The ultrastructure of the sinus gland of Carcinus maenas (Crustacea: Decapoda)

Summary The sinus gland of Carcinus maenas consists of the swollen axonal endings of the neurosecretory cells of the major ganglia and acts as a storage release centre for the membrane bound neurosecretory material. These neurosecretory granules fall into five different types based on size and electron density. Their contents are released by exocytosis of the primary granules or smaller units budded from the primary granules.I thank Professor E. Naylor for his constant advice and Professor E. W. Knight-Jones, Department of Zoology, University College, Swansea, for the provision of laboratory facilities. I am grateful to the Science Research Council for the financial support. Finally, I thank the Electron Microscope Unit, Southampton General Hospital, where the work was completed.     

Fine structure of syzygial articulations before and after arm autotomy in florometra serratissima (Echinodermata: Crinoidea)

Summary About 1 s after appropriate stimulation, arms of Florometra serratissima break at articulations called syzygies that are specialized for autotomy. The fine structure of unreacted and of newly broken syzygies is described. The unreacted syzygy includes (1) ligament fibers consisting of collagen fibrils interconnected by interfibrillar strands and (2) axons filled with presumed neurosecretory granules. The newly broken syzygy includes (1) ruptured ligament fibers consisting of swollen collagen fibrils associated with interfibrillar globules and (2) axons containing few presumed neurosecretory granules, some of which are fixed in the act of exocytosis; moreover, the calcareous skeleton adjacent to the broken syzygy is partly eroded. The observations before and after breaking suggest that the autotomy mechanism may comprise the following sequence of events: rapid neural transmission from stimulation site to syzygy triggers a massive exocytosis of granules from presumed neurosecretory axons; the released neurosecretions (which could include chelating agents, strong acids, proteolytic enzymes or enzyme activators) etch the skeleton and lower the tensile strength of the ligament fibers by weakening the collagen fibrils and/or the interfibrillar material; breakage of the ligament fibers, the major connective tissue of the articulation, is quickly followed by rupture of all the other tissues at the syzygy.     

Further Evidence that Synaptic and Synaptoid Vesicles Constitute a Single Category of Inclusions: Dense-cored Synaptic and Synaptoid Vesicles in Helix Discharge their Contents by Exocytosis

In the pulmonate mollusc Helix, neurosecretory cells have perikarya that form neurohaemal complexes peripherally beneath the inner surface of the neural lamella and give rise to axons with varicosities in the neuropile. Two categories of secretory inclusions are present throughout the cytoplasm and these accumulate adjacent to sites of release. Secretory granules invariably have electron-dense contents, whereas smaller vesicles have fairly lucent contents following fixation in O_sO₄, but are dense-cored in material fixed initially with aldehyde. Vesicles (‘synaptic vesicles’) at central sites appear identical to those (‘synaptoid vesicles’) at peripheral, neurohaemal locations. At both neurohaemal and central sites, both granules and vesicles discharge their contents by exocytosis, this process being most clearly visualized in tissues treated with tannic acid.     

Neurosecretory granule formation in ligated axons: additional arguments for a local differentiation from a Golgi apparatus extension

The sorting domain for the different types of granules and small synaptic vesicles in neurosecretion is still largely a matter of debate. Some authors state that an exocytotic process has to precede granule formation. In previous studies, we favoured the idea that neurosecretory packages in terminals are assembled from axonal reticulum membranes simply by differentiation at the axon ending, the axonal reticulum being an extension of the Golgi apparatus. By ligating bovine splenic nerve, a de novo differentiation can be induced. After ligation, granules and granulo-tubular complexes appear. They were immunoreactive for SV2, VMAT2 and synaptobrevin II, which are all known to be highly enriched in large dense granules. Previously the granulo-tubular structures have already been recognized as precursor stadia of neurosecretory granules.It is concluded that at a de novo differentiation, a sorting out and aggregation is taking place of molecules typical for large dense granules. The small dense granules and tubules can be considered unripe, precursor forms of the large dense granules. All this occurs in the absence of signs of exocytosis. The present findings corroborate the view that granule formation occurs via local differentiation at an axon ending.     

Neural control of the corpus allatum in the Colorado potato beetle, Leptinotarsa decemlineata: An electron microscope study utilizing the in vitro tannic acid Ringer incubation method

The release of material from neurosecretory synapses in the corpora allata of the Colorado potato beetle, Leptinotarsa decemlineata was visualised by an electron microscope procedure which involved tissue incubation in tannic acid. Using morphometry, the frequency of exocytosis phenomena was quantified in beetles kept under two different photoregimes. The number of exocytosis phenomena in the neurosecretory synapses in the corpora allata of beetles kept under short days was significantly higher than that of beetles reared under long-day conditions. In addition, the corpus allatum gland cells appeared to be more richly innervated by neurosecretory synapses under short-day than under long-day conditions. Previous studies using the in vitro radiochemical assay showed that the corpus allatum activity of short-day beetles is at least partly restrained by neurally mediated factors. The present morphological data strongly imply that this corpus allatum inhibitory substance is released from the neurosecretory synapses.     

Depletion of neurosecretory granules and membrane retrieval in the sinus gland of the crab

Summary Ultrastructural aspects of hormone release from the sinus gland of the crab Carcinus maenas, have been studied by incubation of glands in vitro (i) in high potassium-containing media to induce hormone release; (ii) in a high potassium-containing calcium-free medium in which depolarisation but no hormone release would be expected; and (iii) in control saline. Uptake of horseradish peroxidase into subcellular organelles was also studied.Many neurosecretory granules could be found in the nerve terminals but, in contrast to mammalian neurosecretory systems, structures resembling microvesicles were extremely scarce. High potassium stimulation in the presence of calcium caused an 18 % loss of granules from the nerve terminals associated with images of single and multiple exocytosis. It further caused an increase in vacuoles which could have accounted for 33 % of the membrane of the granules exocytosed. After incubation in high potassium-containing, calcium-free media there was no evidence either of exocytosis of granules or of an increase in the vacuole population. The population of sparse microvesicle-like structures was not significantly altered by incubation in either high potassium medium. Horseradish peroxidase reaction product could be found only in vacuoles of tissues stimulated by high potassium concentrations in the presence of calcium. It is concluded that this depolarising stimulus produces, in the presence of calcium, the release by exocytosis of about one sixth of all the granules in the sinus gland, and that vacuoles are the organelle responsible for the recapture of membrane after the exocytosis.     

Immunogold labelling of serotonin-like and FMRFamide-like immunoreactive material in neurohaemal areas on abdominal nerves of Rhodnius prolixus

The ultrastructure of neurohaemal areas on abdominal nerves of the blood-sucking bug Rhodnius prolixus was investigated. Four types of axon terminals were found, distinguished by the morphology of their neurosecretory granules. By use of post-embedding immunogold labelling, granules in Type I axon terminals were shown to contain serotonin-like immunoreactive material, and granules in Type II axon terminals were shown to contain FMRFamide-like immunoreactive material. There was no colocalization of these materials. It is suggested that Type III terminals contain peptidergic diuretic hormone, which has previously been reported to be present in electron-dense neurosecretory granules in this neurohaemal area. The identity of material in Type IV terminals is unknown.     

The neurosecretory system of the adult Melanogryllus desertus pall. (Orthoptera,Gryllidae)

Summary The cerebral neurohemal area of Melanogryllus desertus is located posteriorly among the neurons of nervus corporis cardiaci I (NCCI) on the ventral median surface of the protocerebrum where axons penetrate the neural lamella and terminate on its outer surface. Numerous neurosecretory fibers containing three different types of granule occur within and on the outer surface of the neural lamella.The release of neurosecretory granules is accomplished by exocytosis and the formation of synaptoids. It can also take place as a mass release of granules into the stroma.     

Ultrastructural study on the release of neurosecretory material from the sinus gland of the land crab,Gecarcinus lateralis

Summary Sites of release of neurosecretory material were examined in a neurohemal organ of decapod crustaceans, the sinus gland of the land crab, Gecarcinus lateralis. Such discharge into the circulation seems to occur primarily at interfaces between the neurosecretory axons and the acellular stromal sheath which is interposed between parenchyma and hemolymph. The evidence obtained from electron micrographs of adult specimens indicates that more or less intact secretory granules are released into the extraaxonal space primarily by the process of exocytosis. Synaptic-type vesicles are clustered in parts of neurosecretory axons facing the stromal sheath. Such vesicles are thought to result from rearrangement of membranes temporarily fused at the release site and to a minor degree from fragmentation of neurosecretory vesicles within the axon. The presence of nonmarginal vesicles and the occasional appearance of free intraaxonal dense material are interpreted as indications of a second, probably less frequent, mode of release of neurosecretory material.Supported by grants AM-3984, NB-00840, and NB-05219 from the U.S.P.H.S., administered by Dr. Berta Scharrer.I am greatly indebted to Mrs. Sarah Wurzelmann for her excellent technical assistance. I thank Dr. Dorothy E. Bliss for contributing the animals used in this study, and Mr. Murray Altmann for technical advice.     

Release of brain neurosecretory products from the neurohaemal part of the aorta during egg-laying and egg-care in Labidura riparia (Insecta,dermaptera)

The exocytosis level in the neurosecretory terminals of the aorta wall, was studied by electron-microscopy in female Labidura riparia during different stages of the reproductive period: end of vitellogenesis, beginning, middle and end of egg-laying, and during egg-care.The exocytosis level (L. ex) in the terminals which are filled with big dense elementary granules is low at the end of vitellogenesis (L. ex = 0.28), the level increases 8 times when the first egg is passing under the genital plate, reaching a maximum level (2.46) when approximately ten eggs have been laid. The level then decreases at the end of egg-laying (1.8) and abruptly falls when the female begins her egg-care cycle. Throughout this period, the exocytosis level stays between 0.54 and 0.76.These results show a significant release of brain neurosecretory product at the neurohaemal part of the aorta during egg-laying. This suggests that egg-laying could be regulated by a brain hormone.     

Light,fluorescence and electron microscopic studies on “neurosecretion” in tritonia diomedia bergh (Mollusca,Nudibranchia)

Summary Membrane-limited electron-dense inclusions designated as elementary neurosecretory granules have a characteristic distribution in cerebropleural ganglia of the nudibranch snail Tritonia diomedia. They occur in the neuropile and also in individual nerve fibres, connectives and commissures. These granules have been found neither in perikarya of nerve cells nor in proximal segments of their processes.Specific fluorescence obtained in Tritonia preparations with Sterba's pseudoisocyanin method for neurosecretory products has the same pattern of location.The distribution of stainable material in preparations prepared with ordinary neurosecretory procedures (chrome haematoxylin-phloxin after Gomori-Bargmann and paraldehydefuchsin after Gomori-Gabe) is similar to that described by different authors in other gastropods, but strongly differs from the locationof elementary neurosecretory granules and of pseudoisocyanin-positive material. The adequacy of different histological methods for studying neurosecretion in gastropods is discussed.     

Requirements for the identification of dense-core granules

Dense-core granules (DCGs), cytoplasmic organelles competent for regulated exocytosis, show considerable heterogeneity depending upon the specificity of their expressing cells--primarily neurons and neurosecretory cells. DCGs have been mainly identified by detecting their cargo molecules, often members of the granin family, and using conventional electron microscopy and immunocytochemistry. However, by a critical analysis of the various stages of DCG "life" within neurosecretory cells, we have highlighted several specific molecular and functional properties that are common to all these organelles. We propose that these properties be considered as strict requirements for the identification of DCGs.     

Cytologie du lobe anterieur de l'hypophyse du chien

Summary Detailed histochemical studies have been made on the distribution of various enzymes such as phosphatases, cholinesterases, glycolytic enzymes and respiratory enzymes in various components of the hypothalamus with special reference to the supraoptic and paraventricular nuclei of the Squirrel Monkey. Cytological studies have also been made by the McManus, Einarson, Gomori and Bargmann methods.A few neurons of these nuclei showed scanty Gomori-positive material in the cytoplasm for the Gomori and Bargmann methods. Nissl granules were located in the peripheral cytoplasm of most neurons. No glycogen granules were observed in these neurons. For these reasons, the Squirrel Monkey, like the rat, may not be a suitable species for the study of neurosecretory phenomena.The axons of these neurons were negative for the specific cholinesterase test, though the perikaryon and some parts of the processes gave a moderately positive reaction. These neurons may be non-cholinergic and the cholinergic fibers from an unknown nucleus may end in synapses on their cell bodies. Blood vessels and glial cells in the neurosecretory nuclei showed non-specific cholinesterase activity. This enzyme may hydrolyze the acetylcholine which has escaped splitting by specific cholinesterase. Alkaline phosphatase and acid phosphatase in these neurons may be involved in the metabolism concerned with the production of neurosecretory material. The neurons may be physicochemical receptors and may get enough energy and raw material to synthesize the neurosecretory material from the rich blood supply. Neurons of the supraoptic and paraventricular nuclei as well as other hypothalamic neurons, like neurons of other regions of the brain, are well equipped with the enzymes of the glycolytic pathways and the tricarboxylic acid cycle. Since the glial cells of these nuclei have amylophosphorylase activity and glycolytic pathways, they may work as energy donators to the neurons of the neurosecretory nuclei. T. R. Shanthaveerappa in previous publications.     

Neurosecretory cells in Dysdercus similis (Hemiptera)

The neurosecretory cells of Dysdercus similis have been described. "A", "B", "C" and "D" types of neurosecretory cells are present. The "A" type of cells of the pars-intercerebralis show cyclical secretion. When these cells show secretory activity during one to three days of emergence, they have scattered granules. The cells are seen packed with clumps of neurosecretory material when they are not secreting, and this is interpreted as a storage stage. The axons of these cells supply the corpora cardiaca and some neurosecretory material also reaches the corpus allatum. The release of this neurosecretory material can be correlated both with moulting in the young stages and later with reproduction in the adults.     

Cytophysiological study of neurosecretory and pheromonal influences on sexual development in Ophryotrocha puerilis (Polychaeta,Dorvilleidae)

Summary

Prominent secretory nerve endings are found at the posterior margin of the supraesophageal ganglion in the protandric polychaete, Ophryotrocha puerilis. Solitary juveniles developing as primary males, and then as females, accumulate neurosecretory material in the nerve endings which thereby swell and become filled with granules. Females maintained in mass culture have similar terminals, whereas in secondary males (males which had been females before), these axon terminals are very small and contain no material. When such males are isolated, they accumulate neurosecretory material within the nerve endings and become females. When formerly isolated females are put together, their stores of neurosecretory material are rapidly discharged. Subsequently they lay egg masses and switch to the male state. These effects are mediated by a pheromone released during social contact of formerly isolated females. The complexity of the relationship between neurosecretory activity and sexual state is indicated by the situation in animals maintained in pairs, when both male and female partners have swollen nerve endings packed with secretory material.     

Etude histologique,histochimique et ultrastructurale de la pars intercerebralis chez Locusta migratoria L. (Orthoptere)

Summary A histological, histochemical and ultrastrucutral study of the pars intercerebralis (PI) has been made in Locusta migratoria. The acellular neural lamella is made up of an elastic tissue and collagen fibrils. The cells of the perilemma contain numerous lysosome structures and lipid granules.Three different types of neurosecretory cells (NSC A, B and C) have been distinguished in the PI associated with giant neurons.The cells termed A and B seem not to have an activity cycle during the two last larval instars. At the moment of sexual maturity the NSC A show an important accumulation of neurosecretory material and their number increases at the expense of the NSC B. The NSC A, which are characterized by a highly developped endoplasmic reticulum, contain numerous secretory granules which appear to be individualized in the Golgi complex in three different ways. The NSC B, with a reduced endoplasmic reticulum and an almost quiescent Golgi complex, contain abundant lysosome structures and more seldom some neurosecretory granules. In fact, the study of the fine structure shows different intermediate types, linking in a continuous way typical A cells and typical B cells. NSC A and NSC B might correspond to two opposed stages of secretory activity of one single cell type: the A cell representing the activity stage and the B cell the quiescent stage.NSC C show an accumulation of their neurosecretory products in relation to metamorphosis and sexual maturity. Ultrastructural evidence confirms their neurosecretory activity.A mode of regulating neurosecretion in NSC A and B by internal catabolism of the secretion and formation of lysosome like structures is discussed in the present paper.The giant neurons, which are surrounded by a glial envelope (trophospongium), contain several dense granules originated from Golgi complex.     

Ultrastructural demonstration of exocytosis of neural,neuroendocrine and endocrine secretions with an in vitro tannic acid (TARI-) method

Summary The release of neural, neuroendocrine, and endocrine secretory products by exocytosis was ultrastructurally studied by means of tissue incubation in Ringer containing tannic acid (Tannic Acid Ringer Incubation-method; TARI-method), followed by conventional fixation. Tannic acid strongly enhances the electron density of extracellular (secretory) substances. During TARI-treatment of tissues exocytosis proceeds, but the exteriorized contents of the secretory granules are immediately fixed by tannic acid and do not diffuse away into the extracellular space. In this way detection of exocytosis is markedly facilitated since the number of exocytosis phenomena visible at the ultrastructural level considerably increases with progressing incubation time. Studies of the central nervous system of the mollusc Lymnaea stagnalis show that the occurrence of exocytosis during TARI-treatment is calcium-dependent. With the TARI-method exocytosis has been clearly demonstrated in a variety of structures (endocrine cells, neurohaemal axon terminals, synapses) of L. stagnalis, the insect Locusta migratoria, and the rat, including cell types where exocytotic release had not been shown before.     

Ultrastructural studies on the hypothalamic neurosecretory neurons of the rat

Summary The general ultrastructural features of the hypothalamo-neurohypophysial system in rats with hereditary hypothalamic diabetes insipidus (DI-rats, Brattleboro strain) are described. There is no decisively distinguishing difference between the neurons of the supraoptic and paraventricular nuclei. The neurons of both nuclei show signs of active protein synthesis. The perikarya of the neurons are markedly hypertrophic, the nuclei are large and the nucleoli prominent. In the cytoplasm there are numerous ribosomes, abundant rough-surfaced endoplasmic reticulum and extensive Golgi complexes. However, very few neurosecretory granules are to be seen. The axons of the hypothalamo-neurohypophysial tract are likewise enlarged and the paucity of neurosecretory granules is a striking feature also in the area of the tract. The majority of nerve endings in the posterior pituitary of DI-rats are devoid of neurosecretory granules. Microvesicles are abundant in the nerve endings and there are findings which suggest that microvesicles are involved either in endoor exocytosis. The signs of active protein synthesis and the concomitant paucity of neurosecretory granules are interpreted to imply transportation of the secretory proteins in an extragranular phase. The possible mode of release of the secretory proteins from the nerve endings and the role of microvesicles therein are discussed.This study has been supported by grants from the Finnish Cultural Foundation and the Sigrid Jusélius Foundation. The collaboration of Professors Antti Arstila and Tapani Vanha-Perttula is gratefully acknowledged.The Brattleboro-rats were kindly provided by Dr. Heinz Valtin, to whom we express our thanks.     

Neurosecretion in the sinus gland of the fiddler crab,Uca pugnax

The ultrastructure of the sinus gland of the fiddler crab, Uca pugnax, was investigated and found to be similar to that in other crustaceans. Five types of neurosecretory axon terminals were tentatively identified on the basis of the size, shape, and electron density of granules within the axons. Release of neuro-secretory material appears to be by exocytosis.     

Exo-endocytosis in isolated peptidergic nerve terminals occurs in the sub-second range

Exo- and endocytotic processes induced by depolarization of isolated neurosecretory nerve terminals show a close temporal correlation, which suggests a short time of integration of the neurosecretory granule membrane with the plasma membrane. In order to determine minimal time requirements for exocytosis-coupled endocytosis to occur, we have analyzed by electron microscopy uptake of horserdish peroxidase (HRP) as a fluid phase marker at the onset of depolarization. We have applied rapid mixing and sampling (quenched flow) to assess events in subsecond time peroids after stimulation. A significant number of labelled endocytotic vacuoles was observed during the first second of depolarization. This number then further increased by a factor of about 2 (within 5 s) and 4 (within 50s). Thus, as for exocytosis, the rate of endocytosis decreased considerably during prolonged stimulation. These data indicate i) that a substantial proportion of secretory granules undergoes exocytosis very shortly after stimulation, and ii) that, following exocytosis, the minimal time required for consecutive membrane retrieval is in the sub-second range.