Immuno-cytochemical demonstration,in the external region of the amphibian median eminence,of separate vasotocinergic and mesotocinergic nerve fibres

Summary By means of single and double immuno-enzyme cytochemical staining techniques, it was shown that the external region of the amphibian median eminence contains separate vasotocinergic and mesotocinergic nerve fibres. Moreover, it was demonstrated that the vasotocinergic fibres also contain neurophysin. In animals in which the hypothalamic magnocellular neurosecretory preoptic nuclei had been completely removed, the immuno-reactive vasotocinergic and mesotocinergic fibres of the median eminence had disappeared. From this result, it is concluded that, at least the great majority of the vasotocinergic and mesotocinergic fibres of the external region of the amphibian median eminence are processes of neurosecretory perikarya located in the hypothalamic magnocellular preoptic nuclei. On the other hand, our results do not exclude the possibility that a minority of these neurosecretory fibres originate from small immuno-reactive perikarya which were found in the tuber cinereum. The observation that both kinds of processes accumulate around blood capillaries of the hypophysial portal system strongly suggests that they play a role in the control of the activity of the pars distalis of the hypophysis.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Immuno-enzyme cytochemical demonstration of mesotocinergic nerve fibres in the pars intermedia of the amphibian hypophysis

Summary Immuno-enzyme cytochemical investigations showed that the whole amphibian pars intermedia of the hypophysis is innervated by an intercellular network of peptidergic varicose nerve fibres which contain mesotocin or (and) parts of the mesotocin molecule. The pars intermedia does not contain vasotocinergic fibres. The mesotocinergic fibres are branches of axons leaving the pituitary stalk and the neural lobe. In animals of which the hypothalamic magnocellular neurosecretory preoptic nuclei had been completely removed, the immuno-reactive mesotocinergic fibres of the pars intermedia had totally disappeared. From this result, it is concluded that the mesotocinergic fibres of the pars intermedia of the amphibian hypophysis are axons of neurosecretory perikarya located in the hypothalamic magnocellular neurosecretory preoptic nuclei.Dedicated to Professor Berta Scharrer on the accasion of her 70th birthdayThis investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

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     

Immunocytochemical localization of vasotocin and mesotocin in the hypothalamus of lacertilian reptiles

Summary The hypothalamic magnocellular neurosecretory system of lizards was studied with the unlabeled antibody peroxidase-antiperoxidase complex (PAP) technique at the light microscopic level. It was shown that vasotocin and mesotocin are synthesized in separate neurons. The vasotocinergic as well as the mesotocinergic perikarya are of different sizes. Both cell types occur in close juxtaposition, but without a distinct pattern of distribution. The external zone of the lacertilian median eminence contains numerous immunoreactive vasotocinergic fibers and only few immunoreactive mesotocinergic fibers. The general organization of the hypothalamic magnocellular neurosecretory system of lizards, as revealed by immunocytochemistry, is essentially similar to that revealed with unspecific staining methods.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Immunocytochemical investigation of the hypothalamo-neurohypophysial system in birds

Summary The results of an immunohistochemical investigation of the hypothalamo-neurohypophysial system in several species of birds have shown that: (1) mesotocin and vasotocin are synthesized in separate neurons; (2) in all species investigated the distribution of mesotocinergic and vasotocinergic perikarya follows a common pattern; (3) the external zone of the avian anterior median eminence contains exclusively vasotocinergic nerve fibers, originating in supraoptic and ventral paraventricular regions; (4) the distribution of immunoreactive elements in the neural lobe shows a definite species-dependent pattern.     

Electron microscopic immunocytochemical demonstration of separate vasotocinergic and mesotocinergic nerve fibres in the median eminence of the frog hypophysis

Summary With the use of the unlabelled antibody peroxidase-antiperoxidase complex (PAP) technique at the electron microscopic level, it was shown that both the internal and the external regions of the median eminence of the frog contain separate vasotocinergic and mesotocinergic nerve fibres. This observation confirms the results of previous immunocytochemical studies at the light microscopic level. The mean size of the neurohypophysial hormone-containing granules in the external region of the median eminence is significantly smaller than that of the neurohypophysial hormone-containing granules in the internal region of the median eminence. No significant difference could be found between the mean granule size of the vasotocinergic and mesotocinergic fibres of either the internal or the external region of the frog median eminence.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Mesotocin and vasotocin in the brain of the lizard Gekko gecko

Summary The distribution of mesotocin and vasotocin was studied in the brain of the lizard Gekko gecko with antisera specific for either peptide. Both mesotocinergic and vasotocinergic perikarya are found in the paraventricular and supraoptic nuclei of the hypothalamus, whereas vasotocinergic neurons are exclusively present in the bed nucleus of the stria terminalis and in a cell group of the rhombencephalon. The distributional pattern of the mesotocinergic fibers corresponds closely to that of the vasotocinergic fibers. However, throughout the entire brain the mesotocinergic innervation is less dense than the vasotocinergic innervation. No sex differences are present in the mesotocinergic fiber system.Abbreviations acc nucleus accumbens - bst bed nucleus of the stria terminalis - bv blood vessel - dB diagonal band of Broca - dc dorsal cortex - dth dorsolateral thalamic nucleus - lc lateral cortex - me median eminence - oc optic chiasma - ot optic tract - pag periaqueductal grey - pvn paraventricular nucleus - rc rhombencephalic cell group - sep septum - son supraoptic nucleus - tect mesencephalic tectum - vth ventrolateral thalamus     

Electron microscopic immunocytochemical demonstration of separate vasotocinergic and mesotocinergic nerve fibres in the neural lobe of the amphibian hypophysis

Summary Using the unlabeled antibody peroxidase-antiperoxidase (PAP) technique at the electron microscopic level, it was demonstrated that the hormones of the posterior pituitary of Rana temporaria are located in separate vasotocinergic and mesotocinergic nerve fibres. This observation confirms the results of our previous immunocytochemical studies at the light microscopic level.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Development of blood-cerebrospinal fluid barrier to horseradish peroxidase in the avian choroidal epithelium

Summary Four neurons in the brain of the migratory locust were immunohistologically identified with an anti-met-enkephalin antiserum. The perikarya of two of these cells are located in the center of each of the two groups of lateral protocerebral neurosecretory cells. The fibres coming from these perikarya terminate in numerous immunoreactive ramifications visible at the periphery of both tractus I to the corpora cardiaca, through which pass the neurosecretory products of the pars intercerebralis. The other two cell bodies are located at the bases of the two optic lobes; their fibres enter the posterior part of the protocerebrum and ramify around the root of the nervus corporis cardiaci II, another area through which neurosecretory products pass. The topographic distribution of these met-enkephalin arborizations suggests that these four neurons may act as neuromodulators of the acitivity of the major neurosecretory cells in the brain of this insect.     

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.     

Ultrastructural characterization of neurosecretory fibres immunoreactive for vasotocin,isotocin, somatostatin,LHRH and CRF in the pituitary of a teleost fish,Poecilia latipinna

Summary Using the electron-microscopic immunogold method, vasotocin, isotocin, somatostatin (SRIF), gonadotrophin-releasing hormone (LHRH) and corticotrophin-releasing factor (CRF)-like immunoreactivities were localized in separate neurosecretory fibres in the pituitary of a teleost fish Poecilia latipinna. Antigenicities were preserved in sections of conventionally fixed tissue, except in the case of LHRH and CRF-like substances which were sensitive to osmium postfixation. Under the same fixation conditions, ultrastructural differences were observed between the 5 fibre types, and morphometric analysis of their granule sizes revealed significant differences in mean diameter except between vasotocin and isotocin fibres.Terminal-like regions of each type were identified on blood vessels, glial cells or other fibres in the neurohypophysis, on the basement lamina of the adenohypophysis, or directly on adenohypophysial endocrine cells. The fibres containing the two neurohypophysial hormones, originating from separate preoptic perikarya, were intermingled with, and may form endings near all the adenohypophysial cell types except those secreting prolactin. Although both types had similar mean granule diameters, the granules in the vasotocin fibres (mean 135 nm) were markedly less electron dense than those in the isotocin fibres (mean 140 nm). SRIF-immunoreactive fibres (mean 101 nm) appeared to form synapse-like endings on the somatotrophs, and a few thyrotrophs in the proximal pars distalis, and near the pars intermedia cells. An LHRH-positive type (mean 103 nm) contacted only the gonadotrophs of the proximal pars distalis. The rarer CRF-like fibres (mean 116 nm) appeared to project mainly towards the pars intermedia, but a few appeared to terminate rostrally near the adrenocorticotrophic cells.The significance of these observations is discussed in relation to the direct neurosecretory control of adenohypophysial function in teleosts.     

Axonal projections within the brain-retrocerebral complex of the cricket,Teleogryllus commodus

Summary The cerebral origins and axonal trajectories of neurons projecting to the retrocerebral complex of the cricket, Teleogryllus commodus, were examined in silver-intensified nickel preparations. Spatially separate groups of somata in the pars intercerebralis (PI) and in the pars lateralis (PL), commonly accepted as neurosecretory loci, were found to give rise to axons which terminate in the nervus corporis allati 2, the corpus allatum, or the corpus cardiacum. Additional findings demonstrated a distinct group of somata from the PI whose axons run in the esophageal nerve (stomatogastric nervous system), nine somata in the subesophageal ganglion with axons projecting into the nervus corporis allati 2, and also a small cluster of tritocerebral perikarya with axons terminating in the corpus cardiacum. Somata residing in the PI and PL were found to be compartmentally organized based upon the retrocerebral destinations of their axons. Possible functional consequences of these results with respect to the insect neurosecretory system are discussed.     

The reticular formation of lampreys (Petromyzonidae) — A target area for exohypothalamic vasotocinergic fibres

Summary The octapeptide vasotocin, which is formed in the classical neurosecretory nuclei of lampreys (Petromyzonidae), is transported, bound to the carrier protein neurophysin, not only to the neurohypophysis but also to various other regions of the brain via exohypothalamic fibres. A target area of this exohypothalamic vasotocinergic system is, in the brook lamprey (Lampetra planeri Bloch), a relatively well circumscribed area in the isthmus region of the rhombencephalic tegmentum motoricum, which is called area lateralis tegmenti. In this area, which belongs to the reticular formation, the vasotocinergic fibres form synaptic contacts with nerve cell perikarya and processes. The vesicles contained in the fibres were identified, ultrahistochemically, as neurophysin vesicles. They correspond to the neurophysin vesicles observed in the neurohypophysis of the same species. The functional significance of the vasotocinergic supply to portions of the reticular formation in lampreys is open to discussion.Supported by grants from the Ministry for Science and Technology of the German Democratic Republic. Acknowledgements: For skilled technical assistance the authors are indebted to their colleagues Mrs. S. Mehnert, E. Siebert, C. Schneider, and I. Seifert.     

Immunocytochemical demonstration of peptidergic neurons in the central and peripheral nervous systems of the flatworm Microstomum lineare with antiserum to FMRF-amide

Summary The central nervous system (CNS) and the peripheral nervous system (PNS) of the flatworm Microstomum lineare were studied by means of the peroxidase-antiperoxidase (PAP) immunocytochemical method, with the use of antisera to the molluscan cardioactive peptide FMRF-amide. FMRF-amide immunoreactive perikarya and nerve fibres are observed in the CNS and the PNS. In the CNS, immunoreactive perikarya and nerve fibres occur in the brain, in the epithelial lining and the mesenchymal surroundings of the ciliated pits, and positive fibres in the longitudinal nerve cords. In the PNS, immunoreactive fibre bundles with variocosities occur in the pharyngeal nerve ring, in symmetrical groups of perikarya on each side of the pharynx, and in the mouth area. Positive perikarya and meandering nerve fibres appear in the intestinal wall. A few immunoreactive cells and short nerve processes are observed at the male copulatory organ and on both sides of the vagina. Some immunoreactive peptidergic cells do not correspond to cells previously identified by histological techniques for neurosecretory cells. The distribution of immunoreactivity suggests that the FMRF-amide-like substance in CNS and PNS in this worm has roles similar to those of the brain-gut peptides in vertebrates. The status of FMRF-amide-like peptides as representatives of an evolutionarily old family of peptides is confirmed by the positive immunoreaction to anti-FMRF-amide in this primitive microturbellarian.     

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.     

Monoamine oxidase in the hypothalamo-hypophysial region of the teleosts,Anguilla japonica and Oryzias latipes

Summary Distribution of monoamine oxidase (MAO) was histochemically examined in the hypothalamo-hypophysial region of the eel (Anguilla japonica) and the medaka (Oryzias latipes) with a modified Glenner's tryptamine-tetrazolium method. The hypothalamic neurosecretory cells showed very weak MAO activity in their perikarya. MAO-positive fibers were present in close contact with the neurosecretory cells, suggesting that monoaminergic fibers participate in the control of neurosecretory cell activity. The nucleus lateralis tuberis (NLT) contained cells exhibiting strong MAO activity. These cells must be monoaminergic neurons.In the anterior region of the neurohypophysis of both eel and medaka, two bundles of MAO-positive fibers originating from the NLT proceed down along each side of the third ventricle into the pars distalis. This suggests that monoaminergic neurons of the NLT are involved in the release of hormones from the pars distalis. In addition to these tracts, numerous MAO-positive fibers proceed backward from the post-optic area and end around the blood capillaries located between the neurohypophysis and the pars intermedia in both species.I wish to express my gratitude to Prof. H. Kobayashi for his valuable advice during the course of this study. I am indebted to Prof. S. Uchida, Ocean Research Institute, University of Tokyo, for supplying the eels.     

Early immunohistochemical detection of somatostatin-like and methionine-enkephablin-like neuropeptides in the brain of the migratory locust embryo

The two groups of neurosecretory cells producing neuropeptides related to somatostatin (SRIF) and methionine-enkephalin (met-enkephalin), previously high-lighted in the brain of adult migratory locusts, were detected by immunofluorescent techniques during the embryonic development of these insects. The earliest detection of these neurosecretory products occurred firstly in the terminal arborizations, then in the fibres, and finally in the perikarya. SRIF-like material is present in the corpora cardiaca already four days before hatching, i.e. at two-thirds of embryonic life, whereas immunoreactivity can be detected only after hatching in the perikarya located in the pars intercerebralis. The synthesis of met-enkephalin-like neuropeptide starts in the four cells of this system at least two days before hatching as shown by the immunofluorescence in the terminal arborizations along the tractus I to the corpora cardiaca. SRIF-like and met-enkephalin-like neurosecretory products are synthesized and carried to their release areas whilst the formation of brain structures and of the corpora cardiaca has not yet been completed.     

Localization of urotensin I- and corticotropin-releasing factor-like immunoreactivity in the central nervous system of Catostomus commersoni

The distribution of urotensin I (UI) and corticotropin-releasing factor (CRF) immunoreactive (IR) structures was studied in the central nervous system (CNS) of the white sucker using the peroxidase-antiperoxidase immunocytochemical procedure. The close sequence homology between both peptides resulted in a high degree of crossreactivity. This was resolved by saturating the antisera solutions with heterologous antigens and specificity tests were done by adding excess of homologous peptides. UI immunoreactivity was seen in all of the identifiable caudal spinal cord neurosecretory cells, in their processes projecting to the urophysis, in thin beaded fibres coursing along the spinal cord, in brain stem, hypothalamus, proximal pars distalis and, especially, in the telencephalon. Some IR-UI specific and IR-CRF specific parvocellular neurons were also identified in the caudo-ventral tuberal region and ventral telencephalon. The IR-CRF was mainly present in parvocellular and magnocellular perikarya of the nucleus preopticus and in the preoptic-neurohypophysial pathway. Dense networks of IR-CRF reacting beaded fibres were also located in the lateral and posterior recessus nuclei. In the pituitary, IR-CRF fibre bundles were seen mainly in the neurointermediate lobe and in the rostral pars distalis. The cells of origin of the extraurophyseal system of IR-UI fibres in the sucker CNS have not been identified. The distribution of CRF immunostaining correlates well with the documented knowledge of CNS structures involved in the control of ACTH secretion in the goldfish. The probability of the occurrence of two UI-CRF related molecules, or of two different forms resulting from a common precursor molecule, forming two separate neuronal systems in the sucker CNS seems likely.     

Identification of adenohypophysiotropic neurohormone producing neurosecretory cells in Rana temporaria

Summary By means of electron microscopy, in the median eminence of Rana temporaria, the terminal arborizations of axons of six different types of neurosecretory cells, located in the pars ventralis of the tuber cinereum, were identified. In addition, phenomena connected with the release of neurosecretory material from the axon terminals of these neurosecretory cells into the blood capillaries of the median eminence are described.Preliminary results suggested the existence, in the median eminence, of additional different neurosecretory axon types which could also belong to corresponding neurosecretory cell types probably located in the apical part of the pars ventralis of the tuber cinereum. Moreover, in the external region of the normal median eminence, separate monoaminergic nerve fibres were tentatively identified. Arguments are adduced which plead (1) against the assumption that the ependyma or the pituicytes of the median eminence could produce adenohypophysiotropic hormones; (2) against the inference that the ependymal cells of the median eminence might be involved in the transport of adenohypophysiotropic hormones from the cerebrospinal fluid into the blood capillaries of the median eminence.     

The hypothalamic magnocellular system in the domestic fowl

Summary In the rostral hypothalamus of the domestic fowl, the magnocellular neurosecretory nuclei show a peculiar differentiation. Golgi studies of the supraoptic and paraventricular nuclei of the fowl reveal at least two major cell types: 1) large multipolar neurons, and 2) small interneurons. Golgi impregnations provide a detailed cytoarchitectural picture of the large-sized cells; the latter may well correspond to the neurosecretory cells demonstrated in the same regions by selective staining, and immunocytochemical and electron microscopical techniques.Electron microscopically, neuronal perikarya are observed to contain variable amounts of neurosecretory granules (100–200 nm in diameter; mean diameter of 160 nm) scattered throughout the cytoplasm. The diameters of these granules do not differ statistically in the two principal nuclear areas examined. The perikarya of these neurons display only a few axosomatic synapses containing electron-lucent and dense-cored vesicles (70–90 nm in diameter). Numerous nerve terminals of this type also end on the dendritic ramifications in the surrounding neuropil.