Further evidence for a central stimulatory action of catecholamines on adrenocorticotropin release in the rat.

Catecholamines may stimulate ACTH secretion during stress. To investigate the nature and site of such an action, plasma ACTH was measured in four groups of unanesthetized adult female rats with an indwelling carotid cannula. Sequential 300-microliter blood samples were taken 60 min, 30 min, and immediately before an intracerebroventricular (icv) infusion of 2.5 microliter adrenaline or noradrenaline and 5, 15, 45, 60, and 120 min after the infusion. The four groups were: 1) intact rats; 2) rats infused 7 days after undergoing a discrete bilateral lesion of the ventral noradrenergic ascending bundle caused by 6-hydroxydopamine, which depleted their hypothalamic adrenaline and noradrenaline levels by 90% and 80%, respectively; 3) rats infused 30 min after pretreatment via the icv route with either prazosin or propranolol; and 4) rats infused 16 and 2 h after two successive intracarotid injections of an anti-rCRH-41 serum. In another group, the effects of icv catecholamine administration were compared with those of an intracerebral (ic) microinfusion close to a single paraventricular nucleus (PVN). Finally, in two additional groups blood was sampled at the above-mentioned times before and after a 2-min ether inhalation by intact rats or prazosin- and/or propranolol-pretreated rats. In the intact rats (group 1), a stress-like stimulatory dose response was noted after both adrenaline and noradrenaline infusions, with a half-maximal effect at concentrations of about 0.6 nmol and a maximal effect at 2.7 nmol or more. At maximally effective doses, adrenaline was significantly more active than noradrenaline. In the rats with ventral noradrenergic ascending bundle lesions (group 2), 2.7 nM adrenaline or noradrenaline stimulated ACTH release as in the controls without lesions. In group 3, prazosin blocked the ACTH responses to both adrenaline and noradrenaline, whereas propranolol only blocked the response to adrenaline. In group 4, i.e. rats pretreated with an anti-rCRH-41 serum, the amplitude of the ACTH surge after icv adrenaline or noradrenaline infusion was halved. A unilateral ic catecholamine microinfusion next to the PVN (half the icv dose given in group 1) led to a rapid ACTH release that peaked at half the response measured in the icv infused rats. Ether stress-induced ACTH release was decreased by 50-60% after icv pretreatment with 1 or 10 micrograms prazosin, 1 or 6.5 micrograms propranolol, or a combined dose comprising 1 microgram of both. The following conclusions were reached.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of lesions of the suprachiasmatic nuclei and of p-chlorophenylalanine on the circadian rhythms of adrenocorticotrophic hormone and corticosterone in the plasma, and on locomotor activity of 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.

Temporal relationships between the diurnal rhythm of hypothalamic corticotrophin releasing factor, pituitary corticotrophin and plasma corticosterone in the rat.

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.

Inhibitory interactions between alpha 2-adrenergic and opoid but not NPY mechanisms controlling the CRF-ACTH axis in the rat.

Following a series of investigations supporting the concept that the brain stem catecholaminergic (CA) system played a major stimulatory role on both basal and stress-triggered states of the hypothalamic-pituitary-adrenocortical (HPA) axis, across alpha 1 and beta receptors and also via alpha 2 receptors, the present study was designed to gain a deeper insight into the fine mechanism of functional interactions between the alpha 2 receptors mediated CA system and two peptidergic mechanisms, both shown to take part in the stimulatory control of the HPA axis: beta-endorphin and NPY. All experiments were conducted on rats whose noradrenergic bundles, which directly innervate the CRF neurons and are strongly implicated in the ether stress-induced corticotropic response, had been bilaterally obliterated by an intracerebral (i.c.) injection of 6-OHDA (NAB-X). Results showed that: (1) the blockade of the ether-stress induced ACTH response resulting from NAB-X was entirely reversed by an intraventricular (i.c.v.) infusion of the alpha 2 antagonist idazoxan (10 nmol), which appeared ineffective under basal conditions; (2) the restoration of a normal post-stress ACTH surge by i.c.v. idazoxan was itself blunted by an i.c.v. pretreatment with naloxone (10 nmol), whereas an i.c. pretreatment with an anti-NPY serum appeared ineffective. These data suggest that, in addition to a stimulatory control exerted by postsynaptic alpha 2 receptors directly on CRF neurons, other alpha 2 receptors participate, exclusively under the stress conditions above, in a tonic inhibitory control, indirectly mediated to the HPA axis across a stimulatory opioid, but not NPY regulatory component.

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.

Intrahypothalamic infusion of interleukin-1 beta 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.

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

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

Prenatal treatment with 6-hydroxydopa and DSP 4: Biochemical, endocrinological and behavioural effects.

Biochemical, hormonal, and behavioural aspects of the effects of prenatal treatment (foetal days 18 and 19) with 6-hydroxydopa (2 × 40 mg/kg) and DSP 4 (2 × 20 mg/kg) have been studied in the rat. The results of the catecholamine assays suggest that the 6-hydroxydopa treatment produced a transient decrease of noradrenaline concentration in the cerebral cortex, and a long-lasting decrease in the cerebellum, whereas DSP 4 produced a long-lasting decrease in the cerebral cortex and a tendency towards increase in the cerebellum. Assays of adrenocorticotropic hormone and corticosterone indicated a general tendency towards increase in base line as well as in moderate stress situations. The results of behavioural tests indicated hyperactivity and/or hyperreactivity, but not restlessness, and partly supranormal sensorimotor performances.

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.

Increase of thyrotropin-releasing hormone immunoreactivity in the nucleus of the solitary tract following bilateral lesions of the hypothalamic paraventricular nuclei.

The effects of bilateral electrolytic lesions of hypothalamic paraventricular nucleus on thyrotropin-releasing hormone (TRH)-immunoreactive fibers of the nucleus of the solitary tract were studied by both immunocytochemistry and radioimmunoassay. Contrasting with a near disappearance of TRH immunoreactivity in the median eminence, both morphological and biochemical approaches demonstrate that such hypothalamic lesions induced significant increase of TRH immunoreactivity in the nucleus of solitary tract. These results confirm that TRH fibers of the nucleus of the solitary tract do not originate in the hypothalamic paraventricular nucleus (PVN). They further indicate that these TRH neurons projecting to the nucleus of the solitary tract are strongly influenced by neurons located within the PVN area.

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.

Opposite regulation by glucocorticoids of the alpha 1B- and alpha 2A-adrenoreceptor mRNA levels in rat cultured anterior hypothalamic slices.

In this study we investigated whether the expression of alpha1B- and alpha2A-adrenoreceptor mRNAs is differently modulated by glucocorticoids in rat cultured anterior hypothalamus slices. Using a semi-quantitative reverse transcription-polymerase chain reaction assay, the level of the alpha1B-adrenoreceptor mRNA was significantly reduced in slices cultured in steroid free-medium when compared with that measured in standard medium (i.e. containing basal adrenosteroid plasma concentrations). In contrast, the expression of the alpha2A-adrenoreceptor mRNA was markedly increased. Finally, the ratio of alpha1B- versus alpha2A-mRNA levels was about 1.7 and 0.7 in standard and steroid-free medium, respectively. These responses were completely reversed by supplementation with corticosterone. These findings provide the first evidence that in vitro glucocorticoids may regulate, in an opposite manner, the expression of the alpha1B-and alpha2A-adrenoreceptor mRNAs in the hypothalamus. This kind of regulation could be related to steroid-dependent changes in the noradrenergic control of neuroendocrine secretions.

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.

Glucocorticoids, transmitters and stress.

Many kinds of stress stimulate the neuroendocrine systems controlling catecholamine and glucocorticoid secretion. Stress-induced stimulation of CRF-containing neurons appears to be mediated by serotonergic, noradrenergic, and possibly other neuronal pathways. Stress can alter various neurobiological and endocrine functions, two essential components of the neuroendocrine responses being release of adrenalin from chromaffin cells of the adrenal medulla and secretion of glucocorticoids from adrenocortical cells. Activation of adrenal steroid secretion is mainly by a reflex activation of hypothalamic neurons, which stimulate ACTH secretion from the anterior pituitary. While the neuropeptide CRF plays a major role in the neuroendocrine response to stress, the neuronal signals which are responsible for the regulation of CRF neurons have not been completely elucidated. A number of other regulatory substances may also participate, alone or with CRF, in the control of ACTH secretion by pituitary corticotrophs, and there is increasing evidence that classical neurotransmitters or neuropeptides may act directly on adrenocortical cells to modulate corticosteroid secretion. We review the neuronal, neuroendocrine, and humoral pathways which participate in the regulation of stress-induced corticosteroid secretion, and present preliminary data on the effect of the tricyclic antidepressant, tianeptine in the response of the HPA axis to stress.

[Stress. Neurophysiologic studies]. / Stress. Données neurophysiologiques.

The hypothalamic-pituitary-adrenocortical (HPA) axis knowingly plays a key role in the physiological response to various stressing situations, owing to its gluconeogenetic function, and also, possibly, to its large range of modulating effects on a series of more specific defense mechanisms including the immune system, the latter effect serving to protect the organism against overactive defense reactions. It has long been accepted that under most aggressive conditions the CNS is an essential part of the mechanism controlling the subsequent acute stimulation of the HPA axis. In this line of research, the HPA axis reacts within a few minutes after a standard ether-stress, with a 6 fold increase over the baseline of CRH41 secretion, and at the periphery with 20-fold and 14-fold increases, respectively, in plasma concentrations of ACTH and corticosterone in unanesthetized free-moving rats. From a series of additional experiments a few selected brain structures emerged as basic components of the CNS control involved in the HPA axis stress responses: 1) The catecholamine (CA) producing neurons of the medulla oblongata (A1/C1 and A2/C2 nuclei) which directly innervate the CRH41-secreting neurons in the paraventricular nuclei (PVN) via the ventral noradrenergic bundle (VNAB), yield the major stimulatory pathway to the stress-induced CRH-ACTH surge. Not only was this surge dramatically obliterated by a neurotoxic deletion of the VNAB, with a local microinfusion of 6-hydroxydopamine (6-OHDA) but it was restored by intra-cerebroventricular (icv) microinfusions of adrenaline (AD) or noradrenaline (NA).(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoreactivity of hypothalamo-neurohypophysial neurons which secrete corticotropin-releasing hormone (CRH) and vasopressin (Vp): immunocytochemical evidence for a correlation with their functional state in colchicine-treated rats.

The specific immunoreactivity of neurons containing corticotropin-releasing hormone (CRH) or vasopressin (Vp) was studied both centrally, in the parvocellular division of the paraventricular nucleus, and distally, in the external median eminence. Control rats were compared with adrenalectomized rats and with animals supplemented with corticosterone or dexamethasone, either without additional treatment, or 24, and 48 h after an intraventricular injection of colchicine. In all groups of animals, colchicine induced a progressive and parallel decrease in both CRH and Vp immunoreactivity within the axons of the external median eminence. A semi-quantitative estimation of this axonal immunostaining showed that the decrease was clearly correlated with the axons' releasing activity according to the different functional states of the adrenocorticotropic system. Increased rates of hormonal release induced by adrenalectomy could be seen in the accelerated depletion of axonal immunoreactivity whereas corticosteroid supplementation had the opposite effect. Correspondingly, the progressive intensification of the CRH and Vp immunoreactivity within the perikarya following colchicine treatment was further markedly enhanced in adrenalectomized rats and diminished after corticosteroid supplementation. Taken together, these data suggest that in these neurons, perikaryal hormone synthesis may be closely related to the releasing activity of the axon terminals. They further point to appropriate colchicine treatment as useful tool for evaluating the functional state of CRH and Vp neurons of the parvocellular paraventricular nucleus under various experimental conditions.

[Effects of the light level and duration of the photoperiod on the retina of albino rats]. / Effets du niveau lumineux et de la durée de la photopériode sur la rétine du rat albinos

Female albino rats were exposed during 3 weeks to various photoperiodic sequences and levels of illumination. As long as the light span of the photoperiod did not exceed 12L-12D, the damaging effects of the light, even when intese (3,000 lx), on the retinal were relatively limited. On the other hand, the 14L-10D photoperiodic regimen that is usually used in standard rat animaleries produced marked degenerative lesions on the photoreceptors under either 1,200 or 3,000 1x. Under lighting schedules of 21L-15D or 28L-20D, the photoreceptors were heavily injured by 1,200 OR 3,000 1X. The degenerative process affected all the photoreceptive cells as well as their various components: rods, cones, nucleus and synaptic endings.