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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. [ABSTRACT FROM PUBLISHER]

Elevated cortisol evidence in Alzheimer's disease (AD) patients prompted the hypothesis that stress and glucocorticoids are involved in the development and/or maintenance of AD. We investigated the hypothalamic-pituitary-adrenal (HPA) axis activity, functionality, and reactivity for up to 6 weeks after an intracerebroventricular injection of amyloid-β(25-35) peptide (Aβ(25-35)) in rat, a validated acute model of AD. Aβ(25-35) induces memory impairment, alteration of anxiety responses, HPA axis hyperactivity, and glucocorticoid (GR) and mineralocorticoid (MR) receptor increases in brain regions related to HPA axis functions. GR are progressively translocated in neurons nucleus, while membrane version of MR is evidenced in all structures considered. The MR/GR ratio was modified in all structures considered. Aβ(25-35) induces a subtle disturbance in the feedback of the HPA axis, without modifying its functionality. The reactivity alteration is long-lasting, suggesting that amyloid toxicity affects the HPA axis adaptive response to stress. These findings are evidence of progressive HPA axis deregulation after Aβ(25-35), which is associated with an imbalance of MR/GR ratio and a disruption of the glucocorticoid receptors nucleocytoplasmic shuttling, and suggest that elevated glucocorticoids observed in AD could be first a consequence of amyloid toxicity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) is a neurodegenerative pathology associated with aging characterized by the presence of senile plaques and neurofibrillary tangles that finally result in synaptic and neuronal loss. The major component of senile plaques is an amyloid-β protein (Aβ). Recently, we characterized the effects of a single intracerebroventricular (icv) injection of Aβ fragment (25-35) oligomers (oAβ(25-35)) for up to 3 weeks in rats and established a clear parallel with numerous relevant signs of AD. To clarify the long-term effects of oAβ(25-35) and its potential role in the pathogenesis of AD, we determined its physiological, behavioral, biochemical and morphological impacts 6 weeks after injection in rats. oAβ(25-35) was still present in the brain after 6 weeks. oAβ(25-35) injection did not affect general activity and temperature rhythms after 6 weeks, but decreased body weight, induced short- and long-term memory impairments, increased corticosterone plasma levels, brain oxidative (lipid peroxidation), mitochondrial (caspase-9 levels) and reticulum stress (caspase-12 levels), astroglial and microglial activation. It provoked cholinergic neuron loss and decreased brain-derived neurotrophic factor levels. It induced cell loss in the hippocampic CA subdivisions and decreased hippocampic neurogenesis. Moreover, oAβ(25-35) injection resulted in increased APP expression, Aβ(1-42) generation, and increased Tau phosphorylation. In conclusion, this in vivo study evidenced that the soluble oligomeric forms of short fragments of Aβ, endogenously identified in AD patient brains, not only provoked long-lasting pathological alterations comparable to the human disease, but may also directly contribute to the progressive increase in amyloid load and Tau pathology, involved in the AD physiopathology.

Alzheimer's disease (AD) is a neurodegenerative pathology characterized by the presence of senile plaques and neurofibrillary tangles, accompanied by synaptic and neuronal loss. The major component of senile plaques is an amyloid β protein (Aβ) formed by pathological processing of the Aβ precursor protein. We assessed the time-course and regional effects of a single intracerebroventricular injection of aggregated Aβ fragment 25-35 (Aβ(25-35)) in rats. Using a combined biochemical, behavioral, and morphological approach, we analyzed the peptide effects after 1, 2, and 3 weeks in the hippocampus, cortex, amygdala, and hypothalamus. The scrambled Aβ(25-35) peptide was used as negative control. The aggregated forms of Aβ peptides were first characterized using electron microscopy, infrared spectroscopy, and Congo Red staining. Intracerebroventricular injection of Aβ(25-35) decreased body weight, induced short- and long-term memory impairments, increased endocrine stress, cerebral oxidative and cellular stress, neuroinflammation, and neuroprotective reactions, and modified endogenous amyloid processing, with specific time-course and regional responses. Moreover, Aβ(25-35), the presence of which was shown in the different brain structures and over 3 weeks, provoked a rapid glial activation, acetylcholine homeostasis perturbation, and hippocampal morphological alterations. In conclusion, the acute intracerebroventricular Aβ(25-35) injection induced substantial central modifications in rats, highly reminiscent of the human physiopathology, that could contribute to physiological and cognitive deficits observed in AD., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Depression is potentially life-threatening. The most important neuroendocrine abnormality in this disorder is hypothalamo-pituitary-adrenocortical (HPA) axis hyperactivity. Recent findings suggest that all depression treatments may boost the neurotrophin production especially brain-derived neurotrophic factor (BDNF). Moreover, BDNF is highly involved in the regulation of HPA axis activity. The aim of this study was to determine the impact of chronic stress (restraint 3h/day for 3 weeks) on animal behavior and HPA axis activity in parallel with hippocampus, hypothalamus and pituitary BDNF levels. Chronic stress induced changes in anxiety (light/dark box test) and anhedonic states (sucrose preference test) and in depressive-like behavior (forced swimming test); general locomotor activity and body temperature were modified and animal body weight gain was reduced by 17%. HPA axis activity was highly modified by chronic stress, since basal levels of mRNA and peptide hypothalamic contents in CRH and AVP and plasma concentrations in ACTH and corticosterone were significantly increased. The HPA axis response to novel acute stress was also modified in chronically stressed rats, suggesting adaptive mechanisms. Basal BDNF contents were increased in the hippocampus, hypothalamus and pituitary in chronically stressed rats and the BDNF response to novel acute stress was also modified. This multiparametric study showed that chronic restraint stress induced a depressive-like state that was sustained by mechanisms associated with BDNF regulation., (Copyright © 2010 Elsevier Inc. All rights reserved.)

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.

Immobilization stress induces in adult male rats rapid activation of brain derived neurotrophic factor (BDNF) expression in the hypothalamic paraventricular nucleus (PVN) preceding the increases in corticotropin releasing hormone (CRH) and arginin-vasopressin (AVP) expression. The BDNF mRNA signal belatedly co-localizes with CRH and AVP mRNA signals in the PVN, as determined by in situ hybridization. Intracerebroventricular BDNF injections (5 microg/rat) in non-anesthetized adult male rats induce a gradual increase in the CRH mRNA signal whereas AVP mRNA signal progressively decreases in the parvocellular and magnocellular PVN portions. At the same time, the CRH hypothalamic content decreases while the AVP content increases. These variations are accompanied by increases in ACTH and corticosterone plasma concentrations. These results strongly suggest that BDNF could be a stress-responsive intercellular messenger since when it is exogenously administered acts as an important and early component in the activation and recruitment of hypothalamic CRH and AVP neurons.

This study investigates if the serotoninergic system plays a role in chronotoxic effects of the anticancer agent oxaliplatin (l-OHP). Four groups of female rats (120 in total) synchronized with light-dark (12 h:12 h) were treated with: (i) saline, (ii) para-chlorophenylalanine (pCPA, an inhibitor of serotonin biosynthesis: 300 mg/kg/d, i.p. for two consecutive days), (iii) l-OHP (23 mg/kg, i.v.) at three different dosing times, or (iv) both pCPA and l-OHP. The results show pCPA (ii) obliterates the circadian rhythm in plasma ACTH but not in corticosterone or leukocytes, and (iii) l-OHP exerts circadian time-dependent toxic effects (body weight loss, leukopenia, and intestinal lesions) with greatest toxicity coinciding with treatment at the end of the nocturnal activity span (P < 0.05). In rats whose serotonin biosynthesis was blocked (iv), the circadian rhythms in the toxic effects of l-OHP and in ACTH were obliterated, while the rhythms in corticosterone and leukocytes persisted.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin involved in neuronal survival and plasticity that binds to high-affinity receptors named TrkB. In the central nervous system, brain insults, including stress, induce modifications in BDNF messenger RNA (mRNA) expression. The present study attempted to determine in the adult rat pituitary, a peripheral structure relevant for the stress response: (1) whether BDNF and TrkB mRNA expression is influenced by different durations (15, 30, 60, 180 and 300 min) of single immobilization stress; (2) the expression of BDNF transcripts containing the different exons and their possible variations after stress exposure. Plasma corticotropin (ACTH) and corticosterone concentrations were strongly and significantly increased as early as 5 min after the stress stimulus. Using RNAse protection assay and in situ hybridization, a rapid increase in BDNF mRNA occurred at 15 min. This was accompanied by an increase in BDNF protein at 60 min, and by a rapid and significant decrease in TrkB mRNA expression observed at 15 and 30 min after stress application. RT-PCR analysis of BNDF transcripts showed strong basal expression of exons III and IV, whereas transcripts containing exons I and II seemed weakly expressed. After stress application, transcripts containing exons III and IV were rapidly and significantly increased at 30 min, whereas transcripts containing exons I and II remained unchanged. These results show that pituitary BDNF transcripts expression is differentially affected by immobilization stress., (Copyright 2001 S. Karger AG, Basel)

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.

The tail-cast suspension rat model was developed to explore in ground laboratories the physiological effects of some of the stresses prevailing during space flight including and among them those of the headwards body fluid shifts. We recently showed in rats that an acute head-down tilt (45 degrees) from tail-cast orthostatic (OR) to antiorthostatic restraint (AOR) induced within 30 min and for 2 to 4 h an acute stress-like surge in plasma ACTH and corticosterone levels. Considering the proximity of the CRF producing neurons with the 3rd ventricle, we decided to explore the acute and longer-term effects of the OR/AOR tilt on the intra-cerebroventricular pressure (Picv) measured with an indwelling sensor-transmitter catheter stereotaxically implanted in the 3rd ventricle. At 1- or 10-min intervals the unit sent radiotelemetric signals for both Picv and motor activity (MA) to a receiver coupled with an automatic data analyser. The acute AOR-tilt induced within 10 min and for 60 min a 2.5-fold rise in Picv which receded to baseline between 60 and 90 min. During this time, the normally close correlation between Picv and MA was lost, as assessed by Spearman's rank coefficient. In a long-term experimental series we explored the evolution of both Picv and MA in individual rats subjected successively to a 7 day control phase (C). 7 days OR, and 3 days AOR. After the 1-h-long post-tilt rise of the Picv, the mean Picv levels measured for the next 3 days decreased significantly vs. both the preceding OR phase (-30%) and the initial C Phase (-40%). The circadian pattern of the diurnal Picv profile was impaired, as evidenced by a significant fall (i) in the night/day ratio (-25% vs. C). and (ii) even more in the spectral power of the circadian 1 c/24 h frequency (-85% vs. C). The simultaneously recorded MA fluctuations similarly displayed an altered diurnal pattern with a spectral power of the circadian frequency reduced to 7% of controls. However, contrary to the short-term experiment, in the long-term study the large alterations to both Picv and MA were strongly correlated, as during the control phase. The mechanisms involved in the swift post-tilt rise in the Picv together with an aroused corticotropic axis, and in the impact of sustained head-down restraint on CNS-controlled adaptive regulations including their circadian rhythms remain unknown.

The involvement of histaminergic transmission in the rapid and sustained plasma ACTH and corticosterone (CORT) responses induced in conscious rats by intra-arterial infusions of 25 micrograms.kg-1 Escherichia coli lipopolysaccharide (LPS) was investigated. LPS challenge produced a rapid and transient increase (+ 62%) in the amount of histamine (HA) in the median eminence 15 min after LPS administration, which contrasted with constant concentrations of plasma HA throughout the entire study (up to 480 min). Blockade of histaminergic receptors by intra-arterial pretreatment with H1 or H2 antagonists (mepyramine, 1 mg/rat, and cimetidine, 2 mg/rat), administered separately, did not affect either ACTH or CORT responses to LPS. Pretreatment with the same doses of the two antagonists in combination very significantly but transiently impaired the earliest phase (30 min) of the ACTH and CORT responses, without any apparent effect on the late phase of these responses. Pretreatment of the animals with an H3-receptor agonist (R alpha-methylhistamine dihydrochloride, 1 mg/rat) similarly blunted the early corticotropic responses to LPS, and also slightly depressed the long-lasting CORT response. These findings support the view that activated central HA transmission may be a key intermediate mechanism triggering the CRH41-ACTH-CORT responses to LPS, in addition to the previously demonstrated activating role of catecholaminergic afferences to the CRH41 neurons during this early complex phase of corticotropic response to LPS.

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.

This work was undertaken to investigate whether the inhibitory tone exerted by GABA on somatostatin (SRIH) release operates in the control of thyrotropin (TSH) secretion in both basal and cold-stimulated conditions. In a first group of animals (G1) undergoing both carotid and third ventricle push-pull cannulation, i.vt. injection of picrotoxin (10(-5) M) induces a significant decrease in plasma TSH level under basal conditions (0.09 +/- 0.02 versus 0.27 +/- 0.4 ng/100 microliters; P < 0.03, n = 5). In a median eminence (ME) push-pull cannulated group of rats (G2), picrotoxin, peripherally administered, blocks cold-induced inhibition of SRIH release (35.0 +/- 1.8 versus 7.4 +/- 3.3 pg/15 min; P < 0.005; n = 5). In a third group of intact rats (G3), peripheral administration of picrotoxin (2 mg/kg i.p.) blunts the cold-induced TSH release (0.17 +/- 0.03 versus 0.46 +/- 0.04 ng/100 microliters; P < 0.001; n = 5). Our results strongly suggest that a decrease in SRIH release is involved in the GABAergic control of basal and cold-induced TSH secretion.

Previous morphological and physiological studies have suggested that the adrenergic innervation of the dorsal motor nucleus of the vagus nerve (dmnX) is involved in direct synaptic inhibition of parasympathetic preganglionic neurones of the vagus that control secretion of pancreatic insulin. We investigated the effects of bilateral 6-hydroxydopamine (6-OHDA) lesions of adrenergic innervation of the dmnX on pancreatic insulin secretion and glycaemia in normal and vagotomized rats. After two weeks the 6-OHDA lesions produced a marked increase in circulating insulin levels, but no change in glycaemia. Hyperinsulinaemia after adrenergic denervation of the dmnX was more pronounced when a glucose bolus was injected intraarterially. Bilateral subdiaphragmatic vagotomy reversed the observed hyperinsulinaemia. This targeted pharmacological lesion of the adrenergic innervation of dmnX thus causes hypersecretion by pancreatic B cells, an effect which requires an intact vagus nerve.

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.

To explore the interactions between the hypothalamic-pituitary-adrenocortical axis and the immune system under stress conditions, we used an experimental rat model for chronic tail-restraint devised earlier for ground studies in space physiology. The system was used in two positions: (1) the orthostatic restraint position (OR) and (2) the antiorthostatic position (AOR) after the rat hind limbs had been raised by a head-down tilt. After 7 days of either restraint, sequential blood samples were taken via an indwelling aortic cannula, before and at various time intervals between 15 and 300 min after an intravascular infusion of 25 micrograms/kg lipopolysaccharide (LPS). The plasma titers of adrenocorticotropin (ACTH), corticosterone (CORT) and interleukin-1 beta (IL-1 beta) were assayed. Under basal conditions, both OR and AOR restraints induced a 5-fold increase in IL-1 beta with no significant changes in ACTH and CORT levels. A robust increase in all three variables was observed after LPS injection. However, the IL-1 beta response to LPS was significantly higher in both restrained groups than in controls. Both the amplitude and the percentage of individually restrained rats displaying elevated IL-1 beta levels were increased up to 5 h. In contrast, the ACTH and CORT post-LPS responses were normal in the OR group. They were unusually dissociated in the AOR rats, which displayed depressed ACTH levels associated with slightly increased CORT levels. Our results suggest that immune-neuroendocrine responses to chronic restraint stress may differ from those generally observed in acute stress.

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.

The coexistence of ND with CRH 41 was explored in the parvicellular neurons of the PVN, using dual histochemical and radioimmunocytochemical labelling with the light microscope, in rats treated with colchicine. Even though the ND staining was scarce, a clear colocalization was evidenced in the parvicellular part of the PVN. Under these conditions, the ratio of neurons expressing both markers, ND and CRH, amounted about 15% of the CRH-containing neuron population. This result provides a useful tool to study morphological plastic changes in the PVN in response to environmental variations.

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.

Abstract There is evidence that adrenaline stimulates the release of corticotropin-releasing hormone (CRH-41) from hypothalamic neurons. This study was carried out to ascertain the effects of adrenaline on the perikarya and/or distal terminals of these cells. All experiments were performed on unrestrained rats having chronic intracerebroventricular cannulas in the lateral ventricle or intracerebral cannulas in the paraventricular nucleus and, additionally, a push-pull median eminence cannula. Adrenaline (1.4 mug adrenaline bitartrate in 5 muI vehicle for intracerebroventricular infusion, or 0.7 mug in 0.25 muI for intracerebral infusion) was infused over 2 min, and 15-min median eminence perfusate samples were collected over 2 to 3 h. In another experiment the median eminence was directly perfused for 30 min with a total of 1.2 mug (n = 4) or 12 mug (n = 3) adrenaline per rat. Intracerebroventricular or intracerebral adrenaline induced a swift, short-lived (15 min) CRH-41 surge which reached 7 to 10 times the basal level. Direct perfusion of the median eminence with adrenaline (around the nerve endings of CRH-41-producing neurons) did not stimulate CRH-41 release and higher amine concentrations even tended to depress the neuropeptide release. The stimulatory effect of adrenaline on the corticotropic axis appears, therefore, to be restricted to the central dendro-perikaryal region of CRH-41-producing neurons.

In two simultaneous studies on unanesthetized rats implanted 1 week earlier with either an intracerebral (i.c.) cannula adjacent to the paraventricular nucleus of the hypothalamus and an intracarotid cannula, or the same i.c. cannula together with a push-pull cannula in the median eminence (ME), we explored the effect of i.c. infused interleukin-1 beta (IL 1 beta, 5 ng in 0.25 microliter of vehicle within 2 min) on the release of corticotropin-releasing hormone (CRH) 41 and adrenocorticotropic hormone (ACTH). Intracerebral infusion of the vehicle alone had no significant effect on either the pulsatility or the level of CRH 41 release and only a short-lived minor effect on plasma ACTH, whereas i.c. IL 1 beta injection led to a significant and long lasting (1-2 h) rise in CRH 41 release peaking 3 times higher than the mean peaks of basal pulsatility (26.1 +/- 3.5 pg/5 min vs 9.5 +/- 0.7 pg/5 min), and in plasma ACTH culminating 15-20 times higher than basal levels. Simultaneously, body temperature was increased by 2.3 +/- 0.3 degrees C. In another experiment, i.c.v. infusion of IL 1 beta produced a similar increase in plasma ACTH in rats whose catecholaminergic innervation to the hypothalamus had been obliterated by a bilateral injection of 6-hydroxydopamine into the ventral noradrenergic bundle, which appears to rule out modulation of this innervation in the stimulatory effect of IL 1 beta. The precise cellular site of action of IL 1 beta on CRH 41 secreting neurons and the physiological relevance of the study are discussed within the framework of functional interactions between the neuroendocrine and immune systems.

Blinded female rats underwent additional midbrain raphe lesions, in order to explore the role of the raphe in the organization of endogenous circadian rhythms for ACTH, corticosterone (B) and motor activity (MA). Amplitudes and mean levels of rhythms were depressed for ACTH and MA, with persistent free-running circadian periodicity for MA and, in several rats, for ACTH and B as well. Other rats exhibited split circadian and ultradian rhythmicity for ACTH and B, whereas other again displayed no detectable ACTH rhythmicity. These results are discussed in the light of the structure of circadian pacemaker systems.

Fourteen adult male rats were successfully implanted in their median eminence with a push-pull cannula perfusing an artificial fluid at a rate of 13 microliters/min, to measure the release of corticotropin releasing factor (rCRF-41) under physiological conditions assessed by baseline plasma adrenocorticotropic hormone (ACTH) levels. In the basal conscious free-moving state, rCRF-41 release displayed a fluctuating pattern, with peaks about every 45 min at a mean value of 9.0 +/- 0.7 pg/15 min sample (n = 42) vs a mean trough value of 4.1 +/- 0.3 pg/sample (n = 44). Ether stress was followed by a striking rise in rCRF-41 release which generally lasted about 45 min and reached mean peak values of 54.3 +/- 3.2 pg/15 min sample (n = 7). These data constitute the first direct measurements of basal and stress-induced CRF releases in conscious unrestrained rats.

Adrenocorticotrophin (ACTH) and corticosterone in the plasma of adult female rats were measured sequentially at 4 h intervals for 24 h before and after lesions of the suprachiasmatic nuclei or treatment with p-chlorophenylalanine (to inhibit serotonin synthesis). After lesions or p-chlorophenylalanine treatment, the concentrations of ACTH were diminished relative to those in control animals and rhythmic changes could not be detected. However, injection of animals, pretreated with p-chlorophenylalanine, with 5-hydroxytryptophan (60 mg/kg) 8 h before the time when plasma ACTH is maximal in intact animals, stimulated ACTH secretion up to control values. Mean corticosterone concentrations in plasma remained unchanged (after lesions) or increased (after p-chlorophenylalanine). This increase was associated with an increased minimal concentration of corticosterone. After both treatments there was evidence of continued circadian or ultradian rhythms of corticosterone concentration. Locomotor activity of female rats given identical treatment, but without blood sampling, indicated that nocturnal activity was diminished after lesions whereas diurnal activity was enhanced after p-chlorophenylalanine treatment. Periodicity analysis detected the persistence of free-running circadian, and sometimes ultradian activity, rhythms. Adrenalectomy did not alter further the activity pattern observed in rats with lesions. These results therefore support the proposition that both the suprachiasmatic nuclei and the serotoninergic system play an irreplaceable role in the mechanism of ACTH secretory rhythms. The suprachiasmatic nuclei are also important for synchronization of locomotor activity and corticosterone rhythms, which may both persist after the suppression of ACTH rhythms.

Plasma corticosterone (fluorometric assay), pituitary ACTH (bioassay using isolated adrenal cells) and hypothalamic corticotrophin releasing factor (CRF) (bioassay using isolated pituitary cells) were measured singly in groups of six female rats which were killed at 11.00, 15.00, 19.00, 21.00, 23.00, 01.00, 03.00, 05.00, 07.00 and 11.00 h, after 5 weeks of adaptation to a photoperiod of 12 h light: 12 h darkness. Locomotor activity was recorded continuously, using actographic cages, and the waking/sleep pattern was recorded by electroencephalography from chronically implanted control rats during the first hours of the light span. The three hormones msured fluctuated with a 24 h rhythmicity, with extreme values ranging between 4-12+/-1-42 and 31-78+/-194(S.E.M.) microng/100 ml for corticosterone, 4486+/-269 and 16629+/-882 micronu/mg pituitary for ACTH, and 439+/-20 and 1270+/-39 micronu. ACTH production/hypothalamus/10(5) pituitary cells. The onset of the ascending phase of the rhythm started during the first 2 h of light for CRF, 2 h later for ACTH, and again 2 h later for corticosterone. Similarly, the estimated acrophase of the rhythms occurred respectively, 9-4 (CRF), 10-3 (ACTH) and 14-4 h (corticosterone) after onset of light. These phase relationships point to a central origin of the adrenal rhythm. The diurnal activation of CRF at the very beginning of the light phase was concomitant with an almost immediate reduction of the locomotor activity and onset of sleep. These correlations favour the hypothesis of a common temporal control of both the adrenal and the sleep/waking rhythms.

The effects of various CNS impairments on the circadian rhythm of plasma ACTH and Corticosterone (C) were studied in individual cannulated female Sprague-Dawley rats. Pinealectomy had no effect whatever the light perception (intact or blinded rats). Bilateral ablation of paraventricular nuclei did not obliterate the hormonal rhythms, although the rhythms' amplitude were markedly depressed. On the other hand, destruction of suprachiasmatic nuclei (SCN) or systemic blockade of the serotoninergic (5-HT) system by pCPA blocked ACTH rhythm at baseline levels, although circadian or ultradian fluctuations with normal amplitudes persisted for C. Similar effect was observed in several blinded rats with raphe lesions suppressing 5-HT innervation of SCN. Daily 5-HTP injections, if given 4 h after dawn restored normal ACTH rhythm. The respective role of related structures in the rhythms' control will be discussed.

To investigate the possible effect of GABA on the corticotropic system, the potent GABA antagonist picrotoxin was injected into two groups of 14 female rats at 07.00 h and 19.00 h, respectively. A single subconvulsive I.p. injection dramatically raised plasma ACTH and corticosterone, and thereafter suppressed the circadian rhythm of ACTH, but not of corticosterone, for 24 h in the group injected at 07.00 h and for 48 h in the one injected at 19.00 h and increased mean hormonal levels. Results are discussed in the light of the possibility that inhibition by the GABAergic system and stimulation by the serotoninergic system might be components of the mechanism controlling the circadian rhythm of ACTH.

Female rats were bilaterally injected with 3 micrograms of 6-hydroxydopamine (6-OHDA) dissolved in 0.2 microliter saline, via a glass micropipet stereotaxically implanted into the ventral noradrenergic-ascending bundle (VNAB). This bundle conveys most of the catecholaminergic innervation to the paraventricular nuclei and originates from the locus coeruleus and from two medullary groups of neurons (A1 and A2). Two weeks after injection, and 1 wk after the subsequent implantation of an arterial cannula, serial blood samples were taken from each rat over a 36-h period for assay of basal secretion patterns of ACTH and corticosterone (C) by radioimmunoassay and radiocompetition, respectively. Other blood samples were collected at short intervals over a 2-h period to explore the stress-ether responses of both hormones. Effects of 6-OHDA injections on catecholaminergic innervation were attested by the striking decrease in the histofluorescence of hypothalamic catecholamines and by the 86% drop in the hypothalamic noradrenaline concentrations measured by high-performance liquid chromatography at constant dopamine titers. Compared with control, sham-lesioned rats, pharmacological destruction of the VNAB by 6-OHDA led to 1) obliteration of the circadian patterns for ACTH and C and the emergence in their place of ultradian fluctuations of reduced amplitude above base-line levels and 2) 80% inhibition of the ACTH stress response which correlated with a short-lived, depressed C response. These results are discussed within the framework of the controversial literature on the mechanisms by which catecholamines may control corticotropic function.

Changes in plasma ACTH and corticosterone concentrations were measured in individual cannulated rats at stages 1 and 5 of limbic kindling induced by electrical stimulation of the basolateral amygdala or the dorsal hippocampus. At both stages, a stimulation of either structure produced swift surges, first of ACTH and then of corticosterone. At stage 5 of hippocampal stimulation, ACTH baseline concentrations were four times higher than in the controls. The results are discussed in relation to the central control of the adrenocorticotropic system and to the neuroendocrine correlates of the kindling process.

The circadian rhythms for plasma ACTH and corticosterone (B), as well as motor activity, were explored in female rats after ocular enucleation (O-X), stereotaxic lesion of the suprachiasmatic nuclei (SCN-X) or of midbrain raphe nuclei (R-X), or both O-X and R-X, pharmacological blockade of the serotoninergic (5HT) system by pCPA, sometimes bypassed by 5-HTP, or 5-HT denervation of the SCN by local injection of 5,7-DHT. The three circadian rhythms explored responded quite differently to the treatments. In particular, the ACTH and B rhythms lost their usual close correlations. The amplitude and mean level of ACTH fluctuations were depressed after all treatments, but remained normal or were enhanced for B rhythm. ACTH rhythmicity actually was undetectable after SCN-X, pCPA and, in several rats, combined O-X and R-X, whereas persisting circadian and/or ultradian B and locomotor activity rhythms were always measured. The participation of the suprachiasmatic nuclei, the midbrain raphe nuclei and other possible 5-HT components in a complex circadian pacemaker system is discussed.

The importance of 5-HT synaptic transmission for induced or cyclic activation of pituitary secretion now seems widely agreed upon. Raphe structures, the suprachiasmatic nucleus, and, hypothetically, identified mesencephalic 5-HT containing neurons can be assumed to represent parts of the neuronal circuitry of a central clock which synchronizes neuroendocrine rhythms. Cyclic input from the pineal gland may also be involved, though the meaning of this has not yet been clearly established. The primary role of the transmitter may be to modulate transmission to neurosecretory neurons at the output of this clock. This modulation is not hormone-specific and affects, simultaneously, several endocrine functions. 5-HT has also been shown to facilitate the pituitary response to discrete neuroendocrine reflexes, such as the suckling-induced release of prolactin. 5-HT interactions with hormone control provide a good illustration of the neuromodulatory role of the amine.

Adrenocorticotropin (ACTH) was measured in the cerebrospinal fluid (CSF) of male rats implanted with a push-pull cannula in the 3rd ventricle (ICV) and perfused for 3-6 hr. In 5 conscious sham-lesioned controls studied one week after cannulation, CSF ACTH displayed a pulsatile circhoral pattern with a mean concentration of 10.3 +/- 1.1 pg ACTH/15 min, and a peak/trough ratio of about 2. This pattern was not affected by 2 min of ether vapor inhalation or by pentobarbital anesthesia. The high mortality rate following ICV push-pull cannulation in rats hypophysectomized for 2 weeks led us to perform the ICV perfusion just after cannulation in 5 hypophysectomized rats when they were still under pentobarbital anesthesia. Even so the levels and pulsatile pattern of ACTH release into the CSF were unchanged despite the complete disappearance of any detectable amount of ACTH from the plasma. In two of the latter specimens perfused again one week later but without anesthesia, CSF ACTH levels still were indistinguishable from controls. It can thus be concluded that the ACTH detected in the 3rd ventricle CSF is of neuronal origin.

The dynamic pattern of the immunoreactive prolactin (PRL) concentrations in the cerebrospinal fluid (CSF) of the 3rd ventricle was explored by push-pull cannulation during either stimulation or blocking of PRL production in the plasma, which itself was sampled by chronic cannulation of the carotid. Some of the results were compared to the PRL concentrations in CSF samples obtained by 3rd-ventricle puncture. Ether stress, which induced a 4- to 6-fold rise in plasma PRL, altered neither the pulsatile circhoral pattern of PRL in the CSF nor the mean level and amplitude of its pulses. However, the sustained intense hyperprolactinemia induced by haloperidol increased the mean PRL level in the CSF and possibly its pulse rate. Surprisingly, hypophysectomy, which suppressed production of PRL in the plasma, did not alter its baseline level or cycling pattern in the CSF. The possibility that tuberal adenohypophysial cells and/or CNS prolactinergic neurons supply the CSF with PRL is discussed.