Neurosecretory Stains Research Articles

The neurosecretory staining in the pleural-pedal ganglion of the Japanese abalone (Ezoawabi), Haliotis discus hannai, and its relationship to reproduction; with a description of a newly observed neurohemal organ

Neurosecretory staining in the pleural-pedal ganglion of the Japanese abalone (ezoawabi), Haliotis discus hannai, was studied during the reproductive cycle. The variation in stain affinity of the cells and amount of neurosecretory material contained in the neurosecretory cells were measured during the study. The pleural-pedal ganglion contained 7 different cell types. The quantity of different cell types in the pleural-pedal ganglion was unusual for a prosobranch gastropod. Cell Types, α- and β-cells, showed neurosecretory staining and the stain intensity varied during the reproductive cycle. α-cells were the most abundant cells in the pleural-pedal ganglion and the quantity of neurosecretory material inside the cell body clearly correlated with gametogenesis. Neurosecretory material in β-cells showed a strong correlation with the induction of spawning. A neurohemal organ (pleural organ) was found in close proximity to the β-cells in the statocyst tissue. The pleural organ is the first ectodermal neurohemal organ reported in a prosobranch gastropod. A nervous connection (stato-pleural nerve) with a possible sensory function was found between the statocyst and the pleural-pedal ganglion.

Most lepidopteran neuroparsin genes seem functional, but in some domesticated silkworm strains it has a fatal mutation

The primary sequence of the Arthropod neurohormone neuroparsin is so variable that so far no orthologs from moths and butterflies have been characterized, even though classical neurosecretory stains identify cells that are homologous to those producing this hormone in other insect species. Here Lepidopteran cDNAs showing limited sequence similarity to other insect neuroparsins are described. That these cDNAs do indeed code for authentic neuroparsins was confirmed by in situ hybridization in the wax moth, Galleria mellonella, which labeled the neuroparsin neuroendocrine cells. Although in virtually all genome assemblies from Lepidoptera a neuroparsin gene could be identified, the genome assembly from the silkworm, Bombyx mori, has a neuroparsin gene containing a 16 nucleotide deletion that renders this gene nonfunctional. Although only a small number of all silkworm strains carry this deletion, it suggests that the domestication of the silkworm has rendered the function of this neurohormone dispensable.

The Neurosecretory Staining in the Cerebral Ganglia of the Japanese Abalone (ezoawabi), Haliotis discus hannai, and Its Relationship to Reproduction

Neurosecretory staining in the cerebral ganglia of the Japanese abalone (ezoawabi), Haliotis discus hannai, was studied during the reproductive cycle. The variation in stain affinity of the cells and the amount of neurosecretory material contained in the cells were measured during the study. Four cell types were found in the cerebral ganglion. Neurosectory activity was found in Cell Types A and B, and the amount of neurosecretory material in the cells and the stain intensity of cellular material varied with the reproductive cycle. Cell Types C and D were not neurosecretory. The stain intensity of Cell Type A showed a correlation with vitellogenesis in the ovary. The stain intensity of Cell Type A in males showed no apparent correlation with gonad development or maturation. Cell Type B in both sexes did not show a correlation with gametogenesis, vitellogenesis, or spawning.

Characterization of central neurons in bivalves using antibodies raised against neuropeptides involved in gastropod egg-laying behavior

Summary Immunohistochemistry was used to characterize central neurons which react to antibodies raised against neuropeptides known to control reproduction in other molluscs. Antibodies raised against αCDCP (α caudodorsal cell peptide) and CDCH (caudodorsal cell hormone), peptides which control ovulation in the pulmonate gastropod Lymnaea, labelled numerous neurons in Mytilus, Mya and Placopecten. The labelled neurons in the cerebral and visceral ganglia of Mytilus are consistent with descriptions of neurons implicated in the control of reproduction on the bases of earlier neurosecretory staining procedures. This study thus suggests that related peptides might be involved in the reproduction of gastropods and bivalve molluscs. The use of selective immunological markers for peptides which might control bivalve reproduction not only permits the tentative identification of neurosecretory cells in bivalves but also suggests a promising avenue for future research aimed at isolating and characterizing neuropeptides involved in the control of reproduction in these molluscs.

Immunohistochemical localization of some vertebrate-like neuropeptides (SP, NPY, CGRP, CCK) in the central nervous system of the freshwater snail Planorbarius corneus

The localization of the vertebrate-like neuropeptides substance P (SP), neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), and cholecystokinin (CCK8) in the central nervous system of the freshwater snail Planorbarius corneus has been studied using specific antisera and single and double immunohistochemistry. A widespread but precise distribution of immunore-activity (IR) in neurons and fibers of almost all the ganglia is observed for each antiserum. A comparison of the IR with classical neurosecretory staining (AB/AY) shows a partial overlap only for CGRP and CCK8. Whereas CGRP-IR is found in some Yellow Cells in the left parietal ganglion, CCK8-IR is found in Yellow Green, Green and Brown Cells in the viscero-parietal complex. Studies employing double-sequential methods or simultaneous immunofluorescence have shown that, with regard to the tested antisera, CCK8- and NPY-IR are colocalized in a limited number of cells and fibers in the buccal and visceral ganglia, whereas CCK8- and SP-IR are colocalized only rarely in neurons in the left cerebral ganglion. The possible roles in P. corneus of the investigated neuropeptides and the contribution that molluscan models may offer to the knowledge of the basic properties of neuropeptides are discussed.

Bromobimanes--fluorescent labeling agents for histochemical detection of sulfur containing neuropeptides in semithin sections.

Sulfur containing neuropeptides could be demonstrated in semithin sections of invertebrate nervous tissue, especially of gastropods, by using the bromobimanes as fluorescent labeling agents for thiol groups. Semithin sections showed a brilliant fluorescence of labeled peptides and should be used if an excellent resolution is important. The three bromobimanes (MB: monobromobimane, DB: dibromobimane, MQ: monobromotrimethylammoniobimane) gave positive results under our experimental conditions. Dibromobimane (DB) was selected because the application is more convenient. In gastropods, the bromobimane technique seems to be the most specific and sensitive one compared to the classical neurosecretory staining methods. Neuropeptides with sulfur containing amino acids could be demonstrated in perikarya, axons, and axon swellings easily. We suppose that there are neurons--not stainable by the classical methods--which can be identified as peptidergic ones by the bromobimane technique. A slight reduction of fluorescence intensity (fading) was observed. So, the fading rate was determined for dibromobimane reaction products; a decrease in the fluorescence intensity of 50% was only reached after 1 h using a Neofluar objective 10/0.30. Nevertheless, we suppose that a comparative quantification of the labeled neuropeptides should be possible if special parameters are considered.

Immunocytochemistry of luteinizing hormone-releasing hormone during spontaneous and thyroxine-induced metamorphosis of bullfrogs.

Double-bridge peroxidase-antiperoxidase immunocytochemistry was used to compare the developmental appearance of immunoreactive LH-RH (ir-LH-RH) in brains of bullfrog (Rana catesbeiana) tadpoles during either spontaneous or thyroxine-induced metamorphosis. During spontaneous metamorphosis, ir-LH-RH was localized in fibers of the external layer of the median eminence (ME) of stage XIII-XXV animals, while immunoreactive perikarya and other immunostained brain structures were absent. The extent and intensity of ME immunostaining increased concomitantly with measured ME morphological development. Tadpoles induced with thyroxine to metamorphic stages XIX-XXI exhibited ME structural development and neurohypophysial neurosecretory staining similar to spontaneously metamorphosed individuals of equal stages. However, comparable ME ir-LH-RH immunostaining and gonadal size were both less developed in thyroxine-treated animals, although increased relative to non-metamorphic vehicle-injected controls. These results indicate that the hypothalamic LH-RH system changes concurrently with ME structural development during spontaneous metamorphosis. Reduced ME ir-LH-RH staining and gonadal size in thyroxine-treated animals suggest that during prometamorphosis, factors other than thyroxine alone may coordinate the normal maturation of the hypothalamo-pituitary-gonadal axis of the bullfrog.

A Unicellular Endocrine Gland in Cestodes

Unicellular glands are reported from the scolex and anterior neck region of Hymenolepis diminuta and H. nana. Despite positive staining reactions with the presumptive neurosecretory stains, paraldehyde-fuchsin and chrome-alum-hematoxylin, ultrastructurally these glands exhibit many non-neural characteristics. Glandular cell processes are frequently found in close proximity to muscular tissue, particularly in the suckers, suggesting a regulatory role in muscle modulation as a possible function. Two types of putative, neurosecretory cells are reported from the cephalic ganglia and the lateral nerve cords. Neurosecretory regulation of the unicellular endocrine glands is postulated based on the lack of direct innervation of the glands and the frequent close proximity of axons containing putative, neurosecretory granules.

Monoamines in the pars intercerebralis-corpus cardiacum complex of locusts

The median neurosecretory cells and their pathways and the corpora cardiaca of adult and larval Schistocerca gregaria Forsk and adult Locusta migratoria L. were investigated histochemically by means of the fluorescence microscopic method for visualization of biogenic monoamines, and histologically by “neurosecretory staining”. No obvious differences between the two species were found. The larvae have less peptidergic and aminergic material than the adults. Perikarya of different types occur in the anterior pars intercerebralis: (1) several hundred green-fluorescent cell bodies, most of which probably contain dopamine; (2) one yellow-fluorescent cell body; (3) many hundred peptidergic cell bodies (A and B cells); and (4) several hundred cell bodies which are neither aminergic nor peptidergic. The monoamine containing cell bodies are not identical to the peptidergic ones. The Ncc1 contain peptidergic and aminergic fibres. The monoamine-containing fibres appear as typical varicose neurons, beaded fibres with droplet-like swellings, and fibres with droplets containing primary catecholamines and an indolylalkylamine. Besides the catecholamine and indolylalkylamine fluorophores a third type of formaldehyde-induced fluorophore is described and its nature discussed. The peptidergic and monoaminergic materials accumulate in the storage lobe of the corpus cardiacum, but as a rule the two types of substance do not correspond spatially. The great majority of glandular cells of the corpus cardiacum contain no peptidergic neurosecretory material or monoamines.

EFFECT OF A JUVENILE HORMONE ANALOGUE ON MORPHOLOGY, REPRODUCTION AND ENDOCRINE ACTIVITY OF THE COWPEA APHID, APHIS CRACCIVORA KOCH

ABSTRACTTopical application of AltozarTM†, at rates as low as 0.7 nanograms per aphid, caused severe morphological changes and prevented larviposition when applied to third instar Aphis craccivora, although treatment of other instars was less effective. Aphids which failed to reproduce contained fewer oocytes and embryos than untreated aphids, but enormous development of the embryos had occurred, and oocytes within these embryos also exhibited exaggerated development. The reproductive tract of treated aphids was distorted and non‐functional presumably due to pressare from the hyper‐developed embryos. The volume and mean nuclear size of the corpus allatum of treated aphids were significantly smaller than in untreated controls (newly‐moulted adults) and the effect of the chemical on the endocrine system is discussed. Neurosecretory staining revealed no difference between treated and untreated aphids.

Secretory phenomena at the ependyma of the IIIrd ventricle of the embryonic rat.

On the basis of previous observations of the third ventricle, the brains of rat embryos in the 17 to 21 gestation days were examined by light- and electron microscope as well as histochemically. Following results were obtained: 1. Between the 18th and 20th gestation days supraependymal secretion spherules and intraventricular secretion masses were observed in the rostral part of the third ventricular lumen. The intraventricular structures are not demonstrable with neurosecretory stains; they are PAS-negative and do not stain with periodic-acid-bisulfite-aldehydethionine. All histochemical reactions performed were negative. 2. The morphological finding in the same frontal plane is not uniform. Dorso-ventral difference exist between one ependymal region which lies caudal to the subfornical organ and an area in the same plane which is situated further ventrally near the entrance to the foramina Monroi (Fig. 1 a). In the superior portion, the supraependymal structures are spherical or bottle-shaped formations and are easily definable. In the lower ventricular portion densely situated ependymal protrusions are seen which become sperical only in the ventricular lumen. 3. In both instances (the spherical and flask-shaped protrusions of the superior portion and the strongly shaped protrusions of the lower portion of the third ventricle) cytoplasmic protuberances containing more or less dense, fine-grain ground plasma and a differentiated number of polyribosomes can be seen. Using the Richardson stain, chromophobe protrusions are seen to possess a diffuse ground plasma and only a few ribosomes, while chromophile protrusions have a dense ground plasma and numerous polyribosomes rosettes. Only rarely are single vesicles observed in the protrusions. 4. The ependyma cells contain densely packed, rough-walled endoplasmic reticulum whose cisterns are irregularly dilated and filled with electron-dense material; they also contain many ribosomes and dictyosomes. The organelles are accumulated mainly in the apical cell portions. A terminal web spans the cell surface and attaches to the zonulae adhaerentes, preventing the flow of cell organelles into the organelle-free secretory protrusions. 5. The functional implication of the ependymal secretion, which appears in the third ventricle for a few days only, during embryonic development, is still unknown and is the goal of subsequent examinations.

Studies on the neurosecretory system and retrocerebral endocrine organs in the red cotton bug, Dysdercus koenigii Fabr. (Heteroptera : Pyrrhocoridae)

The neurosecretory cells and the retrocerebral endocrine organs of the red cotton bug, Dysdercus koenigii have been studied with the help of two neurosecretory stains. Only one tinctorial type of 9 neurosecretory cells are present in each protocerebral hemisphere. There are three NCCs of which the NCCI innervate the wall of the aorta, the NCCII, the CC, and the NCCIII, the esophageal dilator muscles and the CC. Only the NCCI contain stainable NSM; the NCCII and NCCIII lack NSM and therefore are completely negative to the stains employed. The CC are devoid of cerebral NSM but show the presence of two tinetorially different intrinsic secretions in their anterior and posterior regions. The CA is unpaired and also lacks NSM while the aorta shows large accumulations of NSM and seems to act as a neurohemal organ.

Neurosecretory system of the American dog tick, Dermacentor variabilis (Acari: lxodidae). I. Diversity of cell types.

An arbitrary classification scheme is presented for the thirteen distinct types of secretory cells distinguished within the central nervous system of Dermacentor variabilis by several specific and general neurosecretory staining techniques. Comparisons to classic arthropod neurosecretory cell types are made and the histochemical implications of the chromophilic response of various secretory products are discussed. Dermacentor cells of Types I, VII, IX and X may be considered neurosecretory on the basis of intracellular elaboration and discharge of secretory product. Type II, III, IV, V, VI, XI and XII cells are considered as putative neurosecretory cells although secretory products were detected only within the perikarya. The large Type XII cells are also similar to motor neurones reported from other arachnids. Cells of Types VIII and XIII appear to be glial elements. The secretory products of Type XIIIA are distributed within trabecular processes in the subperineurium. These products may play a trophic role or they may have some endocrine function as a form of "gliosecretion".

Secretory factors in the retrocerebral endocrine-aortal complex of the water-bug, Sphaerodema annulatum (Heteroptera: Belostomatidae)

A histological study of the retrocerebral endocrine-aortal complex of Sphaerodema annulatum, employing conventional neurosecretory stains, reveals the presence of three qualitatively different secretory factors—one present in the corpora cardiaca (CC) and the other two distributed in the corpora allata (CA), aorta wall and nerves going to the pericardial cells. For reasons discussed in the paper, the factor present in the CC is regarded as an indigenous secretion produced by their own intrinsic secretory cells, and the remaining two as cerebral in origin, produced by two tinctorially different neurosecretory cell types.

A simple manoeuver to prevent loss of sections for performic acid–Victoria blue technique and a comparison of various neurosecretory stains

It is shown that whole brains of migratory locusts can be oxidized with performic acid and that paraffin sections of these can be successfully stained with Victoria blue. Either performic acid or acid permanganate oxidants can be used for Victoria blue and aldehyde fuchsin staining techniques thus allowing crisscross comparisons.

Ultrastructure and histochemistry of neurosecretory cells and neurohaemal areas in the pond snail Lymnaea stagnalis (L.).

In the central ganglia of Lymnaea stagnalis neurosecretory cell groups have previously been identified by means of chrome-haematoxylin or paraldehyde-fuchsin stains. In the present study seven types have been distinguished within the class of Gomori-positive cells on the basis of different staining reactions with the alcian blue/alcian yellow technique. Five types are located in the cerebral ganglia and in the lateral lobes, whereas two cell types occur in the ganglia of the visceral ring. No neurosecretory cells have been observed in the buccal and pedal ganglia. The staining technique used proved to be superior to the classic neurosecretory stains, because with this method the secretory substances can easily be distinguished from nonsecretory Gomori-positive tissue constituents. One of the two Gomori-negative neurosecretory cell types of the cerebral ganglia react positively with the alcian blue/alcian yellow technique. In addition, two Gomori-negative neurosecretory cell types, which had not been described before, were identified in the visceral ring. The ultrastructure of the four neurosecretory cell types in the visceral ring is described. The electron microscope revealed that each of the histochemically distinguished secretory substances consists of elementary granules which differ in size and appearance from each other and from the neurosecretory elementary granules which have been described by other authors in the cerebral ganglia and in the lateral lobes. The neurohaemal areas of the neurosecretory cells in the visceral ring are very extensive and include the peripheral parts of the nuchal nerves and of the connectives and nerves of the ganglia of the ring. The perineurium and the adjacent parts of the connective tissue which surround the ganglia, the connectives and the nerves are regarded as additional neurohaemal zones, because in these regions many tiny nerves occur, which consist mainly of neurosecretory axons ending non-synaptically near parts of the vascular system. In the perineurium surrounding the cerebral ganglia and their neurohaemal area a similar network of neurosecretory fibres was observed. Indications of release of the secretory material were regularly observed. Release apparently takes place by exocytosis. A circadian rhythmicity was observed in the release activity of some of the neurosecretory cell types.

Neurosecretory innervation of the pituitary of the eels anguilla and conger II. The structure and innervation of the pars distalis at different stages of the life-cycle

A number of distinct cell types may be recognized in the pituitary of the eel at the level of ultra-­structure by reason of the specificity of the size and electron-density of the granules they contain. The size of the granules and changes in the different cell types at different stages of the life-cycle permit a tentative identification in terms of function. The pars distalis of the eel pituitary receives the greater part of its innervation from the nucleus lateralis tuberis by Type B neurosecretory fibres (Knowles 1965 a ), which do not stain with the so-called neurosecretory stains, but which nevertheless contain elementary neurosecretory vesicles. Type A, or classical, neurosecretory innervation is also present and seems to be of special importance at certain stages of the life-cycle. The possible function of these two forms of neurosecretory inner­- vation is discussed. The relationship between the intrinsic endocrine cells of the pars distalis and their neurosecretory innervation is fundamentally similar, at the level of ultrastructure, to that of the neuro-intermediate lobe. There are no direct contacts between the neurosecretory fibres and the intrinsic endocrine cells, but the proximity of the fibre terminals ( ca. 2000 to 4000 A) to endocrine cells indicates a functional relationship between these two elements of the pituitary of the eel.

Multiplicity of Neurosecretory Cell Types and Groups in the Brain of the Saturniid Moth Hyalophora cecropia (L.)

As part of an extensive investigation of the anatomy and histology of the known endocrine organs of Hyalophora cecropia in the last 2 larval stages and in pupae and adults, we have examined the brain neurosecretory (NS) cells utilizing paraffin sections and the standard NS stains, paraldehyde-fuchsin (PAF) with counterstains and chrome-haematoxylin (CH) with phloxine. Previous workers1,2 had reported the existence of 26 apparently identical NS cells in the brain of this insect. Our results demonstrate the presence of at least 40 such cells and provide histological evidence for the occurrence of several distinct functional types.