Neurosecretory cells in the central nervous system of the giant garden slug,limax maximumus

The neurosecretory system of the giant garden slug Limax maximus was studied using the alcian blue/alcian yellow (AB/AY) staining technique for neurosecretion. Stainable cells could be identified in the paired cerebral, pleural, parietal, and buccal ganglia, and in the visceral ganglion. The cells occur as single cells or in groups of up to 100, with diameters ranging between 10 and 70 μm. Axon tracts could only be traced for a small number of cells; neurohemal areas were not conclusively identified. The morphological similarities of the neurosecretory system of L. maximus is compared with that of other investigated stylommatophoran slugs.     

Immunocytochemical localization of insulin-related peptide(s) in the central nervous system of the snailHelix aspersa müller: Involvement in growth control

1. The presence of insulin-like substances has been demonstrated by immunocytochemistry in the central nervous system of the snail Helix aspersa. 2. The immunopositivity has been observed especially in the large perikarya of the mesocerebral green cells [the cerebral green cells (CeGC) stained in green by the alcian blue:alcian yellow technique]. 3. The removal of either the mesocerebrum or the CeGC stops the growth of the snail and induces the increase of the glycogen content in the mantle edge. 4. Our results show the existence of insulin-like material in the neurosecretory cells. Previous data having demonstrated the presence of specific binding sites to insulin in the cephalic ganglia of Helix aspersa, one may suggest that insulin could play a neuromodulatory or a neurotransmittory role in the central nervous system and might control the growth.     

The structure and cytochemistry of the neurosecretory cells of Fasciola gigantica Cobbold and Fasciola hepatica L.

Histochemical studies of the nervous system of Fasciola gigantica and Fasciola hepatica were undertaken. Neurosecretory cells were detected by Gomori's aldehydefuchsin, Bargmann's chrome hematoxylin-phloxin, Mallory's triple stain, periodic acid-Schiff, Heidenhain's Azan and alcian blue after potassium permanganate oxidation. Two types of neurosecretory cells were recognized and designated as "A" and "B". Type "A" cells occurred in small numbers in the brain and subesophageal mass and type "B" cells ubiquitous in distribution. The reactions of these cells to the standard stains for neurosecretory substance generally, were less intense than the neurosecretory cells of other animals such as crustaceans and insects. The structure, organisation, distribution and cytochemistry of neurosecretory cells in Fasciola gigantica and Fasciola hepatica is discussed.     

Characterization of a new neurosecretory cell type containing gastrin-cholecystokinin-like peptide in the locust pars intercerebralis: ultrastructural and immunocytochemical studies,in situ and in vitro

A new neurosecretory cell type of the locust pars intercerebralis, immunolabelled with an antiserum against a vertebrate peptide related to gastrin-cholecystokinin (CCK-8(s)), was characterized both in situ and in primary cell cultures. Semithin sections of pars intercerebralis were first immunostained in order to identify neurosecretory cells containing CCK-like material and then examined by electron microscopy. The neurosecretory cells containing CCK-like material were paraldehyde fuchsin negative and were unequivocally identified in ultrathin sections adjacent to immunostained semithin sections. They exhibited neurosecretory vesicles of variable electron density, ranging in diameter from 150 to 250 nm. Immunogold labelled ultrathin sections adjacent to unlabelled ultrathin sections allowed for the unambiguous localization of CCK-like immunoreactive material over the neurosecretory vesicles of the cells containing CCK-like material. Immunoreactivity towards CCK-8(s)-like peptide could also be detected in pars intercerebralis neurosecretory neurons grown in vitro. The CCK-like positive neurons showed a multipolar morphology with fine processes radiating from the cell body. The positive cells had the same ultrastructural characteristics as the in situ CCK-like neurons. The pattern of neurite outgrowth on reactive CCK-like neurosecretory cells in vitro and the neuroanatomical pathway of the CCK-like immunoreactive neurosecretory cells in situ could be correlated. On the basis of their number, size and localization in the locust pars intercerebralis, it is possible that the CCK-like neurosecretory cells correspond to neurosecretory cell type C, which has not, to date, been identified at the ultrastructural level.     

Alcian blue-alcian yellow mapping of neurosecretory cells in the central nervous system of the salt marsh pulmonate snail Melampus bidentatus

1. The neurosecretory system of the primitive ellobiid Melampus bidentatus (Pulmonata: Basommatophora) was investigated using Alcian blue-Alcian yellow histochemistry. 2. Putative neurosecretory cells within the central ganglia were distinguished by the criteria of cell size, position, and staining reaction. 3. The cerebral ganglia, with attached lateral lobes, contained the greatest diversity of neurosecretory cell types (at least seven), including single cells and cell clusters ranging from two to 40 cells. 4. Four neurosecretory cell types were identified in the parietal and visceral ganglia, two in the pedal ganglia, and one each in the buccal and pleural ganglia. 5. Neurosecretory system homology among pulmonate gastropods is suggested by the close similarity of the Melampus AB/AY cell map to those reported in the literature for two limnic basommatophorans and for four terrestrial stylommatophorans.     

Firing activity of "diapause hormone" producing cells in the male silkmoth,Bombyx mori

Diapause hormone (DH) originally identified to be a factor originating from neurosecretory cells in the suboesophageal ganglion acts on developing ovaries to produce diapause eggs in a female silkmoth, Bombyx mori. A male silkmoth has homologous neurosecretory cells, but little is known of the physiological nature of the cells and actions of their products. We examined the long-term firing activity of putative DH-producing neurosecretory cells and hormonal activity of their products in male pupae that had been experienced different environmental regimens for diapause induction. Firing activity patterns of male labial cells strongly depended on diapause types of pupae: cells in a diapause-type male were active throughout the pupal period, whereas the same cells in a non-diapause-type male were usually inactive during the early two-thirds of the pupal period. A male pupa with electrically active labial cells could induce diapause eggs in a female pupa connected parabiotically to that male. The firing activity of male neurosecretory cells and hormonal action of their products are qualitatively the same as in the female previously examined. We suggest that there is no evident sexual dimorphism in the physiological and biochemical nature of neurosecretory cells producing DH and the amidated peptide DH has different functions in a male.     

Färberische Beobachtungen zur Frage nach dem primären Bildungsort von Neurosekret im supraoptico-hypophysären System

Zusammenfassung An Embryonen und Jungtieren von Maus und Huhn wurde die Entwicklung des neurosekretorischen hypothalamo-hypophysären Systems untersucht. Dabei gelang es, neurosekretorisches Material in den Perikaryen des Systems zum gleichen Zeitpunkt nachzuweisen, zu dem es in der Neurohypophyse erstmalig auftrat.In vergleichend-anatomischen Untersuchungen an erwachsenen Tieren ließ sich bei einer Reihe von Säugetierarten Neurosekret im Verlauf des gesamten Neurons sichtbar machen. Korrespondierende Neuronabschnitte verschiedener Spezies enthielten dabei vergleichbare Neurosekretmengen.Die Befunde wurden mit verbesserten Verfahren zur färberischen Darstellung von Neurosekret gewonnen. Die von ihnen abweichenden Ergebnisse früherer Untersuchungen beruhen auf unzulänglichen Methoden und können daher nicht gegen die Hypothese eines intraaxonalen Neurosekret-Transportes ins Feld geführt werden.

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Stainability of neurosecretory material and the beginning of its formation in the supraoptico-hypophysial system

Summary The development of the neurosecretory hypothalamo-hypophyseal system of mouse and chick embryos has been studied. Neurosecretory granules could be identified in the perikarya of the neurosecretory cells simultaneously with their first appearance in the neurohypophysis.Subsequently, a variety of adult mammalian species was investigated. In all cases, neurosecretory material could be demonstrated along the whole length of the neurons. Comparison of corresponding parts of the neurons showed them to contain comparable amounts of neurosecretory material.The investigations were carried out with improved techniques for staining neurosecretory material. Results of earlier investigations, inconsistent with these findings, were obtained with inadequate methods and cannot be regarded as contradicting the hypothesis of an intraaxonal transport of neurosecretory material.

     

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.     

Peptidergic neurons in the major ganglia of the opisthobranch mollusc Philine aperta (L.) mapped using the alcian blue/alcian yellow/phloxin histochemical technique

1. The central nervous system of the mollusc Philine aperta (Gastropoda; Cephalaspidea) was studied using the alcian blue/alcian yellow/phloxin histochemical technique to identify putative peptidergic neurons.2. The position, size and staining colour of positively stained somata were mapped by reconstruction from serial sections of the major ganglia.3. Positively stained neurons were found in all of the ganglia studied. Based on staining colour, three cell types were identified in the cerebral ganglia, four in the pedal and supraoesophageal, two in each of the pleural, suboesophageal and visceral ganglia and one in the genital ganglion.4. Some comparisons can be made between the results of this study and other maps of peptidergic neurons derived from alcian blue/alcian yellow histochemical studies of gastropod nervous systems.     

Immunohistochemical localization of LH-producing cells in the adenohypophysis of the Japanese quail,Coturnix coturnix japonica

Summary The cells that produce luteinizing hormone (LH) in the adenohypophysis of the Japanese quail were identified immunohistochemically using anti-chicken LH serum and horseradish peroxidase-labeled goat anti-rabbit gamma globulin serum. The LH cells are localized in the caudal lobe of the pars distalis. They are elongate in shape and are polarized toward the sinusoids, especially in their active states. Alterations in size of LH cells are directly related to changes in circulating LH levels as induced by castration or photostimulation. The LH cells identified immunohistochemically were only stained by alcian blue with periodic acid-Schiff (PAS), alcian blue and orange G.PAS-positive gonadotropic cells in the cephalic lobe were stained immunohistochemically only slightly if at all using anti-chicken LH serum and consequently may be FSH producing cells. In the cephalic lobe another type of basophilic cell was stained with alcian blue. These cells were also stained immunohistochemically with anti-chicken LH serum. These cells may possibly be identified as TSH cells due to the characteristics of the antichicken LH serum used in this study which cross react with LH and TSH but only slightly with FSH, and also on the basis of previous light and electron microscopic studies.We express our gratitude to Professors Hideshi Kobayashi and Katsumi Wakabayashi for their valuable guidance during the experiment. We also express our cordial thanks to Professor B.K. Follett for the gift of anti-chicken LH serum and standard LH. This work was supported in part by Grants from Ministry of Education of Japan and from the Ford Foundation.     

Receptor-Mediated Calcium Influx in Chlamydomonas reinhardtii

ABSTRACT. Alcian blue acts as a secretagogue and chemorepellent in a variety of unicellular eukaryotes. We report that alcian blue stimulates flagellar excision and induction of RNA encoding flagellar proteins in Chlamydomonas reinhardtii . Flagellar excision by alcian blue is dependent on extracellular Ca²⁺ and is blocked by La³⁺, ruthenium red, and neomycin, and so is similar to flagellar excision by acid shock. However, the adf-l mutant excises its flagella following alcian blue treatment, but not following acid shock, thus genetically distinguishing alcian-blue-induced excision from acid-shock-induced excision. Wild-type, but not adf-1, cells regrow their flagella in the continued presence of alcian blue. Wild-type cells that regrow flagella in the presence of alcian blue fail to excise their flagella in response to either increased concentrations of alcian blue or to acid shock. Alcian blue treatment of cells also induces RNA encoding flagellar components, but in a manner distinct from other means of stimulation. These results suggest that treating Chlamydomonas with the secretagogue alcian blue initiates a Ca²⁺ influx pathway and that prolonged treatment with alcian blue desensitizes the acid-shock-activated Ca²⁺ influx pathway to acid treatment. Alcian blue will thus be a useful excitatory ligand in future studies of receptor-mediated Ca²⁺ signaling in the Chlamydomonas flagellar regeneration system.     

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.     

Fine structure of the median eminence and the pars nervosa of the bullfrog,Rana catesbeiana

Summary The hypothalamic neurosecretory system of the bullfrog, Rana catesbeiana, was studied with light- and electron microscopy. The median eminence is roughly divided into two portions. The upper portion mostly consists of ependymal cells, glial cells and preoptico-hypophysial nerve tract, whereas in the lower portion, neurosecretory axons, glial cells, processes of glial and ependymal cells, and fine blood vessels of the hypothalamic portal vein are located. A part of the neurosecretory axons of the preoptico-hypophysial tract proceeds to the lower portion of the median eminence. These axons are arranged perpendicularly to the capillaries of the hypothalamic portal vein. The glial cells are densely located in the area of the median eminence where neurosecretory material is abundant. The neurosecretory material in the neurosecretory cells, their axons, the median eminence and the pars nervosa of the bullfrog shows a positive reaction to PAS treatment.The neurohemal area of the median eminence is occupied by many neurosecretory and non-neurosecretory axons, containing neurosecretory granules and/or synaptic vesicles. The axonal portions with the synaptic vesicles which are considered to be the nerve endings abut on the capillaries of the portal system. The size of synaptic vesicles in the axon terminals containing few neurosecretory granules is larger than those in the endings with many neurosecretory granules. Infrequently glial and ependymal processes are interposed between the nerve endings and the capillary wall.In the hilar region of the infundibulum, synapses are frequently observed between the thin fibers with or without neurosecretory granules and dendrites of non-neurosecretory neurons. The probable functions of these synapses are briefly discussed on the basis of our findings. Both in the hilar region of the infundibulum and in the pars nervosa, electron-dense neurosecretory granules of two different sizes were observed. The median eminence contains only one type of granules.The fine structure of the pars nervosa shows similar structures to those of the median eminence. Both in the median eminence and the pars nervosa, the fenestrated endothelium of the capillaries was frequently observed. The thick perivascular connective tissue space containing fibroblasts and collagen fibrils was observed both in the median eminence and the pars nervosa. Vesicles in the cytoplasm of the endothelial cells which appear to take a part in the transendothelial transport were observed.This investigation was supported in part by United States Public Health Service Research Grant, No. A-3678, to Hideshi Kobayashi from the National Institute of Arthritis and Metabolic Diseases and partly by a grant for Fundamental Scientific Research from the Ministry of Education of Japan. The authors wish to express their thanks to Prof. K. Takewaki for his kind encouragement.     

Histochemical observations on the giant neurosecretory cells of the thoracic ganglion of the adult and juvenile crabs, Potamon magnum magnum (Pretzman).

The giant neurosecretory cells in the thoracic ganglion of the adult and juvenile crab, Potamon magnum magnum (Pretzman) were histochemically investigated. The secretion is mainly proteinaceous in nature, containing considerable amounts of acid mucosubstances, sulphate esters, lipids and a little carbohydrate but no glycogen. The detailed nature of proteinaceous neurosecretory material in the adult crab was further tested. It appears that the neurosecretory material of these cells contains moderate amounts of sulfhydryl groups and few of disulphide bonds. No trace of tyrosine could be observed. The neurosecretory granules were associated with considerable amounts of cytoplasmic RNA. In general, stronger reactions were obtained in summer and winter than in other seasons.     

The neurosecretory system of the adult Calliphora erythrocephala

Summary The histology of the corpus cardiacum (c. card.) and the hypocerebral ganglion of Calliphora has been described from sections mainly stained with paraldehyde-fuchsin (PAF) and counterstained with Halmi's mixture. Concurrently the nervous connections of these organs with the neurosecretory system and the stomatogastric nervous system were studied.Neurosecretory material from the medial neurosecretory cells of the brain (m.n.c.) could be traced through the cardiac-recurrent nerve, and passing through the c. card. it was seen to be abundantly present in the wall of the aorta and the two pairs of nerves leaving the c. card.-hypocerebral ganglion complex posteriorly, i.e. the aortic and the oesophageal nerves. However, in some old, fed flies a considerable amount of neurosecretory material was also observed in anastomosing branches of the cardiac-recurrent nerve inside the c. card. Thus storage of neurosecretory material originating in the m.n.c. may take place both in the aorta wall and in the c. card. This observation is relevant to the interpretation of previous experiments of E. Thomsen (1952).The c. card. cells proper (the c.n.c.) were not stained by the PAF, although they are known to be neurosecretory.This work was supported by grants from the Carlsberg Foundation. I am grateful to Professor C. Overgaard Nielsen for laboratory facilities.     

Distribution of tachykinin-related neuropeptide in the developing central nervous system of the moth Spodoptera litura

Neuropeptides with similarities to vertebrate tachykinins, designated tachykinin-related peptides (TRPs), have been identified in several insect species. In this investigation we have utilized an antiserum raised to one of the locust TRPs, locustatachykinin-I (LomTK-I), to determine the distribution pattern of LomTK-like immunoreactive (LTKLI) neurons in the developing nervous system of the moth Spodoptera litura. A number of LTKLI neurons could be followed from the larval to the adult nervous system: a set of median neurosecretory cells (MNCs) in the brain, a pair of brain descending neurons and a few sets on neurons in the ventral nerve cord. The distribution of LTKLI neurons in the adult brain is very similar to that seen in other insect species with prominent arborizations in the central body, antennal lobes, mushroom body calyces, optic lobe neuropils and other distinct neuropil areas in the protocerebrum and tritocerebrum. A new finding is the presence of LTKLI neurosecretory cells with axon terminals in the anterior aorta and corpora cardiaca, suggesting for the first time a neurohormonal role of tachykinin-related peptide(s) in insects. During postembryonic development the number of LTKLI neurons in the ventral nerve cord decreases somewhat, whereas the number increases in the brain. Thus the functional roles of TRPs may change to some extent during development.     

Retinal illumination produces synaptic inhibition of a neurosecretory organ in the crayfish,pacifastacus leniusculus (Dana)

We have identified a cluster of neurosecretory cells in the crayfish eyestalk that possess dendrites in the second optic neuropil (Medulla) and project axons to the first optic neuropil (Lamina). Illumination of the ipsilateral retina produces a synaptic inhibition of these cells that is mimicked by iontophoresis of gamma-aminobutyric acid within the medullary neuropil. The neurosecretory nature of the cells, the efferent projection of their axons, and the strong inhibition of their spiking activity upon retinal illumination suggest that they may be involved in the feedback control of dark adaptation and/or circadian changes in visual sensitivity.     

Cells containing IgE in the intestinal mucosa of mice infected with the nematode parasite Trichinella spiralis are predominantly of a mast cell lineage

To determine whether IgE+ cells in the intestinal mucosa of nematode-infected mice were of a mast cell or a lymphocyte lineage, the intestinal mucosae of mast cell-deficient w/wv mice were examined for IgE+ cells after inoculation with Trichinella spiralis muscle-stage larvae. Immunofluorescence staining techniques were used to detect IgE associated with cells in the intestinal mucosa. Comparisons were made among four strains of mice, w/wv (mast cell-deficient), +/+ (normal congenic littermates of w/wv), BALB/c, and SJL, that were either uninfected controls or inoculated with T. spiralis. Tissue sections from the small intestine of T. spiralis-infected BALB/c, SJL, and +/+ mice were fixed in ethanol and were stained with an affinity-purified F(ab')2 rabbit anti-mouse IgE followed by FITC goat anti-rabbit IgG. Large numbers of cells in the intestinal mucosa exhibited bright fluorescence. When other sections of intestines from these mice were processed in Carnoy's fixative and were stained with alcian blue at low pH (a metachromatic stain for mast cells) or alcian blue followed by immunofluorescence staining for IgE, large numbers of mast cells were observed in the intestinal mucosa, and 70 to 90% stained positively for IgE. There was a considerable number of cells in the intestinal mucosa which were IgE+ but which did not stain with alcian blue. Few alcian blue-positive cells and no IgE+ staining cells were present in the intestinal mucosa of control, uninfected +/+, BALB/c, and SJL mice. To determine whether these IgE+ alcian blue-negative cells were of a lymphocyte or a mast cell lineage, the mast cell-deficient w/wv mouse strain was examined after infection with T. spiralis. In contrast to BALB/c, SJL, or +/+ mice, few cells in the intestinal mucosa of T. spiralis-infected w/wv mice stained with alcian blue or were positive for IgE. However, when the IgE response in the MLN of the w/wv mice was compared to the IgE response of BALB/c, SJL, and +/+ mice, numerous IgE+ cells, but no alcian blue-positive cells, were observed in the parenchyma of the MLN from all four strains of T. spiralis-infected mice. In addition, flow microfluorometric analysis of MLN cells stained for surface IgE in suspension showed a comparable proportion of IgE-bearing cells, which were mostly B lymphocytes, among all four strains of T. spiralis-infected mice.(ABSTRACT TRUNCATED AT 400 WORDS)