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     

Immunocytochemical study of the hypothalamo-neurohypophysial system

Summary An antiserum cross-reactive against ovine neurophysins-I-II and -III has been used in conjunction with the immunoperoxidase histochemical procedure to localize the cells of the sheep paraventricular (PVN) and supraoptic nuclei (SON). In order to describe the topographical distribution of the SON and PVN a study was made on the serial sections cut (a) transversely from rostral to caudal positions and (b) sagittally from lateral to medial positions of the hypothalamus.The cells of the SON, when examined in the transverse aspect, extended approximately 1900 caudally and when examined in the sagittal plane were contained within a lateral-medial distance of 4830 . In each case the SON cells lay adjacent to the optic chiasm.As sections were cut transversely, the cells of the PVN first appeared in a rostral position defined as 0 and close to the ventral lining of the third ventricle. This general ventral and ventro-lateral distribution of cells maintained up to a caudal distance of approximately 840 . From positions 1260–2310 there was a dramatic dorsal shift of the PVN cells which by this time had also extended laterally. The total rostral-caudal distance occupied by the PVN cells was 3150 . That the lateral-medial distance occupied by the PVN was small (1050 ) was determined on examining the magnocellular nuclei in sagittal section.This work was financed by a grant awarded by the Medical Research Council of New Zealand     

Neurosecretion in the external and internal zone of the median eminence of the cat and dog

Summary The immunoglobulin-enzyme bridge technique, in association with rabbit antiporcine neurophysin-II has been applied to the median eminence of the dog and cat in order to study the distribution of neurophysin-like antigens throughout this area of the brain and correlate the findings with the corresponding distribution of neurosecretory material (NSM) as revealed by the crotonaldehyde fuchsin stain.Neurophysin and NSM were both present in the hypothalamo-supraoptico-neurohypophysial system—the pathway taken by oxytocin, vasopressin and neurophysin from the hypothalamus to the posterior pituitary lobe.Whereas the tuberoinfundibular tract of the median eminence was almost devoid of NSM, the presence of neurophysin-like material was clearly evident using immunoperoxidase histochemistry. The significance of a protein in the external median eminence possessing determinants cross-reactive against anti-neurophysin serum is discussed.This work was financed by a grant from the Medical Research Council of New Zealand.     

Fine structural characteristics of neurophysin-positive perivascular plexus that develop in the rat hypothalamus following interruption of the hypothalamo-neurohypophysial tract

Summary Transection of neurosecretory axons of the hypothalamo-neurohypophysial tract within the hypothalamus by stereotactic grafts of various tissues or knife cuts induced the development of neurophysin-positive plexus around arterioles, venules and capillaries in the vicinity of these grafts or cuts. These plexus ranged from single axons to densely woven networks and tended to increase progressively with time after experimental intervention. At the fine structural level, typical neurosecretory axon profiles were either abutting the perivascular connective tissue space or located within it. They were usually-accompanied by astrocyte processes or microglial cells. Many of these axons had extensive contact with the surrounding basal lamina at which point clusters of microvesicles reminiscent of axon terminals in the neural lobe were present.     

Localization of neurophysin in the neurosecretory elements of the hypothalamus and neurohypophysis of the normal and osmotically stimulated guinea-pig as demonstrated by immunofluorescence histochemical techniques

Summary Antisera to porcine neurophysin-II and ovine neurophysin-III were used to localize neurophysin-like material in the hypothalamus and neurohypophysis of guinea-pigs with an immunofluorescence technique. Although the guinea-pig appears to have only one major neurophysin it was found to be localized in both of the bilateral magnocellular nuclei. Neurophysin-like material was also present in extreme rostral portions of the hypothalamus, in cells lying between the third ventricle and the supraoptic nucleus and in a cluster of cells dorsomedial to the fornix. Immunofluorescence was observed in neurosecretory fibres that followed pathways previously characterized with classical histological stains for neurosecretory material.The immunofluorescence in the hypothalamic elements of a normal guinea-pig was not greatly different from that in fluorescent structures present in an animal that had been severely dehydrated. In contrast, there was a marked depletion of neurophysin from the posterior pituitary gland of the dehydrated guinea-pig. The lack of graded fluorescence in the hypothalamus of the dehydrated animal is discussed.This work was financed by a grant from the New Zealand Medical Research Council and the Auckland Medical Research Foundation.     

Immunocytochemical identification of dynorphin-containing vesicles in Brattleboro rats

Vasopressin and its carrier protein, vasopressin-associated neurophysin, are co-packaged together with an opioid peptide, dynorphin, into 160 nm diameter neurosecretory vesicles in the normal rat hypothalamo-neurohypophysial system. The homozygous Brattleboro rat lacks vasopressin and vasopressin-associated neurophysin, but contains substantial amounts of dynorphin in the vasopressin-deficient neurosecretory cells. We used post-embedding electron microscopic immunocytochemistry to determine the subcellular location of dynorphin in Brattleboro rats. The results show that dynorphin is present within 100 nm neurosecretory vesicles in homozygous Brattleboro cell bodies and axons, and within 160 nm vesicles in heterozygous (control) neurosecretory cell bodies and axons. Oxytocin-associated neurophysin is present in a separate population of magnocellular neurons in both homozygous and heterozygous rats, and is contained within 160 nm vesicles in both cases. Therefore, the absence of synthesis of the vasopressin prohormone results in a dramatic reduction of neurosecretory vesicle size, despite the continued synthesis and packaging of dynorphin peptides.     

Immunocytochemical study of the hypothalamo-neurohypophysial system

Summary Antibodies raised against porcine neurophysin-I and porcine neurophysin-II using an injection regime in rabbits over a short time period, were used to localize neurophysin-I and neurophysin-II in hypothalamic neurosecretory elements of the domestic pig.In transverse section, neurophysin-II containing cells were more abundant in the dorsal medial region of the rostral supraoptic nucleus (SON) as compared with the distribution of neurophysin-I neurons. The main bulk of the cells of the SON were heavily stained for neurophysin-I with neurophysin-II containing cells positioned dorsal from the edge of the optic chiasma.Neurosecretory cells of the SON as seen in sagittal section also showed a differential staining for neurophysins-I and -II.Rostral regions of the pig paraventricular nucleus (PVN) contained magnocellular elements near the third ventricle which were stained predominantly for neurophysin-II. In regions corresponding to the caudal PVN there appeared two populations of neurosecretory neurons: (a) an area of cells adjacent to the third ventricle which contained neurophysin-II antigen and (b) a group of densely populated cells in the dorsal-lateral region which was stained for neurophysin-I.The results support the existence in the pig of at least two distinct populations of neurosecretory neurons corresponding to the neurophysin-I and neurophysin-II neurosecretory system.This work was financed by the Medical Research Council of New Zealand     

Presence of neurophysins I and II in the human pineal gland: Comparison with the content of neurohypophyseal hormones

We have previously measured the individual content of immunoreactive vasopressin (AVP), oxytocin (OT) and vasotocin (AVT) in 155 human pineal glands, and report here identification and measurement of the neurophysin (Np) content of the same glands, using specific homologous human neurophysin I (HNp I) and neurophysin II (HNp II) radioimmunoassays. Median values for HNp I were for men 47 ng/gland (range, 5 to 1360) and for women 24 ng/gland (range, 5 to 1000); median values for HNp II were respectively 7 ng/gland (range, 2 to 191) and 15 ng/gland (range, 2 to 356) with no significant difference between men and women for HNp I and HNp II but a significant difference (p<0.001) between HNp I and HNp II for both sexes. Gel filtration showed that pineal neurophysins were eluted at the same volume as both standard Np and Np from human posthypophyses used as controls. HNp I correlated both with AVP (r_s=0.54 for men and 0.55 for women) and OT (r_s=0.86 for men and 0.57 for women) but not with AVT, while HNp II correlated with AVP (r_s=0.52 for men and 0.53 for women) and OT (r_s=0.92 for men and 0.50 for women) but not with AVT. This study thus confirms the presence of two neurophysins in the human pineal gland and further indicates that they are related to AVP and OT concentrations in the same gland. The results also imply, however, that the presence of immunoreactive AVT (more probably a closely related peptide) is independent of the neurophysins.     

Effects of tunicamycin on the hypothalamo-neurohypophysial system of the rat

Summary Intracisternal injections of tunicamycin, an inhibitor of glycosylation, decreased the incorporation of [³⁵S]cysteine into the neurophysins in the rat neurohypophysis. Histochemical and immunocytochemical studies showed that there was no concomitant decrease in the amount of secretory product in the perikarya of the hypothalamo-neurohypophysial neurones. Indeed there was an increase, although this was not associated with neurosecretory granules as judged electron-microscopically. Tunicamycin led to the formation of socalled colloid droplets which were immunopositive and of which the ultrastructural correlates appeared to be product-filled dilatations of the rough endoplasmic reticulum. The observations are interpreted to suggest that glycosylation plays a rôle in the packaging of secretory material in the hypothalamo-neurohypophysial system.Supported by grants from the Medical Research Council and The Wellcome Trust     

Immunochemical evidence for the transport of neurophysin in the hypothalamo-neurohypophysial system of the dog

Summary 1. An immunohistochemical study has been made of the hypothalamo-neurohypophyseal system of the dog, 20h after crushing the pituitary stalk.2. By use of a cross-species-reactive neurophysin antiserum it was shown that neurophysin is a component of the axons which originate in the supraoptic and paraventricular nuclei and terminate around blood vessels in the posterior pituitary.3. Neurophysin specific fluorescence accumulated in axons proximal to the constriction but was absent from the axons immediately distal to the site of injury.4. In dogs left for six days it was shown by radioimmunoassay that the amount of neurophysin in the hypothalamus and stalk proximal to the constriction increased twofold while that remaining in the posterior pituitary and stalk distal to the constriction decreased five-fold over the same period.5. The results are interpreted as evidence for a rapid axonal transport of neurophysin from its site of synthesis in the cell bodies of the hypothalamus to the posterior pituitary. Acknowledgements: This work was supported by a research grant to D. B. Hope from the Medical Research Council. L. O. Uttenthal was supported by a Medical Research Training Award and B. G. Livett by a Nuffield Dominions Trust Demonstratorship (Australia). We thank Mrs. Marion Martin for radioimmunoassay of neurophysin, Miss Wendy Jones for technical assistance and the U.C.L.A. Brain Information Service for help with the bibliography.     

Immunocytochemical evidence for the presence of oxytocin in rat testis

Summary The presence of oxytocin, vasopressin and neurophysin in the testis of adult Wistar and Brattleboro rats has been examined immunocytochemically. After fixation in modified Bouin's solution, or Bouin's sublimate fixative, immunostaining was accomplished with the peroxidase-antiperoxidase method. The presence of immunoreactive oxytocin was demonstrated in 80% of the interstitial cell population of both rat strains while no staining was observed for vasopressin or neurophysin.     

Presence of vasopressin, oxytocin and neurophysin in the retina of mammals, effect of light and darkness, comparison with the neuropeptide content of the neurohypophysis and the pineal gland

Arginine vasopressin (AVP) and oxytocin (OT) as well as their CNS carrier neurophysins (Np) have been found in the pineal gland. In view of the analogy between the pineal gland and the retina, the contents of these neuropeptides in rat, human and bovine retinae were determined. AVP, OT and Np were detected by specific radioimmunoassay (RIA) and their presence confirmed by RIA measurements (1) in rat and human retinae on HPLC fractions and (2) by the detection of the C-terminal portion of the precursor to AVP and its associated Np = propressophysin (CPP). The AVP and OT content in the retina of the rat was modified by light: AVP and OT content was smaller at 2 a.m. than at 2 p.m., but was increased by a 7 day constant exposure to darkness. In contrast, pituitary content was decreased after 7 days of constant darkness. If one optic nerve was cut we observed a decrease in retinal AVP content compared to the contralateral side and a decrease in pituitary AVP content. Our data clearly demonstrated the presence of AVP, OT and Np in the retina and their variation induced by light. It is probable that these peptides are of central origin.     

Immunocytochemical localization of the vasopressinergic and the oxytocinergic neurons in the human hypothalamus

Summary The human hypothalamic-neurohypophysial hormone-producing nuclei were investigated with the unlabeled antibody peroxidase-antiperoxidase complex (PAP) technique at the light microscopic level. The size, shape and location of the supraoptic, paraventricular, accesssory supraoptic and suprachiasmatic nuclei were determined. It was demonstrated in the human hypothalamus, as well as in the hypothalamus of other mammals, that vasopressin and oxytocin are synthesized in separate neurons. In each of the nuclei of the magnocellular neurosecretory system, the distribution, ratios and structural features of the vasopressinergic and oxytocinergic neurons were determined. It was shown that the human suprachiasmatic nuclei contain numerous neurophysin-vasopressin-producing neurons.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Immunocytochemical evidence for the presence of oxytocin and neurophysin in the large cells of the bovine corpus luteum

Summary The presence of neurophysin, oxytocin and vasopressin in the bovine corpus luteum was examined immunocytochemically. Tissue blocks of corpora lutea from pregnant and non-pregnant animals were fixed with glutaraldehyde/paraformaldehyde fixative and immunostained by the peroxidase-antiperoxidase (PAP) method. The simultaneous presence of immunoreactive oxytocin and immunoreactive oxytocin-neurophysin was demonstrated in large luteal cells of non-pregnant animals, while no staining for vasopressin or vasopressin-neurophysin was observed. None of the peptides were detected in the corpus luteum of pregnant animals. The small luteal cells were not found to be stainable at any time.     

Immunocytochemical investigation of the hypothalamo-neurohypophysial system in birds

Summary The results of an immunohistochemical investigation of the hypothalamo-neurohypophysial system in several species of birds have shown that: (1) mesotocin and vasotocin are synthesized in separate neurons; (2) in all species investigated the distribution of mesotocinergic and vasotocinergic perikarya follows a common pattern; (3) the external zone of the avian anterior median eminence contains exclusively vasotocinergic nerve fibers, originating in supraoptic and ventral paraventricular regions; (4) the distribution of immunoreactive elements in the neural lobe shows a definite species-dependent pattern.     

Immunohistochemical and electron microscopic study of the rat hypothalamic nuclei and cell clusters under various experimental conditions

Summary In untreated, pregnant and thirsting rats the neurosecretory hypothalamic areas were investigated by means of the immunoperoxidase technique in order to demonstrate vasopressin- and oxytocin containing elements at the light- and electron microscopic level. In addition, chromalum-hematoxylinphloxin (CHP) staining and conventional double staining of ultrathin sections were used. The areas investigated included the anterior and posterior supraoptic nuclei, the paraventricular nuclei, the numerous accessory cell clusters in the region between the tractus opticus and the third ventricle as well as the median eminence. In all nuclei and in the accessory cell clusters, the number of vasopressin-reactive neurons exceeds that of oxytocin-reactive neurons. Compared with the anterior supraoptic nucleus, the posterior supraoptic nucleus and the accessory cell clusters react more heavily to prolonged thirst. In the median eminence the neurosecretory axons display close contacts with the portal vessels not only in its lateral portion but in thirsting animals also around the mid-line. There the internal layer is broadened and vasopressin-positive tanycytic processes reach the external zone. Parasagittally, fine vasopressin-positive material can be traced from the internal layer to small deposits at the portal vessels. In long term thirsting animals the typical feature of swollen axons exhibits a characteristic distribution in the median eminence and renders a distinct positive reaction to anti-vasopressin. The release of peptide hormones from the perikarya and from the axons within the nuclei as well as the mode of release within the median eminence are discussed. The significance of the positive immunostaining of the ependymal tanycytes and of some perikarya of the suprachiasmatic nucleus must be reconsidered by further studies.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/1) and Stiftung VolkswagenwerkDedicated to Professor Berta Scharrer on the occasion of her 70th birthdayThe author wishes to express her special gratitude to Dr. L.A. Sternberger for supplying the peroxidaseantiper oxidase-complex and to Dr. H. Stein (Pathologisches Institut der Universität Kiel) for supplying Anti-IgG. The skilful technical assistance of Mrs. H. Prien and Mrs. H. Schöning is thankfully acknowledged     

The distribution of vasopressin-, oxytocin-, and neurophysin-producing neurons in the guinea pig brain

Summary The location, cytology and projections of vasopressin-, oxytocin-, and neurophysin-producing neurons in the guinea pig were investigated using specific antisera against vasopressin, oxytocin or neurophysin in the unlabeled antibody enzyme immunoperoxidase method. Light microscopic examination of the neurons of the supraoptic and paraventricular nuclei shows that hormone is transported not only in axons, but also in processes having the characteristics of dendrites. Neurons were found to contain only vasopressin or oxytocin; all neurons containing neurophysin appear to contain either vasopressin or oxytocin. In the neural lobe, vasopressin and oxytocin terminals are intermingled. In the median eminence, vasopressin and oxytocin fibers are intermingled in the internal zone. In a caudal portion of the median eminence, a number of vasopressin and neurophysin (but few oxytocin) axons enter the external zone from the internal zone, and surround portal capillaries. In the supraoptic nucleus, vasopressin neurons outnumber oxytocin neurons with a ratio of at least 5:1. The paraventricular nucleus is separated into two distinct groups of neurons, a lateral group consisting of only vasopressin neurons, and a medial group consisting of only oxytocin neurons. In addition to axons passing to the neurohypophysis, a number of axons appear to interconnect the supraoptic and paraventricular nuclei.Supported by the Deutsche Forschungsgemeinschaft (SFB 51, C/21 and C/27), (We 608/3)Acknowledgements. The authors are greatly indebted to Mmes. R. Köpp-Eckmann, B. Reijerman, A. Scheiber, I. Wild and Mr. U. Schrell for technical assistance, to Mmes. P. Campbell and U. Wolf for editorial assistance, and to Dr. R.R. Dries and Ferring Pharmaceuticals, Kiel, for the generous provision of high quality peptides     

Response of Substances Co-Expressed in Hypothalamic Magnocellular Neurons to Osmotic Challenges in Normal and Brattleboro Rats

The intention of this review is to emphasize the current knowledge about the extent and importance of the substances co-localized with magnocellular arginine vasopressin (AVP) and oxytocin (OXY) as potential candidates for the gradual clarification of their actual role in the regulation of hydromineral homeostasis. Maintenance of the body hydromineral balance depends on the coordinated action of principal biologically active compounds, AVP and OXY, synthesized in the hypothalamic supraoptic and paraventricular nuclei. However, on the regulation of water–salt balance, other substances, co-localized with the principal neuropetides, participate. These can be classified as (1) peptides co-localized with AVP or OXY with unambiguous osmotic function, including angiotensin II, apelin, corticotropin releasing hormone, and galanin and (2) peptides co-localized with AVP or OXY with an unknown role in osmotic regulation, including cholecystokinin, chromogranin/secretogranin, dynorphin, endothelin-1, enkephalin, ferritin protein, interleukin 6, kininogen, neurokinin B, neuropeptide Y, vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, TAFA5 protein, thyrotropin releasing hormone, tyrosine hydroxylase, and urocortin. In this brief review, also the responses of these substances to different hyperosmotic and hypoosmotic challenges are pointed out. Based on the literature data published recently, the functional implication of the majority of co-localized substances is still better understood in non-osmotic than osmotic functional circuits. Brattleboro strain of rats that does not express functional vasopressin was also included in this review. These animals suffer from chronic hypernatremia and hyperosmolality, accompanied by sustained increase in OXY mRNA in PVN and SON and OXY levels in plasma. They represent an important model of animals with constantly sustained osmolality, which in the future, will be utilizable for revealing the physiological importance of biologically active substances co-expressed with AVP and OXY, involved in the regulation of plasma osmolality.     

A 3-dimensional reconstruction of the hypothalamo-neurohypophysial system of the rat

Summary A three-dimensional reconstruction of the rat's hypothalamo-neurohypophysial system, depicting the neurons that project to the neurointermediate lobe of the pituitary, has been made by using retrograde transport of horseradish peroxidase injected into the neuro-intermediate lobe and by using immunocytochemistry with antisera to vasopressin and somatostatin. The overall picture illustrates the neurons situated in the walls of a pair of ill-defined cone-shaped tunnels, the apices pointing anteriorly. Among the neuronal aggregates in the tunnel wall two, the paraventricular and forniceal nuclei, appear similar in shape but clearly separated by a gap of at least 150 m. Many of the vasopressin-positive neurons lie in the same nuclear aggregates with two notable exceptions: the suprachiasmatic nucleus contains many vasopressin-positive cells but does not project to the pituitary, and the forniceal aggregate, which does project to the pituitary, contains no vasopressin-positive cells. Somatostatin-positive cells are situated close to the third ventricle, and their size is intermediate between parvocellular and magnocellular. Cell counts show only half the cells in the system lying in the supraoptic and paraventricular nuclei, the rest being in accessory nuclei.Supported in part by MRC Canada, Alberta Mental Health     

Immuno-electron microscopical demonstration of vasopressin and oxytocin synapses in the limbic system of the rat

Summary The extensive distribution of exohypothalamic vasopressin or oxytocin containing nerve fibres is thought to be the anatomical basis for the involvement of these neuropeptides in central processes. Following light microscopic observations suggesting that these fibres terminate on other neurons, the present study was undertaken to demonstrate the existence of such endings in the limbic system, which is one of the main target areas for these peptides. For immunoelectron microscopy glutaraldehyde-paraformaldehyde perfused brains of male Wistar rats and Brattleboro rats, homozygous for diabetes insipidus, with and without postfixation in OsO₄, were used. Post-embedding staining revealed false positive reaction product on all dense core vesicles, e.g., in the lateral septum. With pre-embedding staining, however, intense and specific reactions were observed for both vasopressin and oxytocin at their sites of production, as well as the neurohypophysis and in the extrahypothalamic limbic brain regions.In the lateral septum and habenular nucleus only vasopressin-containing synapses could be demonstrated, while in the medial nucleus of the amygdala synapses containing either vasopressin or oxytocin were observed. These peptide containing synapses do not seem to differ in any fundamental way from the classical transmitter-containing synapses in the brain.Supported by the Foundation for Medical Research FUNGOThe authors wish to thank Prof. Dr. A.H.M. Lohman for having made the vibratome available, and Miss C. de Raay for her expert technical assistance