Studies of the hypothalamo-hypophyseal corticoliberin system. VII. Effect of ether stress and dexamethasone on the hypothalamo-hypophyseal-adrenal axis]

The effect of ether stress and dexamethasone on hypothalamo-hypophyseal-adrenal axis was investigated in sexually mature male Wistar rats. Separate group of rats was subjected to ether stress during 2 minutes. The remaining animals were treated with dexamethasone during 7 days. CRF-immunoreactive and vasopressin-immunoreactive neurons were detected within paraventricular nuclei and median eminence by using specific antibodies. Body weight of the rats as well as the weights of pituitary and adrenal glands were also measured. The levels of ACTH and corticosterone were determined in blood serum. It was found that the ether stress caused a considerable decrease in the amount of CRF-immunopositive substances in the outer layer of median eminence and a decrease in the amount of vasopressin-immunoreactive neurocytes in the parvocellular fragment of paraventricular nuclei. Dexamethasone administration caused an increase in the amount of CRF-immunopositive perikaryons within paraventricular nuclei and also an increase in vasopressin-immunopositive nerve fibers in median eminence.     

Monoamine fluorescence in the median eminence of the Japanese quail,Coturnix coturnix japonica,following medial basal hypothalamic deafferentation

Summary Monoamine fluorescence was examined in the ventral hypothalamus of the Japanese quail, Coturnix coturnix japonica after medial basal hypothalamic deafferentation. In sham-operated control birds, numerous yellow-green fluorescent fibers were observed in the median eminence and the nucleus tuberis. In the area of the paraventricular organ, a number of fluorescent fibers and cell bodies were observed. In birds with deafferented hypothalami, fluorescence disappeared both in the median eminence and the nucleus tuberis. In the area of the paraventricular organ, which was within the area of deafferentation, fluorescence of neuronal perikarya did not change, but fluorescent fibers decreased markedly in number. Disappearance of monoamine fluorescence in the median eminence and the nucleus tuberis is discussed in relation to the tanycyte absorptive function and gonadal development.Supported by Grants from the Ministry of Education to Professors T. Bando and H. Kobayashi, and a Grant from the Ford Foundation to Prof. H. Kobayashi.     

The origin of the vasopressinergic and oxytocinergic fibres of the external region of the median eminence of the rat hypophysis

Summary The origin of the vasopressinergic and oxytocinergic nerve fibres of the external region of the rat median eminence was investigated by means of hypothalamic lesions, adrenalectomy and immunocytochemistry. The results obtained in bilaterally adrenalectomized animals with complete, or incomplete, destruction of the suprachiasmatic nuclei showed that, at least, the great majority of the vasopressinergic and oxytocinergic nerve fibres of the external region of the rat median eminence do not originate from the suprachiasmatic nuclei. From the observations obtained in bilaterally adrenalectomized animals with total or subtotal destruction of both paraventricular hypothalamic nuclei, it appears that the paraventricular nuclei must be the origin of (nearly) all the vasopressinergic and oxytocinergic nerve fibres of the external region of the rat median eminence. The results strongly suggest that both types of fibres originate from all parts of the paraventricular nuclei.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk OnderzoekThe authors are much indebted to Prof. Dr. E. Kühn (Leuven) in whose laboratory the stereotactic operations were done     

Immunoreactive substance P content in median eminence and pituitary gland during oestrus, dioestrus and after anterior hypothalamic deafferentation

The aim of the present study was to find a relation between the Substance P (SP) content in the median eminence and the pituitary gland in rats and hypothalamic regulaton of the oestrus cycle. SP content was measured by radioimmunoassay in female rat pituitary and median eminence during oestrus, dioestrus and after anterior hypothalamic deafferentation. SP content in rat's pituitary did not vary significantly among individual groups of animals. The greatest average SP content in rat's median eminence was found in dioestrus. Both during cyclic and constant oestrus resulting from anterior hypothalamic deafferentation SP content in median eminence was lower than in dioestrus. This fact indicates the possibility of SP involvement in the control of cyclic LH-RH release from hypothalamus.     

Effect of morphine on hypothalamic corticotropin-releasing factor (CRF) and pituitary-adrenocortical activity

The effects of intraperitoneal and intra-third ventricular administration of morphine on the hypothalamic corticotropin-releasing factor (CRF) and the pituitary-adrenocortical activity were examined in unanesthetized, freely moving rats. Hypothalamic CRF was measured by rat CRF radioimmunoassay. Intraperitoneal or intra-third ventricular administration of morphine increased blood concentrations of ACTH and corticosterone while intraperitoneal administration tended to increase CRF concentration in the whole hypothalamus including the median eminence and intra-third ventricular administration increased CRF concentration in the hypothalamus excluding the median eminence. However, morphine seemed to inhibit the increase in CRF concentration in the hypothalamus induced by the ether-laparotomy stress. The main site of morphine action on the hypothalamo-pituitary-adrenocortical system seemed to be in the hypothalamic area.     

Cyto-immunological study of the somatostatin hypothalamic peptidergic system in the fox (Vulpes vulpes)]

Immunocytological investigations on the hypothalamus of the red fox with rabbit anti-SRIF showed large or small perikarya in the supra-optical and paraventricular nuclei. In the median eminence SRIF-positive nerve fibres and their endings have a characteristic distribution in areas different from those of LH-RH containing nerve fibres.     

Corticotropin-releasing hormone and pituitary-adrenocortical responses in chronically stressed rats

Brain corticotropin-releasing hormone (CRH) concentration and pituitary adreno-cortical responses were examined in chronically stressed rats: body restraint stress (6 h/day) for 4 or 5 weeks. Stressed rats showed a reduction in weight gain. CRH concentration in the median eminence and the rest of the hypothalamus were not different between control and chronically immobilized rats. The anterior pituitary adenocorticotropic hormone (ACTH) concentration was elevated in chronically stressed rats, whereas plasma ACTH and corticosterone levels did not differ from the control values. The median eminence CRH concentration was reduced to the same extent at 5 min after onset of ether exposure (1 min) in chronically immobilized rats and controls. However, plasma ACTH and corticosterone showed greater responses to ether stress in chronically immobilized rats than in control rats. Plasma ACTH and corticosterone responses to exogenous CRH were not different between control and chronically immobilized rats, while the response to arginine vasopressin (AVP) was significantly greater in chronically immobilized rats. These results suggest that chronic stress caused an increase in the ACTH-secreting mechanism and that pituitary hypersensitivity to vasopressin might at least be partly responsible for this.     

The influence of CRF and alpha-helical CRF(9-41) on rat fear responses, c-Fos and CRF expression, and concentration of amino acids in brain structures

In the present study we have examined the influence of intracerebroventricullary administered CRF, and a non-selective CRF receptor antagonist, α-helical CRF_(9–41), on rat conditioned fear response, serum corticosterone, c-Fos and CRF expression, and concentration of amino acids (in vitro), in several brain structures. Pretreatment of rats with CRF in a dose of 1μg/rat, enhanced rat-freezing response, and further increased conditioned fear-elevated concentration of serum corticosterone. Moreover, exogenous CRF increased aversive context-induced expression of c-Fos in the parvocellular neurons of the paraventricular hypothalamic nucleus (pPVN), CA1 area of the hippocampus, and M1 area of the frontal cortex. A different pattern of behavioral and biochemical changes was present after pre-test administration of α-helical CRF_(9–41) (10μg/rat): a decrease in rat fear response and serum corticosterone concentration; an attenuation of fear-induced c-Fos expression in the dentate gyrus, CA1, Cg1, Cg2, and M1 areas of the frontal cortex; a complete reversal of the rise in the number of CRF immunoreactive complexes in the M2 cortical area, induced by conditioned fear. Moreover, α-helical CRF_(9–41) increased the concentration of GABA in the amygdala of fear-conditioned rats. Altogether, the present data confirm and extend previous data on the integrative role of CRF in the central, anxiety-related, behavioral and biochemical processes. The obtained results underline also the role of frontal cortex and amygdala in mediating the effects of CRF on the conditioned fear response.     

Identification,in the external region of the rat median eminence,of separate neurophysin-vasopressin and neurophysin-oxytocin containing nerve fibres

Summary Immuno-enzyme cytochemical investigations, using single and double staining techniques, showed that the external region of the rat median eminence contains separate neurophysin-vasopressin fibres and neurophysinoxytocin fibres. These neurophysin-hormone containing nerve fibres are influenced by bilateral adrenalectomy and by colchicine treatment. The external region of the median eminence of the homozygous Brattleboro rat contains neurophysin-oxytocin fibres. It does not contain immuno-reactive neurophysin-vasopressin fibres. Bilateral adrenalectomy also influences the neurophysin-vasopressin containing neurons of the suprachiasmatic nuclei. In the neurons of the parvicellular part of the rat hypothalamic paraventricular nuclei, staining for vasopressin and for oxytocin is completely absent.     

Immunocytochemistry of luteinizing hormone-releasing hormone,vasopressin, and corticotropin following deafferentation of the basal hypothalamus of the male rat brain

Summary Luteinizing hormone-releasing hormone (LHRH), vasopressin, and corticotropin systems were examined by immunocytochemical methods in male rats 2 to 20 days after deafferentation of the basal hypothalamus. Axonal degeneration of the vasopressin system (whose perikarya lie rostral to the island) and the corticotropin system (whose perikarya lie within the island) was examined and compared with the response of the LHRH system.Vasopressin immunoreactive staining was absent in the internal zone of the median eminence 10 and 20 days after deafferentation. Disruption of the efferent projections of the opiocortin system caused the loss of almost all fiber staining outside the island by the 5th postoperative day. LHRH staining in the median eminence was modestly reduced in 5 days, considerably reduced in 10 days and negligible 20 days after deafferentation. At 10 and/or 20 days after deafferentation densely stained fibers of all three systems were observed on both sides of the cut. Invasive vasopressinergic fibers reached the lateral median eminence by the 20th postoperative day.This study reports on the response of three neuropeptide systems after complete deafferentation and demonstrates that regeneration can occur across the knife cut.Supported by: NIH Grants AM-22029 and Program Project NS-15345, and USPHS grant 5T32 GM-07136-06The authors wish to express their appreciation to Ms. Barbara Dolf for her technical assistance.     

Expression of corticosteroid-binding protein in the human hypothalamus, co-localization with oxytocin and vasopressin.

Corticosteroid-binding globulin, a specific steroid carrier in serum with high binding affinity for glucocorticoids, is expressed in various tissues. In the present study, we describe the immunocytochemical distribution of this protein in neurons and nerve fibers in the human hypothalamus. CBG immunoreactive perikarya and fibers were observed in the paraventricular, supraoptic, and sexual dimorphic nuclei in the perifornical region, as well as in the lateral hypothalamic and medial preoptic areas, the region of the diagonal band, suprachiasmatic and ventromedial nuclei, bed nucleus of the stria terminalis and some epithelial cells from the choroid plexus and ependymal cells. Stained fibers occurred in the median eminence and infundibulum. Double immunostaining revealed a partial co-localization of corticosteroid-binding globulin with oxytocin and, to a lesser extent, with vasopressin in the paraventricular and the supraoptic nuclei. Double immunofluorescence staining showed coexistence of these substances in axonal varicosities in the median eminence. We conclude that neurons of the human hypothalamus are capable of expressing corticosteroid-binding globulin, in part co-localized with the classical neurohypophyseal hormones. The distribution of CBG immunoreactive neurons, which is widespread but limited to specific nuclei, indicates that CBG has many physiological functions that may include neuroendocrine regulation and stress response.     

Glucagon-related peptides in the rat hypothalamus

Summary Immunohistochemically, nerve fibers and terminals reacting with anti-N-terminal-specific but not with anti-C-terminal-specific glucagon antiserum were observed in the following rat hypothalamic regions: paraventricular nucleus, supraoptic nucleus, anterior hypothalamus, arcuate nucleus, ventromedial hypothalamic nucleus and median eminence. Few fibers and terminals were demonstrated in the lateral hypothalamic area and dorsomedial hypothalamic nucleus. Radioimmunoassay data indicated that the concentration of gut glucagon-like immunoreactivity was higher in the ventromedial nucleus than in the lateral hypothalamic area. In food-deprived conditions, this concentration increased in both these parts. This was also verified in immunostained preparations in which a marked enhancement of gut glucagon-like immunoreactivity-containing fibers and terminals was observed in many hypothalamic regions. Several immunoreactive cell bodies were found in the ventromedial and arcuate nuclei of starved rats. Both biochemical and morphological data suggest that glucagon-related peptides may act as neurotransmitters or neuromodulators in the hypothalamus and may be involved in the central regulatory mechanism related to feeding behavior and energy metabolism.     

Tanycyte absorption affected by the hypothalamic deafferentation in Japanese quail,Coturnix coturnix japonica

In Japanese quail, Coturnix coturnix japonica the tanycytes of the median eminence absorbed peroxidase injected into the third ventricle. The number of tanycytes showing peroxidase reaction was greater in the posterior median eminence than in the anterior median eminence. Following hypothalamic deafferentation, the tanycyte absorption was augmented both in the posterior and anterior median eminence. These findings suggest that axons of some neurons, which have inhibitory action on the tanycyte absorption, were transected by deafferentation resulting in augmentation of tanycyte absorption. A considerable number of ependymal cells lining the upper portion of the third ventricle and those of the pars nervosa also absorbed peroxidase. In birds with a deafferented hypothalamus, photostimulated ovarian growth was completely inhibited.     

The distribution of monoamines in the hypothalamus of the Japanese quail Coturnix coturnix japonica

Summary The distribution of monoamines in the hypothalamus of the Japanese quail (Coturnix coturnix japonica) has been studied using a histochemical fluorescence technique. In the posterior hypothalamus catecholamine-containing nerve fibres are localised in the nucleus tuberis and nucleus hypothalamicus posterior medialis and are linked by fluorescent tracts running in the stratum cellulare internum. Further tracts may be traced from the nucleus tuberis around the base of the third ventricle to the sub-ependymal layer of the median eminence, where they then appear to pass through the hypothalamo-hypophysial neurosecretory tract to terminate in the palisade zone on the portal vascular bed. The innervation of the palisade layer by catecholamines is sparse. The fluorescent terminals are spread evenly throughout both the anterior and posterior divisions of the median eminence. There is no monoamine innervation of the pars nervosa. The paraventricular organ has both 5-hydroxytryptamine- and catecholamine-containing cell bodies and axons may be traced into the region of the nucleus hypothalamicus posterior medialis. In the anterior hypothalamus the neurosecretory paraventricular nucleus contains many catecholamine nerve fibres and terminals. These are linked by fibre tracts to the nucleus basalis and to the nucleus hypothalamicus posterior medialis. The supraoptic nucleus is less well innervated although a dense accumulation of fibres lies in the preoptic recess. The latter is thought to give rise to long axons which pass in association with the neurosecretory tract to end in the nucleus tuberis.Supported by a Grant (AG 24/36) from The Agricultural Research Council. We are indebted to Dr. G. A. Clayton, Institute of Animal Genetics, University of Edinburgh, for supplying the birds.     

Immunoreactivity to vasoactive intestinal polypeptide (VIP) and thyreotropin-releasing hormone (TRH) in hypothalamic neurons of the domesticated pigeon (Columba livia). Alterations following lactation and exposure to cold

Summary The distribution of VIP- and TRH-immunoreactivity in neurons and processes within the hypothalamus of the pigeon was investigated with light-microscopic immunocytochemical techniques. Most of the VIP-containing neurons are concentrated in the middle and caudal parts of the hypothalamus, with the greatest concentration of perikarya occurring in the medial and lateral part of the ventromedial hypothalamic nucleus and the infundibular nucleus. These cells give rise to axons that seem to extend into the median eminence. An extensive network of VIP-immunoreactive fibers and varicosities occupy the external layer of the median eminence. The majority of TRH-containing neurons is found in the anterior hypothalamus with the greatest concentration of cells in the magnocellular preoptic, medial preoptic, suprachiasmatic and paraventricular nuclei. TRH-immunoreactive fibers and varicosities form a dense arborization in the external layer of the median eminence. Lactation seems to induce substantial changes in VIP as well as in TRH-immunostaining in the median eminence and other hypothalamic regions as compared to control, sexually active animals. Furthermore, TRH-immunoreactivity decreased in the median eminence following 60-min exposure to cold. These results suggest that VIP- and TRH-containing pathways in the pigeon hypothalamus are involved in the mediation of neuroendocrine responses.     

Corticotropin-releasing hormone-like immunoreactivity in the brain of the snake Bothrops jararaca

The distribution of corticotropin-releasing hormone in the brain of the snake Bothrops jararaca was studied immunohistochemically. Immunoreactive neurons were detected in telencephalic, diencephalic and mesencephalic areas such as dorsal cortex, subfornical organ, paraventricular nucleus, recessus infundibular nucleus, nucleus of the oculomotor nerve and nucleus of the trigeminal nerve. Immunoreactive fibres ran along the hypothalamo-hypophysial tract to end in the outer layer of the median eminence and the neural lobe of the hypophysis. In general, immunoreactive fibres occurred in the same places of immunoreactive neurons. In addition, immunoreactive fibres were observed in the septum, amygdala, lamina terminalis, supraoptic nucleus, nucleus of the paraventricular organ, ventromedial hypothalamic nucleus and interpeduncular nucleus. These results indicate that, as for other vertebrates, corticotropin-releasing hormone in B. jararaca brain, besides being a releasing hormone, may also act as a central neurotransmitter and/or neuromodulator.     

Medianosomes as integrative units in the external layer of the median eminence. Studies on grf/catecholamine and somatostatin/catecholamine interactions in the hypothalamus of the male rat

Somatostatin/catecholamine as well as growth hormone releasing factor/catecholamine interactions have been characterized in the hypothalamus and the preoptic area using morphometrical and quantitative histofluorimetrical analyses.

• 1.(1) The morphometrical analysis of adjacent coronal sections of the rat median eminence demonstrated a marked overlap of somatostatin and tyrosine hydroxylase immunoreactive nerve terminals as well as of growth hormone releasing factor and tyrosine hydroxylase immunoreactive nerve terminals in the medial and lateral palisade zones of the rostral and central parts. Furthermore, the studies on codistribution of growth hormone releasing factor and tyrosine hydroxylase immunoreactivity indicate that only a limited proportion of the growth hormone releasing factor and the dopamine nerve terminals may costore dopamine and growth hormone releasing factor respectively in the medial and lateral palisade zones (see Meister et al., 1985).
• 2.(2) Intravenous injections of somatostatin 1–14 (100 μg/kg, 2 h) into the hypophysectomized male rat produced an increase in dopamine utilization in the medial and lateral palisade zones of the median eminence.
• 3.(3) Intravenous injections of rat hypothalamic growth hormone releasing factor (80 μg/kg, 2 h) in the hypophysectomized male rat did not change dopamine utilization in the median eminence but increased noradrenaline utilization in the ventral zone of the hypothalamus and produced a depletion of noradrenaline stores in the paraventricular hypothalamic nucleus.
• 4.(4) Intravenous injections of human pancreatic growth hormone releasing factor 1–44 (80 μg/kg, 2 h) in the hypophysectomized male rat did not change dopamine utilization in the median eminence, but reduced noradrenaline utilization in the subependymal layer and increased noradrenaline utilization in the suprachiasmatic preoptic nucleus.

The combined results of the present and previous studies have led us to put forward the medianosome concept. The medianosome is defined as an integrative unit, which consists of well defined aggregates of transmitter identified nerve terminals interacting with one another in the external layer of the median eminence. Our present data indicate the existence of putative medianosomes consisting predominantly of growth hormone releasing factor nerve terminals costoring dopamine as well as of somatostatin and dopamine nerve terminals, which interact locally to control growth hormone secretion. A complementary control of growth hormone secretion may be exerted by noradrenaline mechanisms in the subependymal layer, in the ventral zone and/or in the suprachiasmatic preoptic nucleus. However, further analyses in view of the differential effects seen with the present doses of rat hypothalamic and human pancreatic growth hormone releasing factor have to be done. The results also indicate the possible existence of growth hormone releasing factor receptors in the median eminence which may participate in the feedback control of the growth hormone releasing factor immunoreactive neurons in the ventral zone of the hypothalamus.     

Adrenomedullin-like immunoreactivity in the rat hypothalamo-neurohypophysial tract.

Adrenomedullin-like immunoreactivity in the hypothalamo-neurohypophysial tract in colchicine-treated and hypophysectomized rats was examined by immunohistochemistry. Adrenomedullin-like immunoreactive (AM-LI) neurons were localized in the hypothalamic areas, including the paraventricular nuclei and the supraoptic nuclei. Abundant AM-LI fibers and varicosities were found in the hypothalamoneurohypophysial tract and the internal zone of the median eminence in the colchicine-treated and hypophysectomized rats, whereas in control rats few AM-LI fibers were observed. These results suggest that the axons of the AM-LI neurons in the hypothalamus may terminate in the neurohypophysis.     

Stress and Colchicine do not Induce the Release of Galanin from the External Zone of the Median Eminence

The aim of the present study was to verify the hypothesis that stress exposure modifies the content and release of galanin in the hypothalamic paraventricular nucleus and the median eminence. Colchicine and immobilization served as stress stimuli, and the changes in galanin immunoreactivity were compared with those in corticotropin-releasing hormone and vasopressin. In control animals, a limited number of galanin perikarya were identified in the paraventricular nucleus. The high dose (75 g) of colchicine enhanced galanin in both parvicellular and magnocellular subdivisions, as analysed 72 h later. In the median eminence, galanin accumulated only in the external zone. High- dose colchicine did not affect galanin, while corticotropin- releasing hormone and vasopressin were depleted from the median eminence. Immobilization (120 min) neither alone nor in combination with colchicine influenced galanin immuno-reactivity in the external zone. The low dose of colchicine induced an unexpected accumulation of galanin in the internal zone of the median eminence, which was further increased by subsequent immobilization. In the external zone, low-dose colchicine induced a complete disappearance of vasopressin, substantial depletion of corticotropin-releasing hormone and no changes in galanin immunoreactivity. The present studies demonstrate that galanin in the external zone of the median eminence is not influenced by colchicine or by immobilization stress.