Co-transport of sex hormone-binding globulin/SHBG with oxytocin in transport vesicles of the hypothalamo-hypophyseal system.

The intrinsic expression of sex hormone binding globulin (SHBG) in magnocellular hypothalamic neurons, in part co-localized with either vasopressin or oxytocin, was recently described. This study is focused on the ultrastructural localization of SHBG in the hypothalamo-neurohypophyseal pathway in rats. Immunostaining for SHBG in the hypothalamic perikarya was increased by colchicine treatment, indicating that the steroid-binding globulin is subject to rapid axoplasmic transport along with the classical posterior lobe peptides. With immunoelectron-microscopic double labeling, we found co-localization of oxytocin and sex hormone binding globulin in a portion of the large dense-core vesicles in paraventricular and supraoptic perikarya and in axonal varicosities in the median eminence and in the posterior lobe. Our observations show that SHBG is processed, transported and stored along with oxytocin suggesting that SHBG is released from nerve terminals in the posterior lobe, the median eminence and possibly the brain similarly to and in conjunction with oxytocin.     

Coexistence of NADPH-diaphorase with vasopressin and oxytocin in the hypothalamic magnocellular neurosecretory nuclei of the rat

Coexistence of NADPH-diaphorase with vasopressin and oxytocin was studied in the magnocellular neurosecretory nuclei of the rat hypothalamus by use of sequential histochemical and immunocytochemical techniques in the same sections. Coexistence was found in all the nuclei examined (supraoptic, paraventricular, circular, fornical, and in some isolated neurons located in the hypothalamic area between the paraventricular and supraoptic nuclei). The ratios of neurons expressing both markers (NADPH-diaphorase and vasopressin, NADPH-diaphorase and oxytocin) in each of the nuclei were very similar. Although further studies must be carried out, the partial coexistence found in all nuclei suggests that NADPH-diaphorase is probably not related to general mechanisms involving vasopressin and oxytocin, but rather in specific functions shared by certain hypothalamic neuronal cell populations.     

Coexistence of intestinal trefoil factor (hITF) and oxytocin in magnocellular neurons in the human hypothalamus.

Human intestinal trefoil factor hITF, a polypeptide of the P-domain family, was found to occur in hypothalamic neurons. With combined immunofluorescence and immunoperoxidase technique we investigated the coexistence of hITF with the neurohypophysial peptide oxytocin and the associated neurophysin I in sections of the human hypothalamus. In the supraoptic nucleus, 39.2% of magnocellular oxytocinergic perikarya show hITF immunoreactivity. A similar distribution was observed in perivascular hypothalamic oxytocinergic neurons, whereas in the paraventricular nucleus, 99% of the oxytocinergic neurons show hITF coexpression. In the periventricular nucleus (PEV), single, scattered neurons with both immunoreactivities occur. Our findings indicate that hITF and oxytocin are coexpressed in a portion of the magnocellular neurons in the human hypothalamus, and that hITF is among the neurohypophysial peptides.     

Relationship of CRF-immunostained cells and magnocellular neurons in the paraventricular nucleus of rat hypothalamus

The distribution of corticotropin-releasing factor (CRF), vasopressin (VP) and oxytocin (OXY) containing neurons within the magnocellular and parvocellular divisions in the paraventricular nucleus (PVN) of rat hypothalamus is described in brains from normal untreated, colchicine treated and adrenalectomized animals. Double immunostained preparations using glucose oxidase-antiglucose oxidase (GAG) complex combined with PAP complex to visualize two antigens with contrasting colors in the same tissue sections were employed. Separate and distinct populations of cells containing the immunoreactive (ir) elements were seen. Immunostained CRF neurons present in the ventral medial portion of the posterior magnocellular division were juxtaposed to oxytocin-ir perikarya in colchicine treated and adrenalectomized animals. CRF-ir cells were for the most part concentrated in the medial parvocellular component of PVN. An intimate anatomical proximity between CRF-ir and VP-ir perikarya was evident in this medial parvocellular division in brains of adrenalectomized animals; this area is normally VP-ir poor except in the adrenalectomized rats. This extension of VP-ir cells into this CRF rich region and the very close approximation between the two cell bodies suggests potential cell to cell communication following perturbation of the brain-pituitary-adrenal axis. No evidence for the co-existence of two peptidergic systems in the same neuron was apparent in the present study.     

The hypothalamic and neurohypophysial vasopressin and oxytocin content under various states of adrenergic transmission in dehydrated and subsequently rehydrated rats

Rats dehydrated for 8 days and subsequently rehydrated were given intracerebroventricularly (i.c.v.) methoxamine hydrochloride (MX) or dihydroergotamine methanosulphonate (DHE), each in a daily dose of 10 micrograms dissolved in 10 microliter of 0.9% sodium chloride. A single dose of MX injected to normally hydrated animals increased the release of hypothalamic and neurohypophysial vasopressin but did not affect significantly the oxytocic activity in the hypothalamus as well as in the neurohypophysis. Under conditions of dehydration MX did not influence the hypothalamic vasopressin content but it stimulated the neurohypophysial vasopressin depletion. On the contrary, MX distinctly inhibited the decrease of hypothalamic and neurohypophysial oxytocin content in dehydrated animals. In rehydrated animals MX restrained some what the renewal of hypothalamic vasopressin and oxytocin storage but intensified this process in the neurohypophysis. A single dose of DHE decreased the vasopressin content in the hypothalamus as well as the oxytocin content both in the hypothalamus and neurohypophysis. Under conditions of dehydration DHE stimulated the depletion of hypothalamic vasopressin and oxytocin. On the contrary, DHE strongly inhibited the depletion of oxytocin in the neurohypophysis of dehydrated rats. DHE restrained the renewal of hypothalamic vasopressin and oxytocin stores as well as intensified this process in the neurohypophysis of subsequently rehydrated rats.     

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 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.     

Relationship of ACTH1-39-immunostained fibers and magnocellular neurons in the paraventricular nucleus of rat hypothalamus

Dual antigen immunocytochemical staining procedures were used in the same tissue section to determine the distribution of ACTH immunostained fibers and varicosities within the magnocellular and parvocellular divisions in the paraventricular nucleus (PVN) of rat hypothalamus and elucidate its anatomical relationship to vasopressin (VP) and oxytocin (OXY)-containing neurons. Double immunostained preparations using glucose oxidase-antiglucose oxidase complex combined with PAP complex to visualize two antigens with contrasting colors in the same tissue section were employed. ACTH-immunoreactive (ir) fibers were distributed throughout the periventricular stratum and the parvocellular component of the PVN; in the latter area fibers were particularly dense in the ventral medial portion of the medial parvocellular division. Dual immunostained sections revealed a close anatomical association between opiocortin fibers and oxytocin and vasopressin parvocellular neurons. ACTH immunostained fibers were present in the anterior and medial magnocellular component of PVN and in the ventral medial portion of the posterior magnocellular division; these immunoreactive fibers were in intimate proximity to oxytocin-ir perikarya. The very close approximation between the ACTH-ir fibers and oxytocin-containing cell bodies suggests potential cell to cell communication between the two peptidergic systems in PVN. Few ACTH immunostained fibers were seen in the dorsal lateral portion of the posterior magnocellular division in which vasopressinergic neurons predominate. The present anatomical study supports pharmacological and physiological studies which indicate that opioids can influence the activity of magnocellular PV neurons. This study also elucidates an anatomical relationship between opiocortins (ACTH1-39) and parvocellular PV neurons which suggests that the opiocortin system may play a role in the regulation of both the neuroendocrine and autonomic activities of specific PV neurons.     

Identification of the vasopressin producing and of the oxytocin producing neurons in the hypothalamic magnocellular neurosecretory system of the rat

Immuno-enzyme histochemical investigations showed that, in the magnocellular hypothalamo-hypophysial neurosecretory system of the rat, vasopressin and oxytocin are synthetized in separate neurons. Both the vasopressin neurons and the oxytocin neurons are present in both the supraoptic and the paraventricular nuclei in about the same number. Preferential location of the two kinds of rat neurosecretory neurons is not as obvious as in the bovine hypothalamus. Their perikarya do not show distinct morphological differences. The two kinds of neurosecretory perikarya are the origin of separate vasopressin-containing and oxytocin-containing axons respectively. In the neural lobe, the distribution of the two different types of axons is described.     

Tunicamycin,puromycin and brefeldin A influence the subcellular distribution of neuropeptides in hypothalamic magnocellular neurones of rat

Summary Magnocellular neurones in the supraoptic nuclei of normal Long Evans and homozygous Brattleboro rats were examined electron-microscopically after intracisternal injections of tunicamycin, puromycin, or brefeldin A. Moderate (50 g) or high (200 g) doses of tunicamycin caused the formation of electron-dense filamentous accretions in the endoplasmic reticulum (ER) cisterns of vasopressin neurones, but only the high dose of tunicamycin also caused accretions to form in the ER of some oxytocin neurones. Immunogold labelling of ultrathin sections from tunicamycin-treated rats revealed that, in about 5% of vasopressin neurones, the accretions could be immunogold-labelled for vasopressin and its associated neurophysin. However, in the majority of vasopressin neurones, the sections required trypsinisation before immunolabelling of the accretions could be detected. Small accretions in the ER of oxytocin neurones did not label for oxytocin or its neurophysin without prior trypsinisation, whereas larger accretions in other oxytocin cells could be labelled without prior trypsin treatment. Administration of puromycin resulted in the formation of small ER accretions in both vasopressin and oxytocin neurones. These accretions were immunolabelled with antisera, respectively, to vasopressin and oxytocin, but neurophysin-immunoreactivity was in most cases absent and was not revealed by treatment with trypsin, suggesting that neurophysin-immunoreactive epitopes were absent from truncated peptides forming the accretions. Brefeldin A caused dilatation of ER cisterns and disruption of the Golgi apparatus in both oxytocin and vasopressin neurones, but did not cause accretions to form in the ER.     

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.     

Immunocytochemical identification of vasopressinergic and oxytocinergic neurons in the hypothalamus of the cat

Our immunocytochemical investigation of the magnocellular neuroendocrine cells in the cat hypothalamus reveals a mixture of vasopressin (VP)- and oxytocin (OT)-containing neurons in the supraoptic (NSO), the paraventricular (NPV) and in five accessory nuclei (NAC). We describe the lateral hypothalamic nucleus (NLH), a new accessory nucleus, lying at the junction of the internal capsule and pallidum, and possibly involved in drinking behavior. Previously characterized incompletely in mammals, the four other accessory nuclei consist of the circularis (NC), anterior fornical (NAF), posterior fornical (NPF) and retrochiasmatic (NRC). The two peptidergic cell types, VP and OT, are equally mixed in the NPV and the NAC, but in the NSO VP neurons predominate. The perikarya of these VP and OT neurons do not show distinct morphological differences at the level of light microscopy. The organization of magnocellular neuroscretory neurons in the cat hypothalamus closely resembles that described in other mammals with the exception of the unique presence of the lateral hypothalamic accessory nucleus.     

Expression of corticosterone-binding globulin in the rat hypothalamus.

We observed coexistence of corticosteroid-binding globulin (CBG) with vasopressin (VP) and oxytocin (OT) in magnocellular neurons in rat hypothalamus by combined immunoperoxidase staining and immunofluorescence. A portion of the supraoptic and of the paraventricular neurons showed double immunostaining of CBG with either VP or with OT. CBG staining was intensified by pretreating animals with colchicine to block axonal transport. CBG was also observed in widespread axonal projections throughout the lateral hypothalamus, the median eminence and the posterior pituitary lobe. Single ependymal cells and some of the endocrine cells in the anterior lobe contained specific CBG immunoreactivity. IN SITU hybridization of semithin sections with a synthetic oligonucleotide probe to CBG mRNA provided staining of magnocellular hypothalamic neurons, but not ependymal cells or anterior lobe cells. Western blots of CBG extracted by affinity chromatography from hypothalamus homogenates showed a band at approximately 50 kDa. Our observations indicate the intrinsic expression of CBG in peptidergic hypothalamus neurons in rat. The multiple locations of CBG-expressing neurons indicate multiple functional properties, probably exceeding the role of a mere steroid transporter. CBG is likely to be subject to axonal transport and secretion in a neuropeptide-like fashion, perhaps involved in neuroendocrine regulation, which may include stress responses.     

Xylazine activates oxytocinergic but not vasopressinergic hypothalamic neurons under normal and hyperosmotic conditions in rats

Role of central alpha2-adrenoceptors in the regulation of hypothalamic magnocellular cells was studied under hyperosmotic challenge elicited by hypertonic saline (HS). Rats pretreated with receptor agonist, xylazine (XYL), were injected intraperitoneally with different (low: 0.375, moderate: 0.75, high: 1.5 M) HS 30 min later. The activity of the paraventricular (PVN) and supraoptic (SON) vasopressin and oxytocin perikarya was established by Fos-dual-immunohistochemistry 60 min after HS administration. Results showed that 1/XYL is a potent stimulus for oxytocin but not vasopressin magnocellular cells under basal and weak hyperosmotic conditions 2/highHS completely overlaps the effect of XYL. In addition, XYL partially suppressed Fos expression in the parvocellular PVN cells activated by highHS. The data suggest that alpha2-adrenoceptors may play an important role in the regulation of oxytocinergic PVN and SON neurons under basal and weak hyperosmotic conditions and that alpha2-adrenoceptors may also participate in the control of PVN parvocellular cells under intense osmotic challenge.     

The hypothalamic and neurohypophysial vasopressor and oxytocic content as influenced by alpha-adrenergic blockade in stressed rats

The hypothalamic and neurohypophysial vasopressor and oxytocic content as influenced by alpha-adrenergic blockade in stressed rats. Acta physiol. pol., 1985, 36 (3): 193-200. The effects of phenoxybenzamine (PBA; an alpha-adrenergic blocker) on hypothalamic and neurohypophysial vasopressin and oxytocin were investigated in stressed rats. Immobilization resulted in a decrease of both vasopressor and oxytocic activities in the hypothalamus and neurohypophysis, whereas in rats, exposed to cold the vasopressin and oxytocin content in the hypothalamo-neurohypophysial system was increased. Under treatment with PBA the vasopressin and oxytocin content in the neurohypophysis was diminished in stressed (both immobilized and cold-exposed) rats when compared to respective groups of untreated animals subjected to appropriate kind of stress. The response of the vasopressinergic and oxytocinergic neurones seems, therefore, to be dependent on the type of stress. The alpha-adrenergic transmission is probably in some way involved in the mechanisms of modified neurohypophysial function in stressed animals.     

Immunohistochemical identification of neurons containing corticotropin-releasing factor in the rat hypothalamus

A specific rabbit anti-CRF serum and the immunoperoxidase technique were used to show that CRF-containing neurons are mainly distributed in the paraventricular and supraoptic nuclei of the rat hypothalamus. In addition, immunoreactive neurons are scattered in other hypothalamic regions. These neurons are 20--30 micrometers in diameter. From the present and previous investigations it may be concluded that the hypothalamic magnocellular nuclei, i.e., paraventricular and supraoptic, and other hypothalamic accessory nuclei, are the producing sites not only for vasopressin and oxytocin, but also for corticotropin-releasing factor.     

Co-localization of putative vasopressin receptors and vasopressinergic neurons in rat hypothalamus

Summary Vasopressin and oxytocin are synthesized by neurons in the paraventricular and supraoptic nuclei of hypothalamus. Dense concentrations of vasopressin binding sites have also been localized in these nuclei. Using a vasopressin anti-idiotypic antiserum, a dual immunocytochemical labeling procedure has been employed to elucidate the distribution of putative vasopressin receptors in anatomical relation to vasopressin and oxytocin immunoreactive cells in rat brain. Putative vasopressin receptors are observed in relation to magnocellular neurons in hypothalamus that are vasopressin immunoreactive. They do not appear to be associated with parvocellular vasopressinergic cells or oxytocin immunoreactive neurons. The presence of these presumed autoreceptors would support evidence that vasopressin may autoregulate the activity of magnocellular vasopressinergic neurons in hypothalamus.     

Co-localization of putative vasopressin receptors and vasopressinergic neurons in rat hypothalamus

Vasopressin and oxytocin are synthesized by neurons in the paraventricular and supraoptic nuclei of hypothalamus. Dense concentrations of vasopressin binding sites have also been localized in these nuclei. Using a vasopressin anti-idiotypic antiserum, a dual immunocytochemical labeling procedure has been employed to elucidate the distribution of putative vasopressin receptors in anatomical relation to vasopressin and oxytocin immunoreactive cells in rat brain. Putative vasopressin receptors are observed in relation to magnocellular neurons in hypothalamus that are vasopressin immunoreactive. They do not appear to be associated with parvocellular vasopressinergic cells or oxytocin immunoreactive neurons. The presence of these presumed autoreceptors would support evidence that vasopressin may autoregulate the activity of magnocellular vasopressinergic neurons in hypothalamus.     

The suprachiasmatic nucleus of the mink (Mustela vison): apparent absence of vasopressin-immunoreactive neurons

The hypothalamic suprachiasmatic nucleus is centrally involved in generation of several circadian rhythms. Neurons of the mammalian suprachiasmatic nucleus express a number of neuropeptides including vasopressin. The suprachiasmatic nucleus of the mink (Mustela vison) is easily distinguished from neighbouring hypothalamic areas and the underlying optic chiasm as a small nucleus containing densely packed parvocellular neurons. A dorsal and ventral subdivision were clearly recognized within the midportion and caudal part of the nuclcus. Using immunohistochemistry, we have identified vasopressin-, neurophysin-, and vasoactive intestinal peptide-immunoreactive neuronal elements in the hypothalamus of the mink. Vasoactive intestinal peptide-immunoreactive neurons can be observed in the ventral aspect of the suprachiasmatic nucleus, but to our surprise, no vasopressin immunoreactive perikarya are found within the suprachiasmatic nucleus, this absence being independent of the experienced annual cycle. The hypothalamic paraventricular and supraoptic nuclei contain large numbers of vasopressin-, neurophysin-and vasoactive intestinal peptide-immunoreactive magnocellular neurons with extensive projections towards the infundibulum and neurohypophysis. A comparative analysis of the distribution of vasopressin-immunoreactive elements in a number of conventional laboratory animals has demonstrated that, in contrast to the rat, golden hamster and Mongolian gerbil, neither vasopressin-containing perikarya in the suprachiasmatic nucleus nor fine calibered immunoreactive fibres entering the adjacent subparaventricular zone are present in the mink. The mink is a photodependent seasonal breeder, and thus vasopressin-immunoreactive neurons in the suprachiasmatic nuclei may not be essential for the photoperiodic regulation of reproduction and seasonal events experienced by this species.     

The vasopressor and oxytocic activities of the hypothalamus and neurohypophysis influenced by stimulated alpha-adrenergic transmission during dehydration and subsequent rehydration in the white rat

Under conditions of equilibrated water metabolism a single dose of methoxamine increased the content of vasopressin in the hypothalamus as well as that of oxytocin both in the hypothalamus and neurohypophysis. During dehydration the depletion of hypothalamic and neurohypophysial vasopressin was more marked in methoxamine-treated animals; this effect, however, was absent in the neurohypophysis on the 2nd day and in the hypothalamus on the 8th day of water deprivation. After two days of dehydration methoxamine inhibited the decrease of oxytocin content in the hypothalamus; simultaneously (2nd and 4th day of dehydration) it intensified this process in the neurohypophysis. During rehydration methoxamine impaired the renewal of vasopressin both in the hypothalamus and neurohypophysis; this effect was most marked on the 8th day of rehydration. On the contrary, it favoured somewhat the renewal of hypothalamic oxytocin in rehydrated rats (such an event was not found on the 8th day of rehydration). Moreover, methoxamine restrained initially (on the 2nd and 4th day of rehydration) the restoration of neurohypophysial oxytocin stores; following eight days of rehydration an opposite effect was here found. It is concluded that the response of the vasopressinergic and oxytocinergic neurons to alpha-adrenergic stimulation, brought about by using methoxamine as pharmacological tool, seems to be depended on the actual state of water metabolism. Impulses from the osmoreceptors may be therefore of some importance in modifying the change in vasopressin and oxytocin synthesis, transport and release resulting from stimulation of alpha-adrenergic transmission through neural chains including units susceptible to methoxamine.