Electron microscope immunohistochemical localization of neurophysin in the rat with hereditary diabetes insipidus

In order to study the subcellular distribution of neurophysin in the rat with hypothalamic hereditary diabetes insipidus (DI), an immunoelectron microscopic localization of neurophysin was performed in the hypothalamo-neurohypophysial system of both homozygous and heterozygous DI rats. Whereas in control rats neurophysin was localized in the granules present in the secretory neurons, in the homozygous DI rats neurophysin was found in the granules and outside the granules in the perikarya and axons of neurons of both supraoptic and paraventricular nuclei. In the heterozygous DI rats findings similar to those observed in homozygous DI rats were observed, although in the posterior pituitary, the exgranular material appeared to be less abundant than in homozygous DI rats. These results clearly demonstrated that in hyperstimulated neurons neurophysin was distributed in both granular and extragranular compartments.     

Reaction in vitro of synthetic oxytocin and lysine-vasopressin with the pseudoisocyaninchloride technique used for the demonstration of neurohypophysial neurosecretory material

Summary The performic acid (or acid potassium permanganate) pseudoisocyaninchloride technique (PSICN), when applied to formol-fixed paraffin-embedded sections of the hypothalamo-neurohypophysial system, causes to fluoresce intensely material with the same distribution as chrome-alum haematoxyphil neurosecretory material.It has been claimed that in vitro this technique does not react with oxytocin, but does react with neurophysin, the carrier (or precursor) polypeptide. Now however, using Rodeck's agar technique, which mimics closely conditions applying in tissue-sections, we have demonstrated that the PSICN technique will react in vitro with the octapeptide neuro-hormones, oxytocin and vasopressin.     

Ultrastructural immunocytochemical localization of neurophysin and vasopressin in the median eminence and posterior pituitary of the guinea pig

Summary With the use of tissue prepared by freeze-substitution and the unlabelled antibody enzyme technique, neurophysin and vasopressin were localized at the ultrastructural level in the posterior pituitary and median eminence of the guinea pig. In the posterior pituitary neurophysin was found in the large neurosecretory granules (1300–1500 Å) of axons, Herring bodies, and nerve terminals. In some of these axons immunoreactive neurophysin was found outside of granules in the axoplasm. By light microscopy neurophysin was found in both the zona interna and zona externa of the median eminence; this was confirmed by electron microscopy. In the zona interna as in the posterior pituitary, neurophysin was localized both inside and outside the large neurosecretory granules. In the zona externa, immunoreactive deposit was primarily located in granules with a diameter of 900–1100 Å in nerve terminals abutting on the primary portal plexus. The distribution of vasopressin paralleled that of neurophysin except that the hormone was rarely extragranular. These results demonstrate for the first time that both neurophysin and vasopressin are present in granules of axons that are in contact with the hypophysial portal vasculature.The authors wish to thank Dr. Alan Robinson for the gifts of antiserum to bovine neurophysin I and for purified bovine neurophysin I; Dr. Ludwig Sternberger for the peroxidase-anti-peroxidase complex; and Dr. Robert Utiger for antiserum to lysine vasopressinSupported in part by U.S. Public Health Service grant RR-00167 to the Wisconsin Regional Primate Research Center from the National Institutes of Health. Primate Center publication No. 14-017.Recipient of NIH, NINDS Teacher-Investigator Award NS-1108.     

Electron-microscopic immunocytochemistry of neuropeptide Y immunoreactive innervation of vasopressin neurons in the paraventricular nucleus of the rat hypothalamus

The neuropeptide Y (NPY) immunoreactive synaptic input to neurons containing neurophysin II (NP II), the carrier protein of vasopressin (VP), was observed in the paraventricular nucleus (PVN) of the rat hypothalamus by double-labeling immunocytochemistry combining the preembedding peroxidase-antiperoxidase (PAP) method with the postembedding immunogold staining method at the electron-microscopic level. NPY-like immunoreactivities were detected by the PAP method in the dense granular vesicles (70-100 nm in diameter) in the immunoreactive presynaptic axon terminals. NP II-like immunoreactive large neurosecretory granules labeled with gold particles were found in the neurons receiving synaptic input of the NPY-like immunoreactive terminals. This suggests that NPY may be a neurotransmitter or neuromodulator and that NPY neurons may, through synaptic contacts, regulate the secretion of VP neurons.     

Use of the unlabeled antibody immunohistochemical technique for the detection of human antibody.

Two methods have been developed which permit use of the unlabeled antibody immunohistochemical technique for detection of human antibody, without the need for immunization of humans with peroxidase. Human antibody to herpes simplex virus (HSV) reacted with human cell cultures infected with HSV was the experimental system. In the first method an attempt was made to employ rabbit peroxidase-antiperoxidase (PAP) soluble complexes in connectin with human antibody. This was done by sequential addition to the HSV-infected cells of (a) human anti-HSV, (b) rabbit antihuman globulin, (c) guinea pig antirabbit globulin (the bridging reagent) and (d) rabbit PAP. Strong specific staining of HSV-infected cells was obtained; however, difficulties were encountered with nonspecific reactions on uninfected cells. In the second method PAP soluble complexes prepared with baboon antiperoxidase were bridged to the human anti-HSV antibody by rabbit antihuman globulin. Because of the phylogenetic relatedness of human and baboon globulins this resulted in firm binding which gave strong specific staining of HSV-infected cells without significant reaction in uninfected cells.     

Ostrich MSEL-neurophysin belongs to the class of two-domain "big" neurophysin as indicated by complete amino acid sequence of the neurophysin/copeptin

Mammalian neurohypophyseal hormones, oxytocin and vasopressin, are known to be synthesized as part of two larger precursors containing, respectively, a VLDV-neurophysin and a MSEL-neurophysin together with its associated glycopeptide. Starting from ostrich neurohypophyses, a "big" neurophysin was isolated and chemically characterized. Following sequence determination of the CNBr-derived fragments and of peptides obtained from trypsin and V8-protease digestion of the oxidized protein, this "big" neurophysin was found to contain an MSEL-neurophysin moiety (94 residues) still covalently associated with the COOH-terminal glycopeptide (38 residues, copeptin). This study demonstrates that the ostrich MSEL-neurophysin sequence closely resembles all known MSEL-neurophysin sequences and that, furthermore, it does not contain the single amino acid insertion shown previously in the ostrich VLDV-neurophysin. It is also shown that the stretch of amino acids, linking the MSEL-neurophysin and the copeptin, is clearly different from its mammalian homologues and lacks the Arg residue normally recognized by the cleaving enzyme. This study also demonstrates that the ostrich copeptin is more closely related to the amphibian copeptin sequence than to its mammalian homologue, leading to the hypothesis that two families of copeptin molecules might exist. Thus, the ostrich MSEL-neurophysin-copeptin molecule is the first "big" neurophysin reported in birds and, together with the guinea pig and amphibian homologues, represents the third example of partial or no neurophysin-copeptin cleavage.     

Conditions for the immunohistochemical demonstration of complement factor C3 in formaldehyde-fixed and paraffin-embedded renal tissues

Summary Paraffin sections of formaldehyde-fixed renal biopsies were labeled for complement C3 by a polyclonal rabbit antibody to human complement C3, by the peroxidase-antiperoxidase complex (PAP) and the avidin-biotin peroxidase complex (ABC) techniques, respectively. All tissues had C3 deposits according to direct immunofluorescence on fresh frozen sections. Staining for muramidase was introduced as an intrinsic control for the degree of tissue proteolysis after the necessary trypsin digestion prior to the immunoenzyme labeling. The results indicated that even minute deposits of C3 could be detected in paraffin sections by the ABC method, which was more sensitive than the PAP technique; the ABC method allowed a maximal dilution of 12,400 of the primary antibody as compared to 1800 for the PAP technique.     

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.     

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.     

Protein carboxymethylation: effects of 2% sodium chloride administration on protein carboxymethylase and its endogenous substrates in rat posterior pituitary

Protein carboxymethylase and methyl acceptor proteins have been studied in the posterior pituitary of control and NaCl treated rats. Salt loading stimulates the hypothalamo-neurohypophysial axis causing the secretion of neurohypophysial peptides. Under these conditions there was a progressive decrease of methyl acceptor proteins with the greatest fall occurring between 1 and 2 days after salt loading. Electrophoretic analysis of the methyl acceptor proteins showed a single major peak of methylated proteins accounting for up to 80% of the total radioactivity. The molecular weight (11,000) and the disappearance of this peak after salt loading suggested that this methyl acceptor protein is neurophysin. After prolonged stimulation of the hypothalamo-neurohypophysial axis there was a progressive increase in protein carboxymethylase specific activity.     

Immunocytochemistry with monoclonal anti-immunoglobulin as a developing reagent: application to immunoperoxidase staining and radioimmunocytochemistry

The development of two monoclonal antibodies for use as second antibodies in immunocytochemistry is described. The antibodies are produced by mouse X mouse hybrid myelomas, and are both of the IgG type. The two antibodies, RB23 and ND13, were used to detect neurophysin by three-step peroxidase-antiperoxidase (PAP) immunostaining, and were "internally labeled" with 3H-lysine for the radioimmunocytochemical localization of neurophysin, substance P, and tyrosine hydroxylase using rabbit first antibodies. The binding sites of RB23 and ND13 on the rabbit IgG antibodies were determined by solid-phase radioimmunoassay, using allotype-specific rabbit serum to compete with RB23 and ND13. It was found that both RB23 and ND13 are directed against the B4 kappa-light-chain allotype. The immunocytochemical localization of adrenocorticotropic hormone and somatostatin with rabbit primary antibodies was not achieved with RB23 or ND13, and it is proposed that these antibodies are not of the B4 allotype. The findings demonstrate that monoclonal second antibodies can be useful general reagents for conventional immunocytochemistry as well as for radioimmunocytochemistry. Furthermore, allotype-specific monoclonal second antibodies may prove useful in the simultaneous immunohistochemical localization of more than one antigen in a given tissue section.     

Immunoreactive material resembling vertebrate neuropeptides and neurophysins in the brain,suboesophageal ganglion,corpus cardiacum and corpus allatum of the dictyopteran Periplaneta americana L.

Summary The peroxidase-antiperoxidase technique (Vandesande and Dierickx 1976) with antibodies raised against several vertebrate neuropeptides and neurophysins, was applied] to 4-m Paraplast-embedded serial sections of in situ fixed brains and adjacent suboesophageal ganglion (SOG), corpora cardiaca (CC) and corpora allata (CA) of the blattarian insect Periplaneta americana L. Substances immunologically related to bovine neurophysin I (NPI) and II (NPII), synthetic arginine vasopressin (AVP) and synthetic oxytocin (OT) were found to be differentially distributed in the central nervous system. The differences among all four antigens demonstrated became clearly evident by immunohistochemical double-staining procedures (Vandesande 1983); no overlapping was observed. The same double-staining technique revealed that these vertebrate-type substances occur in other neurosecretory cells and axons than those containing CRF- and ACTH-like material as reported earlier (Verhaert et al. 1984).     

Chemical and physical properties of the disulfides of bovine neurophysin-II.

Bovine neurophysin-II is shown to be very susceptible to partial reduction in the absence of urea. Reduction of an average of one disulfide leads to major changes in conformation and disulfide optical activity, manifest in part by pronounced far-uv ellipticity changes, complete loss of the 248-nm ellipticity band, and a shift of the 278-nm ellipticity band to shorter wavelengths with loss of half its intensity; the reduction process generates a mixture of products and appears to be accompanied by disulfide interchange. The circular dichroism data indicate that the disulfide(s) most susceptible to reduction or interchange are either the principal contributors to the 248- and 278-nm ellipticity bands or that the optical activity of other disulfides is dependent on their integrity. Peptides that bind to the hormone-binding site of neurophysin-II protect against reduction. On reoxidation of partially reduced neurophysin-II there is only a partial return of the native circular dichroism spectrum and electrophoretic behavior. The percentage of native protein in samples reoxidized following different degrees of reduction was estimated by comparison of the circular dichroism spectra of these samples with those of the fractionated native and denatured components of monoreduced-reoxidized neurophysin. Under our reoxidation conditions, less than 50% native protein was found in monoreduced-reoxidized neurophysin and less than 10% native protein was found in completely reduced-reoxidized neurophysin. The results are interpreted with qualified reference to a model in which one or more disulfides are "strained" in the native state and in which the native protein is unstable relative to species in which the disulfides are differently paired.     

Immunophenotypic evidence for distinct populations of microglia in the rat hypothalamo-neurohypophysial system

The morphology, distribution and immunophenotype of microglia throughout the adult rat hypothalamo-neurohypophysial system was examined. Four macrophage-associated antibodies (OX-42, F4/80, ED1 and ED2) were used; the expression of major histocompatibility complex antigens was investigated by use of antibodies against OX-6, OX-17 (MHC class II) and OX-18 (MHC class I). Three distinct types of microglia were identified. The first was located in the magnocellular nuclei; these radially branched (ramified) microglia had round cell bodies and long branched processes, and were strongly immunoreactive only for OX-42. The second was located outside the blood-brain barrier in the median eminence, pituitary stalk and neurohypophysis often close to blood vessels; these compact microglia had irregular cell bodies and shorter processes, and were strongly labelled by OX-42 and F4/80, weakly labelled by OX-18, and generally unlabelled by ED1, ED2, OX-6 and OX-17. The third type was found in small numbers throughout the system at the surface of the neurvous tissue or around blood vessels; these perivascular microglia were elongated cells with no branching processes, and were strongly labelled by ED1, ED2, OX-18, OX-6, OX-17 and F4/80 antibodies but showed variable OX-42 immunoreactivity. Cells in a perivascular location were heterogeneous with respect to their immunophenotype. The presence in the normal adult rat hypothalamo-neurohypophysial system of MHC class-II molecules (OX-6 and OX-17) on a sub-set of perivascular microglia suggests that these cells are capable of presenting antigen to T lymphocytes. The microglia, which lie on either side of the blood-brain barrier, are well placed to facilitate interaction between the immune and neuroendocrine systems.     

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     

High-performance liquid chromatographic mapping and structural characterization of neurophysin isoforms

Both ion-exchange and reverse-phase HPLC protocols for micromapping of neurophysins have been examined and the structural relationships among the major isoforms identified in the maps have been characterized. Reverse-phase HPLC was found to be especially useful for obtaining fingerprints of the isoforms within each of the two major families of neurophysins, I (oxytocin-related) and II (vasopressin-related), for both bovine and human neurophysins from posterior pituitary sources. From fractionation of the bovine proteins on octylsilyl columns, at least four neurophysins I were identified, one of which corresponds to the intact sequence of 93 residues and three of which vary from the parent by various degrees of carboxyl-terminal truncation. For bovine neurophysin II, two isoforms were identified in the reverse-phase HPLC maps, both of 95 residues, which vary from one another by the residue, either Ile or Val, at position 89. Isoforms were also detected for human neurophysins, including a carboxyl-terminal truncated form of human neurophysin II. All of the major neurophysin isoforms and several of the minor forms were shown to be functionally active as expressed by their binding to peptide ligand affinity matrices. Reverse-phase HPLC mapping on the octylsilyl matrix allowed neurophysin fingerprinting of crude tissue extracts by providing a narrow "window" within which the neurophysins elute but many other polypeptides expected to be present are excluded. The reverse phase HPLC method provides a useful way to obtain isolated neurophysin isoforms for physicochemical characterizations now usually carried out with mixtures of isoforms obtained by ion-exchange chromatography. The method also has characteristics amenable both for high-sensitivity fingerprinting of neurophysin isoforms, from different species and anatomical sources, and as a prelude to microstructural and -functional characterization of the isoforms so isolated.     

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.     

The extravascular channel system in the rostral pituitary of Mugil cephalus (Teleostei) as revealed by use of horseradish peroxidase

Summary Using the unlabeled antibody peroxidase-antiperoxidase (PAP) technique at the light microscopic level, it was demonstrated that, in the amphibian magnocellular hypothalamo-hypophysial neurosecretory system, vasotocin and mesotocin are synthesized in separate neurons. A tendency to preferential location of the two kinds of neuronal perikarya is described. The neurosecretory perikarya are the origin of separate vasotocinergic and mesotocinergic axons. In the neural lobe, the pattern of distribution of the two types of axons is different. The coarse ventricular dendrites of both kinds of neurons are hormone-containing processes. Staining with anti-bovine neurophysin I serum suggested that the vasotocinergic and the mesotocinergic neurons synthesize different neurophysins.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

A modification of the unlabeled antibody enzyme method using heterologous antisera for the light microscopic and ultrastructural localization of insulin, glucagon and growth hormone.

The requirement of using homologous antisera (primary antiserum and peroxidase-antiperoxidase (PAP) complex raised in the same species) in the unlabeled antibody enzyme method has been investigated at the light and electron microscopic level using the localization of insulin, glucagon and growth hormone as model systems. Optimum immunocytochemical staining for all three antigens was observed when sheep or goat antirabbit gamma-globulin (S-ARgammaG or G-ARgammaG) were used to couple rabbit peroxidase-antiperoxidase complex with either guinea pig antisera to insulin (GP-AIS) or glucagon (GP-AGS), or monkey antisera to rat growth hormone (M-ARGH). The cross-reactivity between S-ARgammaG or G-ARgammaG and immunoglobulins in these primary antisera were substantiated by immunoelectrophoresis and radioimmunoassay. S-ARgammaG was shown to produce precipitation arcs with GP-AIS and M-ARGH that were similar to those seen when the latter were reacted with rabbit antiguinea pig gamma-globulin antiserum and goat antimonkey gamma-globulin antiserum, respectively. Radioimmunoassay results revealed that immunoprecipitation of 6-10% as compared to homologous antisera controls yielded excellent staining localization when S-ARgammaG was used for immunocytochemistry. Thus, heterologous antisera (primary antiserum and PAP complex raised in different species) may be used in the unlabeled antibody enzyme method as long as the coupling antiserum shows cross-reactivity with immunoglobulins of the primary antiserum and the PAP complex.     

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