F. Malaval

The involvement of noradrenergic ascending pathways in the stress-induced activation of ACTH and corticosterone secretions is dependent on the nature of stressors

SummaryThe aim of the present study was to explore in male rats the role of the catecholaminergic innervation of the hypothalamus in corticotropic and adrenal responses to different kinds of stress conditions. For this purpose, 6-hydroxydopamine (3 μg in 0.2 μl saline) was stereotaxically and bilaterally infused at two levels of the main noradrenergic ascending brain stem bundle (NAB-X). The efficiency of catecholaminergic denervation of the hypothalamus was checked by measuring noradrenaline concentrations in paraventricular nuclei punches by HPLC and was confirmed by a 86% fall in noradrenaline levels of NAB-X rats killed after the stress experiments. Seven days after lesioning the NAB, sham operated controls and NAB-X lesioned animals were divided into 4 groups and submitted to 4 different stressors, i.e.: 2 min ether vapors (n = 5), 1 h immobilization (n = 7), i.v. histamine (2 mg/kg; n = 7) or i.v. insuline (10 I.U./kg; n = 8) injections. ACTH and corticosterone were measured in blood samples sequentially taken from a chronic carotid cannula, before stress and at short intervals over the 2 following hours. In comparison to the respective control groups, NAB-X dramatically reduced the ACTH response to ether (-78%) and to restraint (-53%) stress whereas the corticosterone response was affected to a lesser extent. In contrast, NAB-X slightly altered these responses in the histamine-treated group, although, surprisingly, the ACTH response tended to decrease and that of corticosterone to increase. Finally, NAB-X provoked a biphasic response to insulineinduced hypoglycemia, with a very early (5 min) rise in ACTH and corticosterone in comparison to the control group, followed by a trend to low hormonal levels up to 120 min. These results strongly suggest a differential involvement of the hypothalamic noradrenergic innervation upon the hypothalamic-pituitary-adrenal axis according to the nature of stress conditions.

The corticotropin-releasing factor release in rat hypophysial portal blood is mediated by brain catecholamines.

In order to study the involvement of the hypothalamic corticotropin-releasing factor (CRF) in catecholamine-induced adrenocorticotropin (ACTH) secretion, we have measured CRF levels in rat hypophysial portal blood (HPB) after the pharmacological destruction of the ventral noradrenergic bundle (VNAB), using 6-hydroxydopamine (6-OHDA) stereotaxically injected into the VNAB. CRF levels in HPB were measured by radioimmunoassay, and the effects of 6-OHDA injection were controlled by the determination of catecholamine concentrations in the total hypothalamus. VNAB lesions induced a dramatic decrease in norepinephrine and epinephrine hypothalamic concentration. The CRF levels in HPB were also significantly reduced. These results suggest that central catecholamines exert a direct stimulatory control on the CRF release and play a major role in stress-induced ACTH secretion.

Intrahypothalamic infusion of interleukin-1β increases the release of corticotropin-releasing hormone (CRH 41) and adrenocorticotropic hormone (ACTH) in free-moving rats bearing a push-pull cannula in the median eminence

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.

Adrenocorticotropic Regulations after Bilateral Lesions of the Paraventricular or Supraoptic Nuclei and in Brattleboro Rats

The circadian rhythm and ether stress responsiveness of plasma ACTH and corticosterone were explored in chronically cannulated female Sprague-Dawley rats with bilateral lesions of either the paraventricular nuclei (PVN) or supraoptic nuclei and also in female Brattleboro rats. 3 weeks after PVN lesions, the circadian ACTH rhythm had dropped by at least half. Less drastic alterations were measured for the corticosterone rhythm, and only its diurnal maximum and amplitude diminished by one third. Although the kinetics of plasma ACTH and corticosterone stimulation after ether stress were essentially normal, the peaks for both hormones, 5–15 min after stress, were only half as high as the control peaks. The effects of bilateral abolition of supraoptic nuclei on the circadian rhythm of the adrenocorticotropic system were slight, and only the diurnal minimum and mean level of ACTH dropped significantly. Intriguingly, both ACTH and corticosterone levels in rats with supraoptic lesions rose to twice the control levels after ether stress. In Brattleboro rats, neither the circadian rhythm nor stress-induced stimulation of ACTH and corticosterone differed from those of the controls. These data indicate that vasopressin is not essential for either of the regulations of the adrenocorticotropic system. The production of corticotropin-releasing factor (CRF) involved in both types of regulation clearly originates, at least partly, from the PVN area and no significant contribution is made by the region of the supraoptic nuclei, a finding which conforms to neuroanatomical data. However, it is not yet clear whether the PVN area is the site of origin of the recently characterized 41 amino-acid CRF or of other CRF fractions distinct from vasopressin.

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.

CNS control of the circadian adrenocortical rhythm

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.

Plasma ACTH and corticosterone responses to limbic kindling in the rat

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.

Acute and delayed effects of picrotoxin on the adrenocorticotropic system of rats

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.

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.