Immunocytochemical localization of urotensin I neurons in the caudal neurosecretory system of the white sucker (Catostomus commersoni)

Summary The localization of urotensin I has been investigated in the caudal neurosecretory system of the white sucker (Catostomus commersoni). The peptide is present in all the cells of the system both large and small, in the large axons passing to the urophysis, and in fine beaded fibres not only within the urophysis but also in a fine plexus lateral to the large cells in the spinal cord proper. The possibility that the caudal neurosecretory system is not a functionally uniform system but rather a collection of dissimilar cells of different synaptic inputs with a common entity, urotensin I, is discussed. Moreover, the feasibility of a urotensin I feedback loop is described.Financial support for this investigation was provided in part by MRC (Canada). K.L. is MRC career investigator; K.L.W, was in receipt of an Alberta Heritage Foundation for Medical Research Fellowship. It is a pleasure to record the valuable technical assistance of Mrs. W. Ho and the dedicated assistance in the collection of the experimental animals by Mrs. Helen Wilson of Nanton, Alberta.     

Immunohistochemical localization of urotensin I and other neuropeptides in the caudal neurosecretory system of three species of teleosts and two species of elasmobranchs

Summary In three species of teleosts — carp Cyprinus carpio; grass carp Ctenopharyngodon idella; and crucian carp Carassius auratus — the caudal neurosecretory system displays small, medium-sized and large neurons. Urotensin I (UI)-immunoreactive and UI-nonreactive neurons were found in all three groups; in general, the number of the latter neurons exceeded that of the former. Noteworthy are: (i) UI-immunoreactive fibers in the caudal spinal cord and (ii) dense accumulations of UI-immunoreactive product around the capillaries of the urophysis. In two species of elasmobranchs — cat shark Heterodontus japonicas and swell shark Cephaloscyllium umbratile — neurosecretory neurons decreased in size in rostro-caudal direction. Most of the neurosecretory perikarya, their axons and the corresponding neurohemal areas were UI-immunoreactive, but a small number of secretory neurons was devoid of immunoreaction. Oxytocin, arginine vasopressin, substance P, somatostatin, neurotensin, vasoactive intestinal polypeptide and gastrin-releasing peptide were not detected in the caudal neurosecretory system of the carp.     

Immunoreactive methionine-enkephalin in the caudal neurosecretory system of the carp,Cyprinus carpio

Summary Methionine-enkephalin (Met-enk) was detected by immunocytochemistry and radioimmunoassay in the caudal neuro-secretory system of the carp Cyprinus carpio. Some cells showing urotensin I (UI)-immunoreactivity reacted to Met-enk antiserum, but others did not. Neurons with urotensin II (UII)-immunoreactivity did not react to Met-enk antiserum; neurons with both UI and UII immunoreactivities also displayed a negative Met-enk reaction. Met-enk was detected by radioimmunoassay in the urophysis, although the content was relatively small compared with that found in other parts of the central nervous system and in the pituitary.     

The caudal neurosecretory system

Summary Recent information on the caudal neurosecretory system (urophysis) is collated, with special reference to cellular biology including neural relations, activity and chemistry of the biological principles associated with the urophysis, pharmacological analysis of the receptors for these principles, and their possible functions in a physiological sense. The existence of at least two principles, urotensins I and II, is well established. They differ pharmacologically and chemically and may arise from different cell types. At present, osmoregulation, cardiovascular regulation and reproduction are the most likely aspects of organismal physiology wherein these principles may be involved.Dedicated to Professor Doctor Berta Scharrer on the occasion of her seventieth birthday, with profound admiration and affection     

Brain stem innervation of the caudal neurosecretory system

Summary The innervation of the caudal neurosecretory system of Poecilia sphenops (black molly) was studied by use of the retrograde horseradish peroxidase (HRP) method. The structure of the caudal neurosecretory system in this species was well suited for application of HRP procedures. Acrylamide/HRP gel implants were placed in the nucleus of the caudal neurosecretory system. Two neuronal groups which contained HRP filled cells were found in the brain stem. Bilateral projections originate from the dorsal tegmentum of the midbrain and the reticular nucleus of the medulla.Supported by PHS 5429-19-4 and BNS 8206452The authors wish to thank Drs. R. Parsons, S. Freedman and J. Wells for reading this report and A. Angel for photographic assistance     

Isolation and amino acid sequence of two urotensin II peptides from Catostomus commersoni urophyses

Two peptides with urotensin II (UII = smooth muscle stimulating) activity have been isolated from urophyses of the sucker, Catostomus commersoni. The amino acid sequences of the two peptides, designated UIIA and UIIB, are as follows: UIIA, H-Gly-Ser-Gly-Ala-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH; UIIB, H-Gly-Ser-Asn-Thr-Glu-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH. These peptides have the part sequence Cys-Phe-Trp-Lys-Tyr-Cys-Val in common with the UII peptide from Gillichthys mirabilis and the part-sequence Phe-Trp-Lys in common with somatostatin.     

Immunohistochemical investigation of urotensins in the caudal spinal cord of four species of elasmobranchs and the lamprey,Lampetra japonica

Summary In the four species of elasmobranchs examined (Triakis scyllia, Heterodontus japonicus, Scyliorhinus torazame, Dasyatis akajei), all identifiable caudal neurosecretory cells and their corresponding neurohemal areas showed urotensin II (UII)-immunoreactivity with varied intensity. To localize urotensin I (UI) in the caudal neurosecretory system of the dogfish, Triakis scyllia, h-CRF (1–20) antiserum that cross-reacts with UI was used in place of UI antiserum. CRF/UI-immunoreactivity was demonstrated in the neurosecretory cells and neurohemal areas. A considerable number of neurons showed both UII- and CRF/UI-immunoreactivities, suggesting that UII and UI are produced in the same neurosecretory cells. However, some neurons exhibited UII-immunoreactivity, but no CRF/UI-immunoreactivity. Cells immunoreactive only to CRF antiserum were not detected. At least two populations of neurons exist in the dogfish caudal neurosecretory system: (i) cells immunoreactive for both CRF/UI and UII, and (ii) cells immunoreactive for UII. The dorsal cells of the lamprey, Lampetra japonica, did not react with either UII or CRF antiserum.     

Development of the caudal neurosecretory system of the chum salmon,Oncorhynchus keta,as revealed by immunohistochemistry for urotensins I and II

In order to make an immunohistochemical analysis of the development of the caudal neurosecretory system of the chum salmon, Oncorhynchus keta, we employed the peroxidase-anti-peroxidase technique using antisera specific for urotensins (U) I and II on artificially reared embryos, larvae, and juveniles of this species. Immunoreactivities for UI and UII were first demonstrated in the embryo immediately before hatching, showing labeled perikarya and fibers in the most caudal region of the spinal cord where the presumptive caudal neurosecretory system is located. However, distinct differentiation of the histological neurohemal organ had not yet begun in the embryo. Immunoreactive perikarya and fibers gradually increased in number, and an elaborate urophysis comparable to that of adults was demonstrated in the larvae about 5 months after hatching. At this stage, weak immunoreactivity against UI was detected in the neurohypophysis.     

A double sequential immunofluorescence method demonstrating the co-localization of urotensins I and II in the caudal neurosecretory system of the teleost,Gillichthys mirabilis

Summary A double immunofluorescence method was devised to localize simultaneously urotensin-I (UI) and -II (UII) immunoreactivities in the caudal neurosecretory system of the goby, Gillichthys mirabilis. In a sequential fashion, sections of the posterior spinal cord and urophysis were treated with antiserum to corticotropin-releasing factor (CRF) that cross-reacts with UI, fluorescein-conjugated sheep anti-rabbit IgG, biotinylated anti-UII and rhodamine-conjugated avidin. UI and UII immunoreactivities appeared to coexist in some neurons and in most fibers and urophysial tissue; the remainder of the fibers and urophysis and the majority of neurons were immunoreactive for CRF/ UI only. No convincing evidence of immunoreactivity for UII only was found. A few nonreactive cells were seen, but these may not be neurosecretory neurons. The two immunoreactive cell types were not segregated topographically, and the intensity of perikaryal immunofluorescence for CRF/UI was variable. To explain these results a hypothesis that all caudal neurosecretory cells may synthesize both UI and UII and that immunoreactive differences may reflect different states of cellular activity, is suggested. This sequential double immunofluorescence method offers several advantages over other techniques and is especially useful for co-localization studies when primary antisera from different species are not available.     

Caudal neurosecretory system of the zebrafish: ultrastructural organization and immunocytochemical detection of urotensins

The caudal neurosecretory system is described here for the first time in the zebrafish, one of the most important models used to study biological processes. Light- and electron-microscopical approaches have been employed to describe the structural organization of Dahlgren cells and the urophysis, together with the immunohistochemical localization of urotensin I and II (UI and UII) peptides. Two latero-ventral bands of neuronal perikarya in the caudal spinal cord project axons to the urophysis. The largest secretory neurons (~20 μm) are located rostrally. UII-immunoreactive perikarya are much more numerous than those immunoreactive for UI. A few neurons are immunopositive for both peptides. Axons contain 75-nm to 180-nm dense-core vesicles comprising two populations distributed in two axonal types (A and B). Large dense vesicles predominate in type A axons and smaller ones in type B. Immunogold double-labelling has revealed that some fibres contain both UI and UII, sometimes even within the same neurosecretory granule. UII is apparently the major peptide present and predominates in type A axons, with UI predominating in type B. A surprising finding, not previously reported in other fish, is the presence of dense-core vesicles, similar to those in neurons, in astrocytes including their end-feet around capillaries. Secretory type vesicles are also evident in ependymocytes and cerebrospinal-fluid-contacting neurons in the terminal spinal cord. Thus, in addition to the urophysis, this region may possess further secretory systems whose products and associated targets remain to be established. These results provide the basis for further experimental, genetic and developmental studies of the urophysial system in the zebrafish.     

Adrenergic neurons in the spinal cord of the pike (Esox lucius) and their relation to the caudal neurosecretory system

Summary The lower spinal cord including the caudal neurosecretory system of the pike (Esox lucius) was investigated by means of light and electron microscopy and also with the fluorescence histochemical method of Falck and Hillarp for the visualization of monoamines. A system of perikarya displaying a specific green fluorescence of remarkably high intensity is disclosed in the basal part of the ventrolateral and lateral ependymal lining of the central canal. The area corresponding to the upper half of the urophysis has most cells; their number decreases caudally and cranially. A considerable number of their beaded neurites reach the neurosecretory neurons by different routes but are only occasionally present in the actual neurohemal region. An intensely fluorescent dendritic process is sometimes observed terminating with a bulbous enlargement at the ependymal surface in the central canal. Besides small, electron lucid vesicles in the terminal parts of the axons, the neurons contain numerous large dense-core vesicles which can apparently take up and store 5-hydroxydopa (5-OH-dopa) and 5-hydroxydopamine (5-OH-DA). These neurons are thought to be adrenergic and to contain a primary catecholamine, possibly noradrenaline.The varicosities of the adrenergic terminals are repeatedly observed contiguous to some of the neurosecretory axons, the membrane distance at places of contacts generally ranging from 150–200 Å. Another type of nerve terminals that contain only small empty vesicles, also after pretreatment with 5-OH-dopa or 5-OH-DA, are frequent among the neurosecretory neurons. These axons establish synaptic contacts with membrane thickenings on most of the neurosecretory neurons. Thus it seems that the neurosecretory neurons are innervated by neurons morphologically similar to cholinergic neurons and that part of them receive an adrenergic innervation, which supports the view hat the caudal neurosecretory cells do not constitute a functionally homogeneous population.Supported by the Deutsche Forschungsgemeinschaft and the Joachim-Jungius Gesellschaft zur Förderung der Wissenschaften, Hamburg.Supported by the Swedish Natural Research Council (No. 99-35). This work was in part carried out within a research organization sponsored by the Swedish Medical Research Council (Projects No. B70-14X-56-06 and B70-14X-712-05).Supported by the Deutsche Forschungsgemeinschaft and USPHS Research Grant TW 00295-02.     

Response of caudal neurosecretory cells of Salvelinus fontinalis to variations in the ionic composition of the environment

Summary The response of caudal neurosecretory cells in Salvelinus fontinalis to exposure to media in which one ion (Na, K, Mg, Ca and Cl) was either selectively enriched or depleted was evaluated by morphometric criteria. Morphological changes indicating stimulation of synthetic activity were observed: exposure to sea water with low potassium concentration was the most efficacious in inducing the increase in the average diameter of caudal cells and the number of nucleoli for both cells with lobed and with nonlobed nuclei. The proportion of cells with lobed nuclei was also increased. To a lesser degree and in decreasing order, experimental milieu with modified magnesium contents, either fresh water enriched in this ion or seawater depleted of the same ion, and fresh water enriched in calcium also resulted in significant increases in cell diameter and the number of nucleoli.The two cytological cell types, viz cells with non-lobed nuclei or cells with lobed nuclei, are interpreted as being linked to different levels of neurosecretory activity.Supported by NSERC (Canada)     

Isolation and amino acid sequence of urotensin I, a vasoactive and ACTH-releasing neuropeptide, from the carp (Cyprinus carpio) urophysis

Urotensin I (UI), a 41-residue mammalian hypotensive and fish or mammalian corticotropin-releasing peptide, isolated from 0.1 N HCI extracts of urophyses of the carp (Cyprinus carpio) was purified and the amino acid sequence was determined to be: H-Asn-Asp-Asp-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu- Arg-Asn-Met-Ile-Glu-Met-Ala-Arg-Asn-Glu-Asn-Gln-Arg-Glu-Gln-Ala-Gly-Leu-Asn-Arg-Lys-Tyr-Leu-Asp-Glu-Val-NH2. When the extraction procedure included heating at 100 degrees C for 15 min, UI was cleaved at a highly acid labile Asp-Pro bond to give the fully active UI (4-41). Urotensin I shows close structural and biological homology with the recently isolated ovine hypothalamic corticotropin-releasing factor (CRF) and the frog skin peptide sauvagine and thus may be considered an evolutionary prototype of unique mammalian-hypotensive and vertebrate corticotropin-releasing factors.     

Ultrastructural changes in the urophysis of Mollienesia sphenops following adaptation to seawater

Summary The urophysis or neurohemal contact site of the caudal neurosecretory system of Mollienesia sphenops, the black molly, was studied in animals adapted to an artificial seawater environment. This species of fish was chosen for these studies because of its known ability to osmoregulate and its adaptability to the laboratory aquarium. The urophysis of freshwater acclimated mollys contained an abundance of neurosecretory granules. However, in fish subjected to a seawater environment for one week the number of neurosecretory granules was significantly decreased. In addition, there was an increase in blood cell infiltration of the urophysis.Supported by PHS 5429-16-19 (5-23311)The Author wishes to thank Drs. W. Young and J. Wells for their careful reading of this report and W. Boldosser for technical assistance     

Identification of urotensin II-related peptide as the urotensin II-immunoreactive molecule in the rat brain

Urotensin II (UII) has been reported as the most potent known vasoconstrictor. While rat and mouse orthologs of UII precursor protein have been reported, only the tentative structures of UII peptides of these animals have been demonstrated, since prepro-UII proteins lack typical processing sites for their mature peptides. In the present study, we isolated a novel peptide, UII-related peptide (URP), from the extract of the rat brain as the sole immunoreactive substance to anti-UII antibody; the amino acid sequence of the peptide was determined as ACFWKYCV. cDNAs encoding rat, mouse, and human precursor proteins for URP were cloned and revealed that the sequences of mouse and human URP peptides are the same as that for rat URP. Prepro-URP gene is expressed in several rat tissues such as those of the thymus, spleen, testis, and spinal cord, although with lower levels than the prepro-UII gene. In the human, the prepro-URP gene is expressed comparably to prepro-UII in several tissues except the spinal cord. URP was found to bind and activate the human or rat UII receptors (GPR14) and showed a hypotensive effect when administered to anesthetized rats. These results suggest that URP is the endogenous and functional ligand for UII receptor in the rat and mouse, and possibly in the human. We also describe the preparation of specific monoclonal antibodies raised against UII peptide and the establishment of a highly sensitive enzyme immunoassay system for UII peptides.     

The activated hypothalamic magnocellular neurosecretory system and the one neuron -one neurohypophysial hormone concept

Summary The activated hypothalamic magnocellular neurosecretory system of the rat was studied in tissue sections, double stained with the unlabeled antibody peroxidase-antiperoxidase complex (PAP) technique. The results indicate that in animals with an activated hypothalamic magnocellular neuroendocrine system, as well as in normal animals, vasopressin and oxytocin are exclusively synthesized in separate vasopressinergic and oxytocinergic neurons.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Coexistence of cholecystokinin and oxytocin-neurophysin in some magnocellular hypothalamo-hypophyseal neurons

Summary The existence of cholecystokinin in the posterior hypophysis and its hypothalamic origin have been unequivocally demonstrated. Immunocytochemical evidence is presented for the coexistence of gastrin-cholecystokinin and oxytocin-neurophysin I immunoreactivities in some magnocellular neurons of the supraoptic and paraventricular nuclei both in rat and bovine hypothalamus.     

Responses of the neurosecretory cells of the freshwater snail,Indoplanorbis exustus to hydrothermal stress

Changes in the neurosecretory cell cytology of I. exustus subjected to hypertonic saline (0.1 ml of 1.5%/snail) loading and thermal stress (35°C) for two hours, have been investigated. Of the two types of neurosecretory cells A and B that are present in the central nervous system (CNS) of I. exustus, striking changes were evident only in B cells. After both treatments, there was about 33% decline in NSM (Neurosecretory material) intensity. However, the nuclear diameter of B cells was significantly (P < 0.001) increased in the snails administrated with hypertonic saline unlike in those exposed to 35°C wherein significant (P < 0.005) decline was evident. The adaptive significance of the neuroendocrine system of I exustus is discussed in relation to hydrothermal stress.     

Sauvagine/urotensin I-like immunoreactivity in the caudal neurosecretory system of a seawater fish Diplodus sargus L. in normal and hyposmotic milieu

We report the presence of sauvagine/urotensin I-like immunoreactive (SV/UI-LI) elements in the caudal neurosecretory system of a teleost (Diplodus sargus L.) collected from aquaria tanks of the Aquaculture Center (Talassographic Institut of CNR) of Messina or maintained in an hyposmotic milieu for different periods. In normal specimens, SV/UI-LI material was recognizable in discrete or little amounts both in Dahlgren cell cytoplasm and in their axons that reach the urophysis. On the contrary, the specimens transferred in an hyposmotic milieu showed a fast and dramatic increase of immunoreactivity mainly in neurohemal endings of the urophysis. This suggests a physiological role of caudal neurosecretory products on osmoregulatory mechanisms.     

The caudal neurosecretory system: control and function of a novel neuroendocrine system in fish.

The caudal neurosecretory system (CNSS) of fish was first defined over 70 years ago yet despite much investigation, a clear physiological role has yet to be elucidated. Although the CNSS structure is as yet thought to be confined to piscine species, the secreted peptides, urotensins I and II (UI and UII), have been detected in a number of vertebrate species, most recently illustrated by the isolation of UII in humans. The apparent importance of these peptides, suggested by their relative phylogenetic conservation, is further supported by the complex control mechanisms associated with their secretion. The CNSS in teleosts is known to receive extensive and diverse innervation from the higher central nervous system, with evidence for the presence of cholinergic, noradrenergic, serotonergic, and peptidergic descending inputs. Recent observations also suggest the presence of glucocorticoid receptors in the flounder CNSS, supporting previous evidence for a possible role as a pituitary-independent mechanism controlling cortisol secretion. The most convincing evidence as to a physiological role for the CNSS in fish has stemmed from the direct and indirect influence of the urotensins on osmoregulatory function. Recent advances allowing the measurement of circulating levels of UII in the flounder have supported this. In addition, there is evidence to suggest some seasonal variation in peptide levels supporting the notion that the CNSS may have an integrative role in the control of coordinated changes in the reproductive, osmoregulatory and nutritional systems of migratory euryhaline species.