A. Szafarczyk

Immunocytochemical evidence for stimulatory control by the ventral noradrenergic bundle of parvocellular neurons of the paraventricular nucleus secreting corticotropin releasing hormone and vasopressin in rats

The regulation, by catecholaminergic innervation, of parvocellular neurons of the paraventricular nuclei (PVN) secreting corticotropin releasing hormone (CRH) and vasopressin (Vp) was studied by immunocytochemical visualization of both neurohormones in control rats and in rats given discrete injections of 6-hydroxydopamine in the ventral noradrenergic ascending bundle (VNAB). In both groups, the changes in immunostaining intensities observed in axon terminals of the external median eminence and in PVN perikarya 48 h after a blockade of axoplasmic transport by intraventricular injections of colchicine, served as an index for hormonal release and synthesis. In controls, this treatment induced a strong decrease in CRH and Vp immunoreactivity within the terminals, together with intense labeling of PVN perikarya containing CRH. By contrast, bilateral VNAB lesions strikingly inhibited both the colchicine-induced reduction of the CRH and Vp immunoreactivity in axons and the accumulation of CRH in the perikarya. Unilateral VNAB lesions induced similar alterations but these were restricted to the ipsilateral PVN and median eminence. Comparison of these immunocytochemical data with earlier physiological observations on the effects of VNAB lesions on ACTH secretion indicates that the catecholaminergic afferents to the PVN conveyed by the VNAB stimulate the release and the synthesis of CRH and Vp by parvocellular neurons projecting into the external median eminence.

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.

Radioautographic evidence that axons from the area of supraoptic nuclei in the rat project to extrahypothalamic brain regions

The axonal efferents of neurons of the supraoptic nucleus area were studied by radioautography in the rat after discrete stereotaxic injections of [3H]leucine into this nucleus. Beside a densely labeled pathway running from the nucleus to the posterior pituitary through the internal median eminence, several of the visualized labeled axonal bundles were found to project into various extrahypothalamic regions, including the olfactory bulb, the cortex, the lateral habenula, the subcommissural organ, the amygdala, the mammillary bodies and the locus coeruleus. These results suggest that part of the vasopressin- or oxytocin-containing perikarya located in the supraoptic nucleus constitute the cells of origin of axons which also contain these peptides and which have already been shown to be present in the above extrahypothalamic areas. This also implies that, like the paraventricular nucleus, the supraoptic nucleus is also involved in central extrahypothalamic regulations.

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.

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.

Effects of raphe lesions on circadian ACTH, corticosterone and motor activity rhythms in free-running blinded rats

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.