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.     

The characteristics of the hormonal profile of the central links in the regulation of adrenocortical function in rat postnatal ontogeny]

Radioimmune assay has been made of the ACTH content of the blood and adenohypophysis, as well as the content of vasopressin in the blood, median eminence, the posterior and intermediate hypophysis in 1, 3, 5, 7, and 20 days old rats and adult male rats. Comparison of the hormonal level in neurohaemal areas with that in the blood plasma suggests that transadenohypophyseal path of hypothalamic neurohormonal control of the adrenocortical function becomes essentially important only after formation of capillary plexus in the external zone of the median eminence.     

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.     

Effects of lithium on the hypothalamo-pituitary-adrenal axis

The effect of lithium on the hypothalamo-pituitary-adrenal axis was studied in vivo and in vitro. The levels of plasma vasopressin, ACTH and corticosterone increased after the administration of lithium (LiCl 4 mmol/kg BW, 11 days) in rats, while the tissue vasopressin concentration in the median eminence, the rest of the hypothalamus and the posterior pituitary was decreased. The CRF concentration in the posterior pituitary increased markedly, but it did not change significantly in the median eminence or the rest of the hypothalamus. The elevated plasma ACTH level might be at least partly due to the increased vasopression secretion. Lithium stimulated ACTH secretion per se and also enhanced vasopressin-induced ACTH secretion in cultured pituitary cells and in half pituitary incubations, while it did not affect CRF-induced ACTH secretion. Lithium inhibited CRF-induced cAMP accumulation in half pituitary incubations, while lithium and vasopressin did not affect cAMP accumulation per se or even when administered together. The results suggest that lithium-induced ACTH release is via a cAMP-independent mechanism. Thus, it is possible that lithium stimulates ACTH release by acting directly on the corticotroph, stimulating vasopressin release and potentiating vasopressin-induced ACTH release.     

Dual effects of (D-Ala2,Met5)-enkephalinamide on CRF and ACTH secretion

An intra-third ventricular administration of (D-Ala2,Met5)-enkephalinamide (DALA) did not elevate plasma ACTH and corticosterone levels in unanesthetized freely moving rats, but intra-third ventricular administration of DALA and methionine (Met)-enkephalin potentiated a mild stress (hanging for 10 or 30 sec)-induced plasma ACTH and corticosterone elevations in unanesthetized freely moving rats. DALA and Met-enkephalin seemed to stimulate CRF release from the median eminence to increase plasma ACTH, as the CRF concentration in the median eminence area was reduced after injection in these stressed rats. When hypothalamic tissues were perifused in vitro, DALA (1-100 ng/ml) reduced the release of CRF. These results suggest that the opiates seem to have a dual effect on the CRF-ACTH system depending on which action overrides the other.     

Time course of vasopressin and oxytocin secretion after stress in adrenalectomized rats.

To characterize the participation of vasopressin (AVP) and oxytocin (OT) in hypothalamus-pituitary-adrenal regulation after adrenalectomy (ADX), we evaluated corticosterone, ACTH, AVP and OT plasma concentrations and AVP and OT content of the paraventricular nucleus (PVN) at different periods (3 h, 1, 3, 7 and 14 days) in sham or ADX rats under basal conditions and after immobilization stress. ADX animals showed undetectable corticosterone levels, while sham animals showed a marked increase in corticosterone and ACTH 3 h after surgery, then lowering to basal control levels. ADX rats showed high basal ACTH levels with a triphasic response without changes after immobilization. After three hours, the ADX group showed higher OT levels than the sham group. OT was increased after immobilization stress in sham and ADX groups. AVP plasma levels did not change throughout the basal or stress studies in either group. There was a decrease in hypothalamic AVP content 1 and 3 days after ADX under basal and stress conditions. Plasma osmolality showed a significant decrease in the ADX group at 3, 7, and 14 days. In conclusion, there are different pituitary-adrenal axis set points after removal of the glucocorticoid negative feedback. The role of vasopressinergic and oxytocinergic neurons in the ACTH secretion after ADX or immobilization stress appears to differ. Magnocellular AVP is unlikely to contribute to ACTH secretion in response to ADX or immobilization stress. On the other hand, OT is elicited by immobilization stress and might contribute to the ACTH secretion during short-term ADX.     

Structure of the tail fin in teleosts

The influence of adrenalectomy and administration of hypertonic saline on the amount of vasopressin, oxytocin, and neurophysin contained in the median eminence and the neural lobe of rats was studied by means of the following methods: (i) morphometric and microphotometric analyses of aldehyde fuchsin-stained histological sections of the neurohypophysis; (ii) immunohistochemical demonstration of vasopressin, oxytocin, and neurophysin in the neurohypophysis, and (iii) radioimmunological measurement of vasopressin and oxytocin in extracts of the median eminence and the neural lobe. Adrenalectomy increases the amount of vasopressin and neurophysin in the external layer of the median eminence but does not change the content of oxytocin. It has no influence on the amount of vasopressin, oxytocin, and neurophysin demonstrable in the inner layer of the median eminence and in the neural lobe two weeks after the operation. Hypertonic saline markedly diminishes the vasopressin, oxytocin, and neurophysin content of the inner layer of the median eminence and the neural lobe but reduces only slightly, if at all, the amount of vasopressin and neurophysin in the outer layer of the median eminence. The findings support the concept that osmotic stress reduces only the vasopressin and oxytocin content of the hypothalamus-neural lobe system and has no or only little influence on the vasopressin content of the outer layer of the median eminence.     

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.     

Transitory coexistence of oxytocin and vasopressin in the hypothalamo neurohypophysial system of parturient rats.

We used in situ hybridization and immunocytochemistry to investigate a possible coexistence of vasopressin and oxytocin in hypothalamic neurons of parturient rats. We found that a fraction of magnocellular neurons in the paraventricular and supraoptic nuclei contained immunostaining for both peptides as well as oxytocin and vasopressin mRNA hybridization. Colocalization of immunoreactive vasopressin and oxytocin could be observed in some of the Herring bodies in the median eminence and the posterior lobe. No coexistence of vasopressin and oxytocin was found in pregnant or in lactating animals, indicating that the observed coexistence is transitory, perhaps mediated through changing hormonal conditions peri partum.     

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.     

Immunocytochemical study of neurohypophysial peptides during corticotropic maturation of infant rats

Summary Immunocytochemical localization of vasopressin (VP), oxytocin (OXY) and neurophysins I and II (NI and NII) in the hypothalamus of growing rats revealed several new features. Hormones and neurophysins were present in magnocellular neurons of newborn rats: VP and NII in a first neuronal population, OXY and NI in a second neuronal population. For the first three days after birth, the ratio of detected VP to detected NII increased whereas the ratio of detected OXY to detected NI appeared constant. It was obvious that maturation of the OXY/NI magnocellular system occurred later than maturation of the VP/NII magnocellular system. During the first three weeks of development, VP/NII fibres in the median eminence were separated into two distinct groups: the hypothalamo-neurohypophysial tract in the internal part of the median eminence, and pericapillary endings heavily loaded with VP and NII in the external part of median eminence. OXY/NI fibres did not show this particular distribution. Therefore, during this post-natal period, loading of infundibular VP endings was related to the increase of ACTH synthesis demonstrated by various authors. Therefore, we suppose that simultaneous participation of VP and corticotropin-releasing hormone does not only appear during experimental and chronic stimulation of ACTH synthesis (after adrenalectomy, for example) but that it also exists during physiological stimulation of corticotropic function.At birth, parvocellular suprachiasmatic neurons were devoid of VP and NIL A slight immunological reactivity appeared in 6-to 10-day-old rats and it became equivalent to the immunological staining in the adult rat during the third post-natal week. Thus, the appearance of suprachiasmatic VP preceded the differentiation of the rhythmic activity of the pituitary adrenal axis. These chronological relationships suggest the involvement of the suprachiasmatic VP-related-peptide in the circadian periodicity of the corticotropic function.     

Vasopressin neurons grafted into Brattleboro rats: Viability and activity

Blocks of the anterior hypothalamus containing vasopressin neurons were grafted from normal 17-day-old rat fetuses into the median eminence of adult female rats with a congenital deficiency of vasopressin neurons (Brattleboro strain rats). Immunocytochemical staining of the transplants 40 days after grafting demonstrated the presence of magnocellular neurons which stained positively for vasopressin and neurophysin. Axons from these neurons could be traced into the median eminence and the primary capillary plexus of the hypothalamo-hypophyseal portal system. Water consumption decreased by as much as 63% in animals carrying viable grafts. The observation that water consumption decreased and remained depressed in hosts carrying viable grafts along with the immunocytochemical data suggest that the transplanted neurons are synthesizing, storing, and releasing biologically active VP.     

Cardiovascular responses to V1-vasopressinergic antagonism in conscious versus anesthetized rats

Experiments were performed to compare the possible effect of endogenous arginine vasopressin on renal hemodynamics between anesthetized, surgically stressed rats and conscious rats. Animals were instrumented with arterial and venous catheters as well as with a pulsed Doppler flow probe on the left renal artery. The rats were studied under the following conditions: (1) conscious and unrestrained; (2) anesthetized only; (3) anesthetized with minor surgical stress; and (4) anesthetized with major surgical stress. Two anesthetic agents were also compared, a mixture of ketamine (110 mg/kg i.m.) and acepromazine (1 mg/kg i.m.), and sodium pentobarbital (50 mg/kg i.p.). Baseline mean arterial blood pressure was significantly higher in pentobarbital-anesthetized rats following surgical stress compared with conscious animals, but blood pressure was not affected by ketamine-acepromazine anesthesia. After baseline measurements of blood pressure, heart rate, and renal blood flow, a specific V1-vasopressinergic antagonist (d(CH2)5Tyr(Me) arginine vasopressin, 10 mg/kg i.v.) was administered to each group. Mean arterial blood pressure, heart rate, and renal blood flow were monitored for an additional 15 min. Mean arterial blood pressure and renal blood flow decreased after V1 antagonism in ketamine-acepromazine-anesthetized rats with major surgical stress, but were not affected in pentobarbital-anesthetized animals. Heart rate and renal vascular resistance were not affected following V1 blockade with either anesthetic agent. These data suggest that arginine vasopressin plays a role in maintaining blood pressure and renal perfusion in ketamine-acepromazine-anesthetized rats following surgical stress, but does not have a significant effect on renal hemodynamics under pentobarbital anesthesia.     

Nitric oxide mediates the interleukin-1beta- and nicotine-induced hypothalamic-pituitary-adrenocortical response during social stress.

We investigated the role of nitric oxide (NO) in the interleukin 1beta (IL-1beta) and nicotine induced hypothalamic-pituitary-adrenal axis (HPA) responses, and a possible significance of CRH and vasopressin in these responses under basal and social stress conditions. Male Wistar rats were crowded in cages for 7 days prior to treatment. All compounds were injected i.p., nitric oxide synthase (NOS) inhibitors, alpha-helical CRH antagonist and vasopressin receptor antagonist 15 min before IL-1beta or nicotine. Identical treatment received control non-stressed rats. Plasma ACTH and serum corticosterone levels were measured 1 h after IL-1beta or nicotine injection. L-NAME (2 mg/kg), a general nitric oxide synthase (NOS) inhibitor, considerably reduced the ACTH and corticosterone response to IL-1beta (0.5 microg/rat) the same extent in control and crowded rats. CRH antagonist almost abolished the nicotine-induced hormone responses and vasopressin antagonist reduced ACTH secretion. Constitutive endothelial eNOS and neuronal nNOS inhibitors substantially enhanced the nicotine-elicited ACTH and corticosterone response and inducible iNOS inhibitor, aminoguanidine, did not affect these responses in non-stressed rats. Social stress significantly attenuated the nicotine-induced ACTH and corticosterone response. In crowded rats L-NAME significantly deepened the stress-induced decrease in the nicotine-evoked ACTH and corticosterone response. In stressed rats neuronal NOS antagonist did not alter the nicotine-evoked hormone responses and inducible NOS inhibitor partly reversed the stress-induced decrease in ACTH response to nicotine. These results indicate that NO plays crucial role in the IL-1beta-induced HPA axis stimulation under basal and social stress conditions. CRH and vasopressin of the hypothalamic paraventricular nucleus may be involved in the nicotine induced alterations of HPA axis activity. NO generated by eNOS, but not nNOS, is involved in the stress-induced alterations of HPA axis activity by nicotine.     

Degradation of CCK-8 by rat synaptosomal peptidases

By use of an antiserum raised against conjugated ovine corticotropin releasing factor (CRF_1–41), nerve fibres can be stained immunocytochemically in the external zone of the median eminence of rats. The presence of CRF-immunoreactive (CRF_i) nerve fibres and the plasma corticosterone response to ether stress were studied in rats 6–7 days after making various types of lesions in the hypothalamus. Complete anterolateral deafferentation of the mediobasal hypothalamus caused complete disappearance of CRF_i fibres from the median eminence and blocked the corticosterone response to stress. Incomplete anterolateral hypothalamic deafferentation did not prevent the stress-induced increase of corticosterone and in these rats, part of the CRF_i nerve fibres remained intact. A horizontal cut placed ventral to the paraventricular nuclei, completely prevented the corticosterone response in those rats that showed a complete disappearance of CRF_i nerve fibres from the median eminence. Some rats however, still exhibited CRF_i nerve fibres and these animals responded to stress with increased corticosterone levels. A similar horizontal cut made just dorsal to the paraventricular nuclei affected neither the corticosterone response to stress nor the appearance of CRF_i nerve fibres in the median eminence. We conclude that the presence of CRF_i nerve fibres in the median eminence is a prerequisite for rats to show a pituitary-adrenal response to ether stress and therefore represents the first functional evidence for the role of these hypothalamic CRF_i-neurons.     

Neural lobe function in aged Wistar/Tw strain rats showing polydipsia and polyuria

Neural lobe function in male rats of the Wistar/Tw strain was studied at 3, 7 and 16-18 months of age. A significant rise in the serum arginine vasopressin (AVP) level was noted in 16-18-month-old rats showing polydipsia and polyuria. The content and concentration of AVP in the neural lobe of aged rats were significantly less than those of younger animals (3 and 7 months). These results point out an enhancement of AVP release from the neural lobe of aged rats. The reduction in urinary volume in aged rats subjected to 24 hours of water deprivation was less than those in younger animals. No increase in urinary sodium, potassium and chloride concentrations was observed in aged rats, and the decrease in electrolyte excretion from urine during the dehydration period was less in aged rats than younger ones. These results suggest that the antidiuretic response to osmotic stimuli was reduced in aged rats. The administration of AVP to aged rats resulted in a significant decrease in water intake and urinary volume, but AVP administration did not induce any change in the electrolyte balance. Therefore, it is concluded that the main cause of the development of polydipsia and polyuria is the decline in renal function but not in neurosecretory activity, although exogenous AVP can effectively reduce water intake and urinary output in aged rats.     

Long-term ACTH induced diminished responsiveness of prolactin secretion to morphine

The effect of morphine on plasma prolactin level and on dopamine turnover in the median eminence was studied using adult male rats chronically treated with ACTH. It was found that the ACTH pretreatment caused a decrease in the effect of morphine on prolactin secretion and prevented the inhibitory effect of morphine on dopamine turnover measured in the median eminence. The prolonged ACTH administration did not influence the prolactin content of the pituitaries and the in vitro dopamine sensitivity of lactotroph cells. Acute dexamethasone injection did not change the morphine-caused prolactin release. These results suggest that chronic ACTH treatment (possibly via corticosterone hyperproduction) elicits an opiate-tolerance like state of tuberoinfundibular dopaminergic neurons.     

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.     

Nighttime immunoreactive somatostatin content of the median eminence in hypo- and hyperthyroid rats

The median eminence content of immunoreactive somatostatin (IRS) was measured by radioimmunoassay in 107 male albino rats, who were either hypothyroid after surgical thyroidectomy (N = 38), hyperthyroid following a subcutaneous implant of 5 mg of L-thyroxine (N = 36), or otherwise untreated (N = 33). Thyroid function was assessed by determining plasma levels of T4 and TSH from trunk blood obtained at the time of decapitation. Subgroups of animals from the 3 groups were killed either before (1800 hr), during (2200, 0200, 0400 hr), or after the dark portion of their 14:10 LD photoperiod. Although no changes in median eminence IRS content were found throughout the period of study within any of the 3 groups, hypothyroid animals (297.23 +/- 13.47 ng per ME; 620.41 +/- 58 ng IRS/mg protein) had a significantly lower median eminence IRS concentration than untreated rats (355.86 +/- 16.55 ng of IRS per ME, P less than 0.01; 906.86 +/- 96.38 ng IRS/mg protein, P less than 0.05) and hyperthyroid animals (384.12 +/- 14.67 ng per ME, P less than 0.001; 874.1 +/- 104.5 ng IRS@mg protein, P less than 0.05). It is concluded, that the feedback of thyroid hormones on the hypothalamic-pituitary axis during thyroid hormone excess in vivo, contrary to what occurs in hypothyroidism, is probably independent of hypothalamic somatostatin.     

Maturation of the hypothalamo-neurohypophysial system

Summary Sections of the hypothalamus, median eminence and pituitary from fetal and neonatal rats were examined with the immunoperoxidase staining technique and light microscopy. Purified antisera raised against vasopressin and oxytocin, and antisera cross-reactive with rat neurophysin were used to localize these antigens in the hypothalamo-neurohypophysial system (HNS). Neurophysin was detected throughout the HNS of the 18-day fetal rat. Vasopressin was present in the hypothalamus and pituitary of the 19-day fetus, and in the median eminence of the 4-day neonate. Oxytocin was not detected in the pituitary until 1–2 days after birth, in the hypothalamus after 4 days, and in the median eminence after 8 days. During the first days after birth the supraoptic nucleus was more mature than the paraventricular nucleus. The HNS did not approach maturity until at least 7 days after birth. The relative maturity of the supraoptic nucleus compared with the paraventricular nucleus, and the detection of vasopressin before oxytocin are evidence for the one-neuron-one-hormone theory. The data do not exclude the possibility that the fetal hypothalamo-neurohypophysial system, and perhaps the fetal hormone, vasotocin, affect the initiation and course of parturition.This work was financed by the Medical Research Council of New Zealand