P. Siaud

A subpopulation of corticotopin-releasing hormone neurosecretory cells in the paraventricular nucleus of the hypothalamus also contain NADPH-diaphorase

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.

Chronic Restraint Enhances lnterleukin-1-Beta Release in the Basal State and after an Endotoxin Challenge, Independently of Adrenocorticotropin and Corticosterone Release

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.

The organization of prolactin-like-immunoreactive neurons in the rat central nervous system

SummaryThe localization and distribution of prolactinlike-immunoreactive perikarya and nerve fibers in the rat central nervous system have been studied by a preembedding immunoperoxidase method using well-characterized specific immunsera to rat prolactin. Although the localization of labeled neuronal structures in a number of brain areas correlates with the data of previous immunocytochemical studies, we found prolactin-immunoreactive neurons in various regions not previously reported. In untreated animals, the highest concentrations of prolactinfibers were observed: (i) in the external layers of the median eminence where they exhibited close contact with blood vessels, and (ii) in the bed nucleus of the stria terminalis and in the central nucleus of the amygdala where they closely surrounded unlabeled perikarya. Dense networks of finely varicose prolactin fibers were also observed in the organum vasculosum of the lamina terminalis, in the subfornical organ, and in the dorsolateral regions of the medulla oblongata and the spinal cord. Lastly, a number of large, varicose, intensely immunoreactive fibers were found in the olfactory bulb, the cingulum, and the periventricular regions of the hypothalamus and central gray, whereas isolated fibers could be detected in the caudate nucleus and in the cerebral cortex. In animals treated with colchicine, prolactin-immunoreactive perikarya were essentially located within the periventricular and perifornical regions of the hypothalamus, and within the bed nucleus of the stria terminalis. Although corticotropin (ACTH 17-39)-immunoreactive fibers could be detected in several regions found to contain prolactin fibers, the distribution and organization of both fiber types clearly differed in numerous brain regions, and the regions containing the corresponding perikarya did not overlap. The ultrastructural organization of the prolactin-immunoreactive fibers revealed by electronmicroscopic immunocytochernistry in various brain regions, allowed the characterization of two main types of prolactinergic neurons including: (i) endocrine neurons, whose axons terminated in close vicinity to portal blood vessels in the external median eminence, and (ii) neurons projecting to extrahypothalamic regions, whose axons formed typical synaptic connections with unidentified neuronal units.

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.

Microinjection of oxytocin into the dorsal vagal complex decreases pancreatic insulin secretion

Microinjections of oxytocin and of an oxytocin antagonist into the dorsal vagal complex of the medulla oblongata were performed in order to study the possible role of the oxytocin containing axons that innervate this region in the regulation of pancreatic insulin secretion. No significant effect was produced by the intramedullary injection of the oxytocin vehicle alone or of 0.04 pM oxytocin. Injections of 4 and 20 pM oxytocin produced a reversible decrease of plasmatic insulin levels which fall to 59% of basal levels 15 min after the injection. Such an effect was abolished when 4 pM oxytocin was injected to animals which have been previously bilaterally vagotomized. In contrast to oxytocin, intramedullary injection of a specific antagonist of oxytocin to intact animals induced a marked increase of plasmatic insulin levels which raised 131% of basal levels 15 min after the injection. In animals receiving such an injection of oxytocin antagonist, a secondary injection of 4 pM oxytocin produced a slight but not significant decrease of plasmatic insulin levels. These data strongly suggest that the hypothalamic neurons producing oxytocin that densely project to the dorsal vagal complex may be involved in an inhibitory control of the vagal preganglionic neurons that innervate the pancreas.

Adrenergic innervation of the dorsal vagal motor nucleus: possible involvement in inhibitory control of gastric acid and pancreatic insulin secretion

SummaryMorphological and physiological approaches were used to investigate the possible role of an adrenergic innervation of the dorsal vagal complex in the control of basal gastric acid and pancreatic insulin secretion in the rat. The use of retrograde-tracing methods with injections of True Blue or of wheat-germ agglutinin into the stomach or pancreas first confirmed that most vagal preganglionic neurons innervating these two viscera are localized in the dorsal motor nucleus of the vagus, a number of them connected to both viscera. Light- and electron-microscopic investigation of the organization of adrenergic neuronal structures immunoreactive to phenylethanolamine-N-methyltransferase within this medullary nucleus further revealed: (i) that adrenergic axons establish profuse synaptic connections of the symmetrical type with perikarya and dendrites of this nucleus, and (ii) that several of these adrenergic fibers are connected with retrogradely labeled neurons innervating the stomach and/or pancreas. Lastly, measurements of basal gastric acid output and plasma insulin clearly indicated that both visceral secretions are rapidly and conspicuously decreased by local infusion of 2 nM adrenaline within the dorsal vagal complex. Taken together, these data strongly suggest that the adrenergic innervation of the dorsal medulla oblongata is involved in direct synaptic inhibition of the parasympathetic preganglionic neurons of the vagus that control secretion of gastric acid and pancreatic insulin.

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‐1β (IL‐1β) were measured before (time 0) and at intervals between 15 and 480 min following intra‐arterial (i.a.) infusions of 25μg/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‐1β levels were studied 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‐1β 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‐1β. 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‐1β 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‐1β 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‐1β release as an intermediary factor in the LPS‐induced recruitment of the corticotropic axis in general infections.

Complex catecholaminergic modulation of the stimulatory effect of interleukin-1β 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 bundles 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.

Axons Containing a Prolactin-Like Peptide Project into the Perivascular Layer of the Median Eminence: An Immunocytochemical Light and Electron Microscope Study in Adult and Infant Rats

A light and electron microscopic immunocytochemical study was undertaken to explore the fine structural organization of prolactin-immunoreactive axons in the rat median eminence. In adult intact males and females and in hypophysectomized females, light microscopic immunocytochemical labeling of the mediobasal hypothalamus revealed a marked concentration of prolactin-like immunoreactive fibers in the perivascular layer throughout the median eminence and the hypophysial stalk. At the electron microscopic level, immunostaining was associated with typical neurosecretory axons located either in the palisade layer where they displayed numerous contacts with tanycyte processes, or in the perivascular layer where they frequently contacted the perivascular space. Within the labeled axonal profiles, immunostaining was essentially located on secretory granules, 90-120 nm in diameter, whereas the microvesicles accumulated in some perivascular profiles constantly remained unlabeled. These data strongly suggest that most prolactin-immunoreactive axons of the median eminence release their content into the hypophysial portal vessels. In 1-day-old infant rats, intensely prolactin-like immunoreactive fibers were similarly localized in the most external layer of the median eminence, in which, contrary to adult animals, very slight if any tyrosine-hydroxylase-immunoreactive fibers were detected. Since earlier studies have provided evidence for a nondopaminergic prolactin-release-inhibiting factor in the hypothalamus of infant rats, and for an inhibitory effect of prolactin on pituitary mammotrophs, we propose that hypothalamic prolactin may contribute, as an additional prolactin-release-inhibiting factor, to the multifactorial control of pituitary mammotrophs.

Early hypothalamic activation of combined Fos and CRH41 immunoreactivity and of CRH41 release in push-pull cannulated rats after systemic endotoxin challenge.

We previously showed that intra-arterial endotoxin infusion (lipopolysaccharide [LPS]: 25 micrograms.kg-1) induced an early (15 min) and sustained (480 min) rise in plasma ACTH associated with delayed (60-120 min) increases in plasma concentrations of TNF alpha, IL-6, and IL-1 beta. In the present study, we followed the post-LPS time-course of immunocytochemical expression of Fos-like activity in CRH41 neurons whose immunolabeling was enhanced by icv colchicine pretreatment 48 h before the LPS, and CRH41 release in the push-pull cannulated median eminence of free-moving rats, in parallel with the ACTH response. The earliest Fos-like activity in IR-CHR41 neurons was detected 30 min post-LPS. Colchicine strongly inhibited the LPS-induced activation of Fos expression in single-labeled paraventricular neurons. CRH41 release in the median eminence displayed a biphasic stimulation pattern, with a first peak (+60%) at 15 min together with the ACTH surge, followed by a second rise beginning at 45 min and lasting more than 2 h. Thus, the early stage of the ACTH surge following a nonlethal endotoxin challenge (< 60 min) already involves the activation of CRH41-producing neurons.