Apelin-immunoreactivity in the rat hypothalamus and pituitary.

With the use of an antiserum against human apelin-36, apelin-immunoreactivity (irAP) was detected in neurons and cell processes of the supraoptic nucleus (SO), paraventricular nucleus (PVH), accessory neurosecretory nuclei (Acc) and suprachiasmatic nucleus. Strongly labeled cells/processes were noted in the internal layer of the median eminence, infundibular stem, anterior and posterior pituitary. Double-labeling the sections with goat polyclonal neurophysin I-antiserum and rabbit polyclonal apelin-antiserum revealed a population of magnocellular neurons in the PVH, SO and Acc expressing both irAP and neurophysin I-immunoreactivity (irNP), the latter being a marker of oxytocin-containing neurons. By inference, the AP-positive but irNP-negative magnocellular neurons could be vasopressin-containing. The presence of irAP in certain hypothalamic nuclei and pituitary suggests that the peptide may be a signaling molecule released from the hypothalamic-hypophysial axis.

Regeneration of neurosecretory axons into various types of intrahypothalamic graft is promoted by the absence of the blood-brain barrier: a neurophysin-immunohistochemical and horseradish peroxidase-histochemical study.

In order to test the hypothesis that neurosecretory axon regeneration occurs only in the presence of specific vascular, perivascular, and glial microenvironments, isografts of neural lobe and optic nerve and autografts of sciatic nerve were transplanted into the hypothalamo-neurohypophysial tract at the lateral retrochiasmatic area of adult male rats. The integrity of the blood-brain barrier (BBB) to intravenously administered horseradish peroxidase (HRP), the regenerative process of neurosecretory axons, and functional recovery from lesion-induced diabetes insipidus were analyzed at 18 hr, 36 hr, 10 days, 30 days, and 80 days postsurgery. Neurophysin-positive axons invaded all grafts, as well as perivascular spaces of the adjacent hypothalamus. Wherever neurosecretory axon regeneration occurred, the BBB was breached. Reestablishment of the BBB was paralleled by a decrease in both density and staining intensity of regenerated neurophysin-positive axons. These observations illustrate that neurosecretory axon regeneration is tributary of the absence of BBB. It is speculated that blood-borne factors, provided when the BBB is breached, initiate and sustain neurosecretory axon regeneration. In addition, products of glial elements may enhance or complement the above stimulatory processes.

Hypophysial and extrahypophysial projections of the neurosecretory system of cartilaginous fishes: an immunocytochemical study using a polyclonal antibody against dogfish neurophysin.

Immunocytochemistry using antibodies against the neurohypophysial nonapeptides has given equivocal results regarding relevant aspects of the classical neurosecretory system of elasmobranchs. The lack of antibodies reacting with the elasmobranch neurophysins (Nps) has prevented the study of this neurosecretory system by Nps immunocytochemistry. This led us to purify Nps from Scyliorhinus canicula, and to use them to raise a polyclonal antibody. This antibody reacted strongly with the elasmobranch neurophysin neurons, revealing their most delicate and distant hypophysial and extrahypophysial projections. A detailed mapping of the neurosecretory system of five elasmobranch species (Etmopterus spinax, Squalus acanthias, Scyliorhinus canicula, Galeus melanostomus, Raja radiata) and one holocephalian species (Hydrolagus colliei) was performed. In elasmobranchs, the magnocellular neurophysin cells formed a distinct preoptic nucleus, whereas in Hydrolagus the immunoreactive cells were scattered. Distinct parvicellular neurophysin cells were present in the preoptic nucleus. In Raja the nucleus "O" contained parvicellular Nps-immunoreactive neurons. The findings at the pituitary level point to the possibility that neurophysin neurons, in addition to releasing nonapeptides into the systemic capillaries of the neural lobe, also participate in the regulation of the function of the rostral, medial and intermediate lobes of the adenohypophysis by a dual mechanism, i.e., a neurovascular pathway and a direct neural input. The extrahypophysial projections of the neurophysin neurons were highly developed to a degree not comparable to any other vertebrate group. The targets of these projections were located in the telencephalon, diencephalon and hindbrain. The evolutionary and functional implications of this phenomenon are discussed.

Combined use of immunocytochemistry and lectin histochemistry for the study of the hypothalamic neurosecretory system of the snake Natrix maura (L.).

Natrix maura snakes were processed for immunocytochemistry and lectin histochemistry at both light- and electron-microscopic levels. Antisera against bovine neurophysins, vasotocin and mesotocin were used as well as concanavalin A, wheat germ and Limax flavus agglutinin lectins. The hypothalamic supraoptic and paraventricular nuclei were studied. Vasotocin neurons should contain a glycopeptide and displayed large colloid droplets consisting of large cisternae filled with packed secretory material. Mesotocin was located in different neurons.

Proto-oncogene c-fos and the regulation of vasopressin gene expression during dehydration.

Secretion of the antidiuretic hormone (ADH) vasopressin is increased when body fluid homeostasis is disturbed by dehydration. Associated with this increased secretion is an elevation of vasopressin mRNA in magnocellular hypothalamic neurons projecting to the posterior pituitary. The proto-oncogene c-fos codes for a nuclear phospho-protein Fos which binds to specific DNA elements and acts as a transcriptional regulator coupling short-term extracellular stimuli to long-term responses by altering secondary target gene expression. This study in rats examined the time courses of dehydration induced c-fos expression and the change of vasopressin gene expression in the magnocellular neurons of the hypothalamus. Immunocytochemical and in situ hybridization study demonstrated that c-fos was induced by acute intracellular dehydration in the hypothalamic magnocellular nuclei of paraventricular (PVN), supraoptic (SON), and accessory groups such as nucleus circularis. Double-label immunocytochemical study co-localized Fos and vasopressin-neurophysin immunoreactivity in the same magnocellular neurons in the SON and PVN. In situ hybridization analysis after acute dehydration revealed a rapid and transient c-fos induction followed by a persistent increase in vasopressin mRNA for up to 2 days even after rehydration. Furthermore, prevention of c-fos translation by pretreatment with protein synthesis inhibitor cycloheximide attenuated this dehydration induced increase in vasopressin mRNA. This study demonstrated that an increase in vasopressin transcription after acute dehydration is dependent on an early phase of protein synthesis.

Heterologous biosynthesis and processing of preprovasopressin in Neuro2A neuroblastoma cells.

To obtain a model for the sorting and processing of preprovasopressin (preproVP), rat VP cDNA was transfected in murine Neuro2A neuroblastoma cells, which do not express VP. The precursor of VP was expressed and processed into the authentic VP gene products VP, neurophysin (NP) and glycopeptide (GP) as determined with reversed phase HPLC and radioimmunoassay. In addition, Neuro2A-specific forms of NP and GP were observed, which may be produced in the constitutive secretory pathway in these cells.

Biosynthesis of vasopressin by gastrointestinal cells of Brattleboro and Long-Evans rats.

The distribution of vasopressin, provasopressin, vasopressin-associated neurophysin, and vasopressin-associated glycopeptide was determined immunohistochemically in the gastrointestinal system of Brattleboro and Long-Evans rats. Cells containing immunoreactivity for vasopressin, provasopressin, neurophysin, and glycopeptide were detected in the same cell types of the stomach and duodenum, while selected cells in the duodenum contained only immunoreactive glycopeptide. Unlike that in the hypothalamus, staining for neurophysin in the gastrointestinal tract was sensitive to fixation. These findings indicate that vasopressin is produced by cells in the rat gastrointestinal system and suggest the existence of synthetic pathways different from those found in hypothalamic neurons.

A tissue culture model of the hypophysiotrophic CRF producing neuronal system.

To investigate functional and chemical properties of anatomically characterized corticotropin-releasing factor-41 (CRF-41) producing neurons in vitro, hypothalamic slices of 6-day-old rats were maintained in culture for up to 6 weeks using a modified roller culture technique. This technique yields thick (100 microns) slices that contained an average of 300-400 CRF-41-immunostained neurons. The majority of CRF-41-positive cells were of small size (12-15 microns in diameter), and contained CRF-41-labeled dense core vesicles of 100 nm diameter as detected by electron microscopic postembedding immunocytochemistry. These cells represented the only CRF-41-positive cell population in the culture. Light microscope double immunolabelling of colchicine-treated cultures kept in a serum-containing media (SCM) indicated that about 60% of these CRF-41-positive neurons contains detectable levels of vasopressin-associated neurophysin (VP-NP). Culturing slices in serum-free, chemically defined media (SFM) resulted in an increased VP-NP immunostaining: parvicellular neurons labeled for both CRF-41 and VP-NP could be detected without colchicine treatment, and practically all CRF-41-positive neurons expressed VP-NP immunoreactivity. At the electron microscopic level there was a significant increase in VP-NP labeling density in the dense core vesicle compartment of CRF-41-positive varicosities. Adding dexamethasone (10 nM) to the SFM restored the staining pattern originally observed in SCM. Hence, the increased VP-NP and CRF-41 immunostaining after culturing CRF-41 neurons in SFM is most likely due to the absence of inhibitory glucocorticoids. The capacity of cultured paraventricular cells to release CRF-41 was assessed using an immunoassay. Unstimulated (basal) secretion of CRF-41 was not altered by five successive samplings at 2-hour intervals and stimulation of the same culture with 56 mmol K+ significantly increased (2-3 times) the CRF-41 content in the medium. The presence of dexamethasone (10 nM) in SFM induced a 6-fold reduction of K(+)-stimulated CRF-41 release and a 5 times reduction in tissue content in relation to cultures maintained in SFM without dexamethasone. In summary, we have demonstrated that cultured CRF-41 cells display morphological and biochemical features, as well as responsiveness to glucocorticoids, that is reminiscent to the situation in vivo. Thus, the model is well suited for studies of hypophysiotrophic CRF-41 cell functions.

Regulation of vasopressin expression in cultured diencephalic neurons by glucocorticoids.

Glucocorticoids have long been recognized as playing a major role in the regulation of vasopressin synthesis. However, the factors determining cellular specificity and molecular mechanisms of glucocorticoid action on the vasopressin gene are not understood. In the present investigation, we used primary cell cultures derived from 14-day-old fetal rat diencephalon to investigate the regulation of vasopressin expression under controlled conditions. The experimental paradigm used ensured that only magnocellular, but not parvocellular neurons grew in the cultures. The following criteria were used to establish this phenotype. (1) Cultures were derived from fetal brain well before the time parvocellular neurons are generated, and neuronal precursors did not proliferate in vitro. (2) Vasopressinergic neurons measured some 18 x 25 microns, being conspicuously larger than the average neuronal population in vitro, and clearly larger than parvocellular neurons in vivo. (3) Neurons did not express corticotropin releasing factor in vitro. Selective neutralization of glucocorticoids contained in the serum-supplemented culture medium by the drug RU 38 486 resulted in an about 2-fold increase of numbers of vasopressinergic cells and about 4-fold increase in vasopressin mRNA, but did not affect numbers of oxytocinergic neurons or expression of general neuronal marker proteins. The effects of RU 38,486 were not dependent on synaptic communication between cultured cells, as the drug was still effective when cells were synaptically isolated by growth is in 14 mM Mg2+. RU was not mitogenic for vasopressinergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration of immuno-bound alkaline phosphatase by a direct lead method. Optimization of reaction parameters by microdensitometry using computer-aided image processing.

Antigens labelled by the immunohistochemical alkaline phosphatase anti-alkaline phosphatase (APAAP) method can be visualized by a lead capture technique. Optimal conditions of the reaction, evaluated by microdensitometry using computer-aided image processing, are described.

Immunological studies with a monoclonal antibody suggest a different conformation of neurophysin in parvicellular neurons of rat hypothalamus.

A monoclonal antibody (mAb L6) to a carcinoma surface antigen has previously been shown to recognize neurophysins (NP), proteins associated with oxytocin and vasopressin. L6-reactivity in rat hypothalamus was confined to magnocellular neuronal systems. No staining was detected in parvicellular suprachiasmatic or paraventricular systems. mAb L6 immunoprecipitated vasopressin-neurophysin only under reducing conditions, and detected it in Western blots only after gel-renaturation and electroblotting in basic buffer. These findings suggest L6-reactivity to NP is conformation-sensitive, and imply NP expression in a unique configurational form in hypothalamic parvicellular systems.

Direct immunoblotting from histological sections of brain onto nitrocellulose: evaluation with the protein neurophysin.

A method for the isolation and localization of proteins and peptides from histological sections of rat and human brain by immunoblotting is described. For validation, the well-characterized protein neurophysin was electrophoretically transferred from formaldehyde-fixed or fresh tissue sections onto a nitrocellulose membrane. Neurophysin on the nitrocellulose membrane was detected by a specific antibody reaction. The antibody against neurophysin was visualized either by using secondary antibodies, conjugated with peroxidase or by protein A gold, followed by enhancement with silver. With this simple and fast method, neurophysin (or other proteins and peptides) can be identified on nitrocellulose membranes in areas that correspond to anatomically defined regions. Since the procedure combines the advantages of precise regional localization of polypeptides with the specificity of antibody-antigen reactions, the method may prove useful for rapid screening of the distribution of peptides or proteins in (brain) tissue.

Identification of neurophysin immunoreactivity in hypothalamus by a monoclonal antibody to a carcinoma cell surface antigen.

Mouse monoclonal antibody (mAb) L6 identifies an antigen expressed on the cell surface of many different human carcinomas. While studying the binding activity of mAb L6 to intracerebral tumor xenografts of human lung carcinoma LX-1 cells in nude rats using immunohistological techniques, we observed that L6 can also bind to a cytoplasmic antigen expressed in the magnocellular component of the hypothalamo-neurohypophysial system. Double-labeling experiments with antisera to vasopressin and oxytocin confirmed the localization of L6 immunoreactivity within both peptide-containing cell groups. L6 immunoreactivity in Brattleboro rats (with genetic deletion in the vasopressin gene) was exclusively localized within oxytocin neurons. Oxytocin and vasopressin failed to block L6 staining which suggested that its target epitope resides within the neurophysin sequence, and this explanation was supported by the finding that adsorption of L6 with porcine neurophysin completely eliminated hypothalamic immunoreactivity. Western blot analysis of bovine neurophysin and human pituitary extracts identified L6-immunoreactive bands which corresponded to the position of neurophysin and pro-pressophysin, confirming that L6 immunoreactivity in hypothalamus is related to neurophysin. Thus, monoclonal antibody L6, which is highly reactive with a membrane antigen of human lung cancer cell line LX-1, recognizes a cytoplasmic epitope in hypothalamic neurons identified as neurophysin by immunohistochemistry and Western analysis.

[The influence of CCNU (lomustine) on the neurosecretion of hypothalamic nuclei and the neurohypophysis during early stages of extrauterine development of the rat]. / Wplyw CCNU (Lomustine) na neurosekrecje jader podwzgórza i nerwowej czesci przysadki szczura we wczesnym okresie rozwoju pozamacicznego.

The hypothalamo-neurohypophyseal neurosecretory system was investigated in 8-, 15- and 30-day-old rats subjected to three intragastric doses of CCNU - 12.5 mg/kg b. wt. each on the 3rd, 5th and 7th day after birth. Neurosecretory neurons in the hypothalamus were visualized in the paraffin sections by the immunoenzyme (PAP) technique using antibodies against neurophysin and by Gomori chrome-hematoxylin staining. Accumulation of neurophysin was observed in these cells after treatment with CCNU. Karyometric measurements showed an increase of the mean nuclear cross-section area in PVN neurons in 8-day-old rats exposed to CCNU. In four experimental rats disseminated intracerebral hemorrhagic foci were present. Plasma osmolality was far below the normal values on the 8th day, on the 15th day of life it shifted to hyperosmolality and returned to normal at the age of 30 days. Discussion of the results leads to the conclusion that the increase of the neurosecretory function observed in this experiment was secondary to vasogenic changes.

Localization of vasopressin mRNA-containing neurones in the hypothalamus of the monkey.

Neuronal perikarya containing vasopressin mRNA were detected in cryostat sections of cynomolgus monkey brains by using an in situ hybridization technique. The neurones were observed in hypothalamic regions (supraoptic nucleus, paraventricular nucleus, suprachiasmatic nucleus and accessory supraoptic nucleus). These findings are in agreement with previous reports using immunohistochemical methods.

Immunocytochemical study of the hypothalamic magnocellular neurosecretory nuclei of the snake Natrix maura and the turtle Mauremys caspica.

An immunocytochemical study of the magnocellular neurosecretory nuclei was performed in the snake Natrix maura and the turtle Mauremys caspica by use of antisera against: (1) a mixture of both bovine neurophysins, (2) bovine oxytocin-neurophysin, (3) arginine vasotocin, and (4) mesotocin. Arginine vasotocin- and mesotocin-immunoreactivities were localized in individual neurons of the supraoptic and paraventricular nuclei, with a distinct pattern of distribution in both species. The same cells appeared to be stained by the anti-oxytocin-neurophysin and antimesotocin sera. The supraoptic nucleus can be subdivided into rostral medial and caudal portions. In N. maura, but not in M. caspica, neurophysin-immunoreactive neurons were found in the retrochiasmatic nucleus. No immunoreactive elements were seen in the suprachiasmatic nucleus of both species after the use of any of the antisera. A dorsolateral aggregation of neurophysin-containing cells, localized over the lateral forebrain bundle, was present in both species. Magnocellular and parvocellular neurophysin-immunoreactive neurons were present in the paraventricular nucleus of both species. In the turtle, the paraventricular neurons were arranged into four distinct layers parallel to the ependyma; these neurons were bipolar with the major axis perpendicular to the ventricle, and many of them projected processes toward the cerebrospinal-fluid compartment. In N. maura a group of large neurons of the paraventricular nucleus was found in a very lateral position. The posterior lobe of the hypophysis and the external zone of the median eminence contained arginine vasotocin- and mesotocin-immunoreactive nerve fibers. The lamina termialis of both species was supplied with a dense bundle of fibers containing immunoreactive neurophysin. Neurophysin-immunoreactive fibers were also present in the septum, some telencephalic regions, including the cortex and the olfactory tubercule, in the paraventricular organ, and the periventricular and periaqueductal gray of the brainstem.

Slice cultures of rat hypothalamus examined by immunohistochemical staining for neurohypophyseal peptides and GFAP.

Organotypic cultures were prepared from slices of neonatal rat hypothalami and were immunohistochemically stained for the neurohypophyseal peptides vasopressin and oxytocin, their associated neurophysins, and for glial fibrillary acidic protein (GFAP). Both glial and neural elements survived and matured within the cultures, expressing cellular morphologies and retaining a topographic organization similar to that found in vivo. Neurones producing peptides were readily identified and such peptidergic neurones elaborated processes with an appearance characteristic of beaded axons. These presumptive axons grew in a selective and specific manner over certain regions in the slice cultures while avoiding other regions in a manner similar to that found in vivo. In cocultures of hypothalamus and neurointermediate lobe tissue, peptidergic axons found and grew over the neurointermediate lobe tissue and elaborated extensive terminal arborizations. Thus, it appears that at least some of the cues used for appropriate axonal guidance are maintained in these cultures. Organotypic cultures retain many in vivo characteristics as regards cellular morphology and cellular interactions, yet provide an in vitro environment useful for the study of morphology, physiology, cell biology and neurone-target interaction of hypothalamic neurones.

Topography of neurophysin-immunoreactive neurons projecting to the neurohypophysis: direct evidence as revealed by a double staining method.

The topographic distribution of neurophysin-immunoreactive (NP-IR) cells projecting to the posterior pituitary gland has been studied in the cat using a double staining method: immunohistochemistry of neurophysin in conjunction with horseradish peroxidase (HRP) retrograde tracer technique. We found that almost all the hypothalamic NP-IR cells project directly to the neurohypophysis except those localized in the suprachiasmatic nucleus and in the caudal part of the paraventricular nucleus.

Distribution of nicotinic binding sites with respect to CRF and neurophysin immunoreactive perikarya within the rat hypothalamus.

These studies determined the differential autoradiographic distribution of [125I]alpha-bungarotoxin versus [3H]nicotine relative to the histochemically defined perikarya for neurophysin and corticotropin releasing factor (CRF). Specific [3H]nicotine binding sites occurred in relatively greater density within the neuropil surrounding PVN and SON compared to within the nuclei. In contrast, the highest density of [125I]alpha-BTX sites codistributed with neurophysin immunoreactive perikarya within these nuclei.

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

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