMC mRNA. In aged females a 24% reduction in the hybridization signal was observed. This reduction was completely reversed by DHEA which induced a 45% increase over the levels observed in vehicle-treated aged animals. In the intermediate lobe, the results were very similar to those obtained in the anterior lobe, although the observed effects induced by ageing and DHEA were less striking. These results together with previous ones indicating an age-related decrease in corticotropin-releasing hormone (CRH) neuronal activity suggest that ageing is associated with a decrease in HPA axis activity. They also demonstrate that a short-term DHEA treatment can exert a beneficial influence by reversing the decrease in pituitary POMC mRNA expression which occurs as a consequence of ageing.

Influence of adrenal glands on the modulation of prolactin gene expression by the endogenous benzodiazepine ligand octadecaneuropeptide in the male rat pituitary gland.

Recently, an 86-amino acid polypeptide with high affinity for diazepam binding sites, termed diazepam-binding inhibitor (DBI), has been found in the rat brain. DBI, as well as a peptide derived from DBI, the octadecaneuropeptide DBI[33-50] (ODN), interacts with the GABA(A) receptor complex. To investigate the role of these endogenous ligands for GABA(A) receptors on prolactin gene expression, we studied the effects of acute intracerebroventricular administration (4 h before sacrifice) of ODN on prolactin mRNA levels in the male rat. Because, in some neuropeptidergic systems, glucocorticoids play a role in the response to ODN, we also studied the influence of adrenal glands and the effect of dexamethasone administration in the response of prolactin gene expression to ODN. ODN injection produced an increase in prolactin mRNA levels. Adrenalectomy performed 5 days before sacrifice resulted in an increase in prolactin gene expression and also potentiated the stimulating effect of ODN. Because castration has been shown to decrease prolactin gene expression in the male rat, we used castrated and adrenalectomized animals to study the role of dexamethasone in the response of lactotrophs to ODN. In these steroid-deprived animals, dexamethasone treatment (for 4 days) decreased prolactin mRNA levels but did not modify the response to ODN. These data indicate that an endogenous neuropeptide interacting with the GABA(A) receptor complex can stimulate prolactin gene expression and suggest that the adrenal glands may produce factor(s) capable of decreasing prolactin mRNA. On the other hand, it does not appear that glucocorticoid hormones play a role in the effect of ODN on lactotroph activity.

Serotoninergic and suprachiasmatic nucleus involvement in the corticotropic response to systemic endotoxin challenge in rats.

We have investigated whether the serotonin system participates in the mechanisms underlying the corticotropic response in experimentally infected rats. Intra-arterial injection of lipopolysaccharide (LPS; 25 microg/kg b.w.) resulted in a slight but significant increase in serotonin (5-HT) metabolism, detectable 60 min after the stimulus and lasting more than 480 min. Adrenocorticotropin (ACTH) and corticosterone (CORT) responses in intact rats conformed to earlier reports, increasing as early as 30 min after LPS injection and reaching maximal concentrations in the circulation 60 min after the bacterial endotoxin injection. Plasma concentrations of interleukin-1beta (IL-1beta) increased only after 60 min, reaching maximal levels 120 min after LPS. Depletion of hypothalamic 5-HT (-93%) by pretreatment of the animals with para-chlorophenylalanine (p-CPA), resulted in a halved ACTH response to LPS, despite an overall unchanged secretory pattern. Neither CORT nor IL-1beta secretory patterns were affected in these rats pretreated with p-CPA. Complete bilateral electrochemical lesions of the suprachiasmatic nucleus (SCN), which is innervated by mesencephalic 5-HT, impaired the early phase of the ACTH (-75% at 30 min) and CORT (-40% at 30 min) responses but did not affect the later increases of the corticotropic and the plasma IL-1beta responses following the LPS injection. These results indicate that serotonin pathways and SCN are involved in the earlier mechanisms of corticotropic axis recruitment following systemic LPS endotoxemia.

Effects of aging and melatonin administration on gonadotropin-releasing hormones (GnRH) gene expression in the male and female rat.

It is well documented that in the rat of both sexes aging is associated with a decline in reproductive functions. We have recently shown that melatonin exerts a positive influence on GnRH gene expression in the adult male rats. In order to evaluate the effect of aging as well as melatonin on GnRH mRNA levels, we have studied the effect of 2.5-day administration of melatonin to young (50-55 day of age) and aged (18 month of age) rats of both sexes. In the young males melatonin induced a 11% increase in the hybridization signal. In the aged males, the GnRH mRNA levels were 13% lower than those observed in the young animals. Melatonin administration to aged animals completely restored GnRH mRNA levels when compared to those observed in the young untreated male rats. In contrast, melatonin did not modify the hybridization signal in young female rats, while aging induced a 20% decrease in mRNA levels. Melatonin administration to aged female induced a 18% increase in GnRH mRNA levels, thus completely reversing the influence of aging. These results indicate that the decrease in GnRH gene expression which is likely involved in the decline of reproductive functions in aging can be totally reversed by a short term administration of melatonin, then suggesting that the pineal hormone may be involved in the decrease of GnRH neuronal activity during aging.

Role of adrenal and gonadal steroids in the response of GnRH gene expression to the endogenous benzodiazepine receptor ligand octadecaneuropeptide in the male rat brain.

We have recently demonstrated that the inhibitory influence of the endogenous benzodiazepine receptor ligand octadecaneuropeptide (ODN) on gonadotropin-releasing hormone (GnRH) gene expression could be prevented by specific inhibitors of 3 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase in adrenalectomized and castrated male rats, then suggesting an involvement of neurosteroids in the action of ODN on GnRH neurons. In order to study in detail the role of circulating steroids in the effect of ODN, we have evaluated the influence of adrenalectomy, castration and the combination of adrenalectomy and castration as well as the effect of dexamethasone administration in the response of GnRH gene expression to ODN in the male rat. The intracerebroventricular injection of ODN (4 h before sacrifice) produced a 36% decrease in the hybridization signal. Adrenalectomy induced a 21% decrease in GnRH mRNA levels. In the adrenalectomized rats, the injection of ODN increased by 11% the amounts of mRNA. As previously reported by our group, castration was found to enhance GnRH mRNA (15% over control values). In castrated animals, ODN produced an inhibitory effect in the hybridization signal which was of the same amplitude as that observed in sham-operated animals. Finally, the combination of adrenalectomy and castration resulted in a small but significant decrease in the hybridization signal, the values being intermediary between those observed after adrenalectomy and those obtained after castration. In these animals, ODN induced a 38% decrease in the amounts of GnRH mRNA. In animals that had been castrated and adrenalectomized, dexamethasone treatment during 4 days produced a 19% increase in hybridization signal. In these dexamethasone-treated animals, ODN produced the usual decrease (33%) in GnRH mRNA. These results demonstrate that gonadal hormones do not play a major role in the activation of the GABAA receptor complex by ODN. On the other hand, it clearly appears that glucocorticoids exert a tonic stimulatory influence on GnRH neuronal activity and are involved in the inhibitory effect of ODN. The mechanism of action of glucocorticoids, which seems complex since the influence of adrenalectomy on the ODN action can be prevented by orchidectomy, remains to be fully elucidated.

Superior cervical ganglionectomy suppresses circadian corticotropic rhythms in male rats in the short term (5 days) and long term (10 days).

Superior cervical ganglionectomy (SCGx) has drastic effects on numerous hormonal circadian rhythms and particularly on pineal melatonin secretion. We investigated the hormonal consequences of ablation of the superior cervical ganglion on the corticotropic circadian rhythms in the male rat. Plasma were obtained by sampling blood every 4 h, using a chronic carotid cannula. Adreno-corticotropin hormone (ACTH) was assayed by radioimmunoassay (RIA) and corticosterone (B) by radiocompetition. Urinary 6-sulphatoxymelatonin (aMT6s), considered as an index of the pineal gland activity, was assayed by specific RIA: a decrease in the aMT6s concentration after ganglionectomy was taken as proof of adequate surgical operation. Control animals showed classical circadian rhythms for ACTH and B with basal values during the light phase and circadian peaks around the light/dark interface. Five and ten days after ganglionectomy, the circadian rhythms of ACTH and B were suppressed. In addition, the mean ACTH concentrations increased significantly 10 days after ganglionectomy compared to those in sham-operated rats and 5 days post-operation group. The mean plasma corticosterone levels were similar in those three groups of animals. This is the first study demonstrating the suppressive effect of superior cervical ganglionectomy on the circadian corticotropic hormonal cycle.

Short-term but not long-term adrenalectomy modulates amplitude and frequency of the CRH41 episodic release in push-pull cannulated median eminence of free-moving rats.

CRH 41 release in push-pull cannulated median eminence (ME) was measured in unanesthetized male rats, 3 and 7 days after adrenalectomy (ADX) and in sham-lesioned controls. Perfusion started at 13.30 h and perfusate samples were collected at 5 min intervals for 3 h to estimate the mean release rate of CRH41. The major parameters of the neurohormone's episodic release pattern were analyzed using the Ultra algorithm. In a parallel study, 3 groups of similarly treated rats were used to measure plasma ACTH and hypothalamic CRH41. Three days after ADX, the plasma ACTH titers had risen 14-fold, the hypothalamic CRH41 content had decreased by 40%, while the CRH41 release in the ME had doubled as a result of a significant increase in most variables of the pulsatile release pattern: pulse frequency (+34%; P < 0.01), mean amplitude (+36%; P < 0.05), mean peak levels (+67%; P < 0.01) and mean pulse nadirs (x2.5; P < 0.01). Seven days after ADX, even though plasma ACTH had further increased to 30-times control levels, hypothalamic CRH41 content and CRH41 release in the ME had returned to almost control levels. The possible mechanisms of the discrepancy between the CRH and ACTH response time-courses following ADX are discussed.

Complex catecholaminergic modulation of the stimulatory effect of interleukin-1 beta on the corticotropic axis.

We recently showed that bilateral neurotoxic microlesions (6-OH-DA) of the ventral noradrenergic ascending bundle (VNAB-X) at stereotaxic coordinates that blocked corticotropic stress responses did not affect the ACTH surge after bilateral intra-paraventricular (i.PVN) injections of interleukin-1 beta (IL-1 beta), and that lesioning at these stereotaxic coordinates obliterated the dorsal axonal populations of the VNAB (dVNAB-X), but spared the bundle's most ventral axons (vVNAB). The present study compares the effects of IL-1 beta given i.PVN (2 x 5 ng) of intra-arterially (i.a.) (100 ng) on plasma ACTH in rats with bilateral 6-OH-DA microlesions placed in the dVNAB or the vVNAB, or in an intermediary central position (cVNAB-X). Unlike our previous results, in which dVNAB-X did not alter the biphasic ACTH response to i.PVN IL-1 beta, both vVNAB-X and cVNAB-X reduced by 50-75% the early and delayed ACTH surges which are typical of the i.PVN route. On the other hand the swift monophasic ACTH surge usually occurring after an i.a. injection of IL-1 beta was 65% smaller after dVNAB-X, but was doubled after vVNAB-X or cVNAB-X. Hence, the release of ACTH after both i.PVN or i.a. IL-1 beta requires brainstem afferences conveyed to the hypothalamus by the VNAB. However, the VNAB appears to include at least two functionally different subsets of axons, the roles of which in the ACTH response to IL-1 beta depend on the route by which the cytokine is given.