Ontogeny of vasopressin and oxytocin in the fetal rat: Early vasopressinergic innervation of the fetal brain

The content and distribution of vasopressin and oxytocin were determined during fetal development in the rat brain and pituitary by means of radioimmunoassay and immunocytochemistry. The vasopressin content in the fetal brain showed a gradual rise from day 16 of pregnancy onwards, while pituitary vasopressin rapidly increased from fetal day 19 until birth. The oxytocin content in the fetal brain was considerably lower than the vasopressin content. A decrease in oxytocin content was seen between day 16 and day 18 while from day 18 of pregnancy onwards a slight increase was found. The pituitary oxytocin content starts to rise between day 17 and 18 of pregnancy, but at term the pituitary oxytocin content was only 1/20 of the vasopressin value. Immunocytochemistry revealed that vasopressin levels in the fetal rat brain were not only due to the presence of the classical hypothalamoneurohypophyseal system, but also to the early development of exohypothalamic fibers. Vasopressin containing cells were seen from fetal day 16 in the supraoptic nucleus, and from fetal day 18 in the paraventricular nucleus. The fiber outgrowth of these cells towards the pituitary and extrahypothalamic brain sites seems to be well synchronized, as on day 17 vasopressin containing fibers could be demonstrated in the olfactory bulb as well as in the median eminence. No positive staining for oxytocin could be obtained in the fetal rat, while during the entire fetal period no positive staining was found in cell bodies in the region of the suprachiasmatic nucleus. The early peptidergic innervation of the brain, which enabled the tracing of the source of some exohypothalamic fibers, might be related to several central processes among which brain development itself is included.     

Ontogeny of vasopressin and oxytocin in the fetal rat: Early vasopressinergic innervation of the fetal brain

The content and distribution of vasopressin and oxytocin were determined during fetal development in the rat brain and pituitary by means of radioimmunoassay and immunocytochemistry. The vasopressin content in the fetal brain showed a gradual rise from day 16 of pregnancy onwards, while pituitary vasopressin rapidly increased from fetal day 19 until birth. The oxytocin content in the fetal brain was considerably lower than the vasopressin content. A decrease in oxytocin content was seen between day 16 and day 18 while from day 18 of pregnancy onwards a slight increase was found. The pituitary oxytocin content starts to rise between day 17 and 18 of pregnancy, but at term the pituitary oxytocin content was only of the vasopressin value. Immunocytochemistry revealed that vasopressin levels in the fetal rat brain were not only due to the presence of the classical hypothalamoneurohypophyseal system, but also to the early development of exohypothalamic fibers. Vasopressin containing cells were seen from fetal day 16 in the supraoptic nucleus, and from fetal day 18 in the paraventricular nucleus. The fiber outgrowth of these cells towards the pituitary and extrahypothalamic brain sites seems to be well synchronized, as on day 17 vasopressin containing fibers could be demonstrated in the olfactory bulb as well as in the median eminence. No positive staining for oxytocin could be obtained in the fetal rat, while during the entire fetal period no positive staining was found in cell bodies in the region of the suprachiasmatic nucleus. The early peptidergic innervation of the brain, which enabled the tracing of the source of some exohypothalamic fibers, might be related to several central processes among which brain development itself is included.     

Immunocytochemical localization of neuropeptides in the hypothalamus of the Japanese long-fingered bat,Miniopterus schreibersii fuliginosus

Summary To elucidate the role of hypothalamic neuropeptides in regulation of reproductive phenomena of seasonally breeding feral mammals, we used Japanese long-fingered bats, Miniopterus schreibersii fuliginosus, for immunocytochemical study of distribution of the following neuropeptides in the hypothalamus: arginin vasopressin, oxytocin, luteinizing hormone-releasing hormone, somatostatin, corticotropin-releasing factor, and growth hormone-releasing factor. The size, shape and location of supraoptic, paraventricular, suprachiasmatic, and arcuate nuclei of the bat were determined. Arginin vasopressin-and oxytocin-immunoreactive magnocellular neurons were found in the supraoptic and paraventricular nuclei, where they exhibited separate distribution into two distinct groups. Parvocellular arginin vasopressin neurons occurred only in the suprachiasmatic nucleus. The hibernating bats exhibited slightly increased numbers of vasopressin and oxytocin neurons in the supraoptic and paraventricular nuclei. The pregnant bat displayed further increased numbers of vasopressin and oxytocin neurons in both nuclei. Somatostatin-immunoreactive neurons in the paraventricular nucleus were also immunopositive to anti-oxytocin serum, while those in the ventromedial and arcuate nuclei reacted solely to anti-somatostatin serum. They projected to the anterior median eminence and infundibular stalk. Luteinizing hormone-releasing hormone-immunoreactive perikarya were scattered throughout the basal hypothalamus, being particularly abundant in the arcuate nucleus. They were larger in size in hibernating bats than those in normal (non-pregnant) and pregnant females. They projected fibers mainly to the internal layer of the median eminence and infundibular stalk. A few luteinizing hormone-releasing hormone-reactive fibers were also observed in the organum vasculosum laminae terminalis, lateral habenular nuclei, pineal stalk, retroflexus fasciculus, and olfactory tubercle. Corticotropin releasing factor-immunoreactive perikarya were distributed in the paraventricular nucleus and medial preoptic area and projected into the external layer of the anterior median eminence, while growth hormone-releasing factor-immunoreactive perikarya occurred only in the arcuate nucleus and projected into the posterior part of the median eminence.     

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     

Distribution pattern in the human pituitary and hypothalamus of a new neuropeptide: The C-terminal glycoprotein-fragment of human pro-pressophysin (CPP)

Summary The distribution pattern of CPP-containing neurons and fibers in the human pituitary and hypothalamus was studied with a specific antiserum to human CPP and the unlabeled antibody technique. Immunoreactive CPP was found in the magnocellular neurons of the supraoptic nucleus (SON), the paraventricular nucleus (PVN) and in neurons scattered in the supraoptic hypophyseal tract. CPP-containing parvocellular neurons were found in the suprachiasmatic nucleus (SCN). The CPP-containing fibers from the magnocellular neurons formed a tract coursing through the median eminence and the pituitary stalk to the posterior lobe of the hypophysis. In contrast, no such fibers from the SCN projected to SON, PVN and the median eminence. This pattern is identical to that of vasopressin and its associated neurophysin-containing neurons and fibers and strongly supports the concept that CPP is a part of the common precursor for vasopressin and neurophysin II. The biological importance of human CPP other than being a precursor fragment remains to be elucidated.To whom requests for reprints should be addressed     

An immunocytochemical comparison of the angiotensin and vasopressin hypothalamo-neurohypophysial systems in normotensive rats.

In the present study we investigated the possibility that angiotensin II/III and vasopressin coexist in the hypothalamo-neurohypophysial pathway. For our experiments 8-week-old male rats not treated with colchicine were used. The anatomical orientation of the entire pathway for angiotensin and vasopressin was facilitated by examining a series of subsequent coronal, horizontal and sagittal sections. Arching fibre tracts are formed mainly by projections emanating from cell bodies in the paraventricular nucleus, the accessory magnocellular nuclei, the supraoptic nucleus and the retrochiasmatic part of the supraoptic nucleus. The majority extend as far as the median eminence and the neurohypophysis, where major terminal fields exist. However, there is a difference between the staining pattern within the suprachiasmatic nucleus and the hypophysis. The results clearly show the colocalization of angiotensin and vasopressin in neurones as well as in fibres of the hypothalamo-neurohypophysial system.     

Changes in pituitary oxytocin and vasopressin during the estrous cycle and after ovarian hormones: evidence for mediation by norepinephrine

Pituitary levels of oxytocin and vasopressin were maximal on the morning of proestrus, declined during estrus, and were lowest on metestrus in cycling female rats. Norepinephrine levels in the paraventricular nucleus were decreased on proestrus and estrus when compared with metestrus-diestrus. Norepinephrine did not vary in the supraoptic nucleus. Administration of estradiol benzoate to ovariectomized rats elevated oxytocin in the pituitary 54 hr later. This elevation was not affected by a subsequent injection of estrogen or progesterone. Estrogen priming did not affect vasopressin levels in the pituitary, but a second injection of estrogen or of progesterone 48 hr later increased vasopressin in the pituitary when measured 6 hr after the second injection. Vasopressin was decreased 30 hr after a second injection of estrogen. The ovarian hormone treatments that elevated pituitary vasopressin decreased steady state levels of norepinephrine in the paraventricular nucleus and reduced the depletion of norepinephrine after administration of the catecholamine synthesis inhibitor α-methyltyrosine, suggesting a decrease in turnover. Ovarian hormones did not affect norepinephrine in the supraoptic nucleus. The present results suggest a role for posterior pituitary hormones in reproductive processes and a role for noradrenergic mechanisms in the paraventricular nucleus in mediating the effects of ovarian steroids on pituitary vasopressin.     

Immunoreactive galanin-like material in magnocellular hypothalamoneurohypophysial neurones of the rat

Summary Immunoreactive galanin-like material was recently shown to co-exist with vasopressin in parvocellular and magnocellular perikarya of the paraventricular nucleus in the anterior hypothalamus of the rat (Melander et al. 1986). Since this distribution pattern differed from our observation of oxytocin-associated galanin-like immunoreactivity (LI) in the neurohypophysis, we compared in series of 0.5-m thick sections the localisation of galanin-LI with the localisation of oxytocin and vasopressin/dynorphin in the hypothalamus, the median eminence and the neurohypophysis. In the oxytocin system, galanin-LI was intense in oxytocin varicosities of the neurohypophysis. Oxytocin perikarya of the hypothalamic supraoptic and paraventricular nuclei exhibited galanin-LI only after intraventricular injection of colchicine and when sections were treated with trypsin prior to application of the antibody. In the vasopressin/dynorphin system galanin-LI was intense in hypothalamic perikarya after colchicine injection and in neurohypophysial varicosities after treatment of the sections with trypsin. In these neurones, galanin-LI was absent or weak in all elements when treatments with colchicine or trypsin were omitted. Galanin-LI in the neurohypophysis was not co-localised with the numerous fine endings showing GABA-LI. These observations indicate that galanin-like material coexists with vasopressin and oxytocin in the respective magnocellular neurones, although not always in an immunoreactive form.     

Immunocytochemical study of the hypothalamo-neurohypophysial system

Summary Antisera, with cross reactive antibodies removed by affinity chromatography, were used in the immunoperoxidase-bridge technique to study the distribution of oxytocin and vasopressin together with neurophysin in the hypothalamo-neurohypophysial system of the rat. The hormones were demonstrated in different areas of the supraoptic nucleus (SON) and paraventricular nucleus (PVN), in neurosecretory fibres of the hypothalamoneurohypophysial tract, median eminence, and in nerve terminals of the neurohypophysis. Intact normal and rats with hereditary hypothalamic diabetes insipidus (Brattleboro strain), and rats dehydrated by the administration of oral hypertonic saline were studied. In dehydrated rats the hormone concentration in the neurons, and the number of neurons containing hormone varied according to the time of dehydration stress.The observations support the hypotheses that: 1) oxytocin and oxytocinneurophysin, and vasopressin and vasopressin-neurophysin are synthesised in different neurons and are transported along different axons; 2) the SON and PVN are functionally indistinguishable in that neurons containing oxytocin or vasopressin are present in both nuclei; and 3) the two types of neurons respond to osmotic stimulation in a way that is qualitatively the same but quantitatively different.This work was supported by a grant from the Medical Research Council of New Zealand     

Microarray analysis reveals interleukin-6 as a novel secretory product of the hypothalamo-neurohypophyseal system

Physiological activation of the hypothalamo-neurohypophyseal system (HNS) by dehydration results is a massive release of vasopressin (VP) from the posterior pituitary. This is accompanied by a functional remodeling of the HNS. In this study we used cDNA arrays in an attempt to identify genes that exhibit differential expression in the hypothalamus following dehydration. Our study revealed nine candidate genes, including interleukin-6 (IL-6) as a putative novel secretory product of HNS worthy of further analysis. In situ hybridization and immunocytochemistry confirmed that IL-6 is robustly expressed in the supraoptic (SON) and the paraventricular (PVN) nuclei of the hypothalamus. By double staining immunofluorescence we showed that IL-6 is largely co-localized with VP in the SON and PVN. In situ hybridization, immunocytochemistry, and Western blotting all revealed IL-6 up-regulation in the SON and PVN following dehydration, thus validating the array data. The same dehydration stimulus resulted in an increase in IL-6 immunoreactivity in the axons of the internal zone of the median eminence and a marked reduction in IL-6-like material in the posterior pituitary gland. We thus suggest that IL-6 takes the same secretory pathway as VP and is secreted from the posterior pituitary following a physiological stimulus.     

Intra- and extrahypothalamic vasopressin and oxytocin pathways in the rat

Summary Perfusion of rat brain followed by immersion fixation with 2.5% glutaraldehyde-1% paraformaldehyde, purification of the first antisera and application of the unlabelled antibody enzyme method were used to specifically identify vasopressin and oxytocin containing cells and fibres. The conventional sites of production of these hormones were confirmed as follows: supraoptic and paraventricular nuclei, suprachiasmatic nucleus (only vasopressin), and other cells and cell groups of the hypothalamus. Fibres from the suprachiasmatic nucleus spread out in various directions, and probably project to the nucleus praeopticus periventricularis, organum vasculosum laminae terminalis and in the direction of the supraoptic nucleus. Oxytocin and vasopressin containing pathways could be traced from the paraventricular nucleus to the lateral ventricle, the stria terminalis and the stria medullaris. Some of the oxytocin and vasopressin containing tracts appear to continue onto the septum. The possible importance of these morphological findings for the behavioural effects of vasopressin and oxytocin is discussed.The authors wish to thank Dr. L. Sternberger for his generous gift of peroxidase-antiperoxidase complex, and Miss M.M. Smidt, Mr. A. Potjer and Mr. P. Wolters for their assistance. This work was supported in part by the Foundation for Medical Research FUNGO     

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.     

Vasopressinergic and oxytocinergic pathways in the central nervous system

Recent data obtained by immunohistochemical and other anatomical tracing methods indicate that oxytocin and vasopressin pathways are much more complex and extensive than previously recognized. In addition to the classic magnocellular neurons that project from the supraoptic and paraventricular (PVN) nuclei to the posterior pituitary gland, generally smaller neurons in various parts of the PVN send vasopressin fibers to the portal capillary bed in the median eminence, or send oxytocin or vasopressin projections to other brain and spinal cord sites. In addition, vasopressin neurons are also found in the suprachiasmatic nucleus and may contribute to extrahypothalamic projection areas. Many of these axonal projections appear to form synapses with other neurons in forebrain, hindbrain, and spinal cord regions, which suggests roles for these peptides in neuronal communication. In brain stem and spinal cord, terminal fields include both parasympathetic and sympathetic regulatory centers. Oxytocin terminals are also found on large intracerebral arteries where the peptide may regulate cerebral blood flow.     

Vasopressin and oxytocin in the neural control of the circulation

Catecholamine innervation originating in dorsal medial and ventral lateral medulla terminates on parvocellular and magnocellular subnuclei, respectively, of the paraventricular nucleus of the hypothalamus. In turn, parvocellular pathways terminate in brain stem and spinal cord, whereas magnocellular pathways terminate in median eminence and posterior pituitary. Consistent with the neuroanatomy, we find that baroreceptor regulation of neuroendocrine (plasma vasopressin) and autonomic (blood pressure) functions can be dissociated. Further, studies indicate that sympathetic vasomotor pathways are activated by injections of vasopressin and oxytocin into the nucleus tractus solitarii and vasopressin into the lateral cerebral ventricles. Also, parasympathetic pathways to the heart and baroreflex function are activated and augmented, respectively, by i.v. administered vasopressin. These results are consistent with at least three central sites of action and suggest a complex role of vasopressin (and possibly oxytocin) in the central neural regulation of the heart and circulation.     

Desmopressin, but not vasopressin, decreases activity of the hypothalamo-neurohypophysial system in Brattleboro rats

The pituitary neural lobe of homozygous Brattleboro rats has high rates of glucose utilization not affected by chronic treatment with exogenous vasopressin, despite attenuation of polydipsia and polyuria. We evaluated whether this effect may result from the inability of vasopressin to affect the hypothalamo-neurohypophysial metabolism or from the development of resistance to chronic vasopressin treatment. We used the [14C]deoxyglucose method to compare 28-h effects of vasopressin treatment (5 U/kg, i.m., twice a day) with that of desmopressin (100 micrograms/kg, i.p., once a day), a long-lasting antidiuretic hormone, on glucose utilization of the hypothalamo-neurohypophysial system and related structures in conscious homozygous Brattleboro rats. Vasopressin and desmopressin reduced water intake, plasma osmolality and plasma Na+ concentration similarly. Vasopressin decreased glucose utilization in the supraoptic nucleus, subfornical organ and median preoptic nucleus, but did not alter activity in the paraventricular nucleus and neural lobe. Desmopressin decreased glucose utilization in all these structures. The results indicate that desmopressin has a more potent inhibitory action on the hypothalamo-neurohypophysial system than vasopressin over this short duration of treatment. The lack of response in the neural lobe from chronic treatment with vasopressin seems to be due to its inability to affect the paraventricular nucleus metabolism. The maintenance of metabolic activity in the paraventricular nucleus of vasopressin-treated Brattleboro rats suggests that this structure contributes importantly to the metabolism of neural lobe.     

The topography of mesotocin and vasotocin systems in the brain of the domestic mallard and japanese quail: Immunocytochemical identification

Summary The neurosecretory systems producing mesotocin (MT) and vasotocin (VT) (the avian homologues of oxytocin and vasopressin, respectively) were characterized in the brains of the domestic mallard and Japanese quail by means of indirect immunofluorescence techniques using specific antisera. In the anterior preoptic region, including the organum vasculosum of the lamina terminalis, and at different levels of the supraoptic and paraventricular nuclei, separate mesotocin- and vasotocin-producing neurons were identified. Mesotocinergic and vasotocinergic neurons were also located in the tuberomammillary area, among the ectomammillary tract fibers. The supraoptico-neurohypophysial tract, formed by vasotocin- and mesotocincontaining axons, enters the internal zone of the median eminence and ends in the posterior lobe of the pituitary. The external zone of the rostral median eminence appears to contain vasotocin and mesotocin fibers, which terminate in close contact with the capillaries of the hypophysial portal system.With contributions by Dr. B. Kerdelhué, Laboratoire des Hormones Polypeptidiques du CNRS, 91190 Gif-sur-Yvette, France     

Ontogeny of the hypothalamo-neurohypophysial system in rats--an immunohistochemical study.

In fetal rats neurophysin has been visualized immunohistochemically first at the 16th gestation day in perikaryons of the supraoptic nucleus, followed by the median eminence and the neurohypophysis at the 17th day, and the paraventricular neurons at the 19th day. The external zone of the median eminence contains abundantly immunoreactive fibres at the first days post partum. In the perikaryons of the suprachiasmatic nucleus immunoreactive material appears after the 3rd day of postnatal development.     

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.     

Biosynthesis and axonal transport of rat neurohypophysial proteins and peptides

35S-cysteine injected adjacent to the supraoptic nucleus (SON) of the rat is rapidly incorporated into proteins. These 35S-cysteine-labeled proteins in the SON (1-24 h after injection) were separated by polyacrylamide gel electrophoresis, and the distribution of radioactive proteins on the gels was analyzed. 1 h after injection, about 73% of the radioactivity appeared in two peaks (both about 20,000 mol wt). With time, these peaks (putative precursors of neurophysin) decreased, as a 12,000 mol wt peak (containing two distinct neurophysins) increased in radioactivity. Both the 20,000- and 12,000-mol wt proteins are transported into the axonal (median eminence) and nerve terminal (posterior pituitary) regions of the rat hypothalamo-neurohypophysial system. Conversion of the larger precursor protein to the smaller neurophysin appears to occur, in large part, intra-axonally during axonal transport. Six distinct 35S-cysteine-labeled peptides (less than 2500 mol wt), in addition to arginine vasopressin and oxytocin, are also synthesized in the SON and transported to the posterior pituitary where they are released together with labeled neurophysin by potassium depolarization in the presence of extracellular calcium. These data provide support for the hypothesis that the neurohypophysial peptides (vasopressin and oxytocin) and neurophysins are derived from the post- translational clevage of protein precursors synthesized in the SON, and that the conversion process can occur in the neurosecretory granule during axonal transport.