Light and electron microscopic studies on the neurosecretory control of diapause incidence in Pieris rapae crucivora

The incidence of diapause was shown to be determined humorally during the larval-pupal ecdysis by means of brain extirpation experiments.On the basis of this observation, light and electron microscopic changes in the neurosecretory type II cells in the pars intercerebralis-corpus cardiacum system during pharate pupal and early pupal stages were examined in insects reared under long day-length (non-diapause individuals) and in insects reared under short day-length (diapause individuals). In the diapause individuals, neurosecretory granules in NS-II cells increased during the pupal instar and large aggregates of granules packed the cytoplasm. Thereafter, inclusion bodies showing cytoplasmic breakdown of the granules appeared.In the non-diapause individuals, on the contrary, electron micrographs suggesting the release of neurosecretory material from axon terminals were obtained just after the pupal ecdysis. There were very few granules, with many Golgi bodies and much rough ER 8 to 12 hr after the ecdysis.It is concluded that adult development is determined by the release of neurosecretory material from the axon terminals of NS-II cells at the larval-pupal ecdysis. If release does not occur, the pupae enter diapause. It is also thought that differences in day-length during the larval stages influence the activities of NS-II cells before pupation.     

Diapause-dependent changes in prothoracicotropic hormone-producing neurons of the tobacco hornworm,Manduca sexta

The prothoracicotropic hormone (PTTH), which stimulates ecdysteroid synthesis in the prothoracic glands, is produced, in the dorso-lateral protocerebrum of Manduca sexta, by paired peptidergic neurons, the lateral neurosecretory cell group III (L-NSC III). Our study revealed ultrastructural features of L-NSC III, identified by immunogold labeling, and compared developing and diapause states. In developing and early-diapause pupae, L-NSC III soma ultrastructure is similar and is characterized by numerous clusters of neurosecretory granules (NSG) and an extensive trophospongium formed by satellite-glial cells. However, as diapause progresses, the ultrastructure changes, with the NSG becoming concentrated into large clusters separated by highly organized rough endoplasmic reticulum. Most conspicuous is a substantial reduction in the number of Golgi complexes and the glial trophospongium, and the presence of stacked plasma membrane separating the glia and neuron somata. The deep-diapause soma also has abundant glycogen deposits and autophagic vacuoles. With diapause termination, this morphology reverts to the nondiapause ultrastructure within three days, i.e. just before PTTH release that evokes development to the adult. During PTTH release the abundance of NSG in the soma does not change, suggesting that NSG depletion in the perikarya is not a marker for neurosecretion by the L-NSC III.     

Thiamine pyrophosphatase activity in the axonal smooth endoplasmic reticulum of neurosecretory neurons

Summary Neurosecretory cells of the supraoptic-neurohypophysial system of normal mice were investigated with the use of the cytochemical reaction for thiamine pyrophosphatase (TPPase) at the ultrastructural level. In the hypothalamic perikarya dense lead precipitates occur within the cisterns of the mature face of the Golgi apparatus, these being the cisterns that give rise to neurosecretory granules (NSG). Smooth endoplasmic reticulum is occasionally confluent with TPPase-positive Golgi cisterns. Along axons, within swellings, and within terminals distinct profiles of TPPase-positive tubules and cisterns are revealed, apparently part of a network of axonal smooth endoplasmic reticulum (AER). Some NSG appear to be confluent with AER. NSG with TPPase-positive tubular protrusions (likely vestiges of AER) are seen. Apart from reaction product (lead precipitate), the AER often contains an electron dense substance optically similar to that of NSG. TPPase-containing AER is often associated with mitochondria. Profiles of electron-lucent, precipitate-free tubules and cisterns are occasionally seen alongside reactive AER. Optimal TPPase activity in the AER occurs at pH 7.0–7.4, whereas in the Golgi complex intense marking is in the range of pH 6.0–8.5. A faint peppering of precipitate occasionally appears in the AER in controls (incubation medium without substrate), but neither in density nor in extent is this comparable to the reaction product seen after incubation in the presence of TPP. Preliminary comparison has been made between the AER revealed by the TPPase reaction, and that visualized after heavy metal impregnation according to the method of Alonso and Assenmacher (1978a). The nature of the close association between NSG and AER, and the possible roles of this membrane system in neurosecretory cells is discussed.Abbreviations AER axonal smooth endoplasmic reticulum - NSG neurosecretory granules - TPPase thiamine pyrophosphatase - SON supraoptic nucleus Research supported in part by a grant from the Israel Academy of Sciences to M.C.We thank Mrs. Ilana Sabnay for excellent technical assistance     

Neurosecretory cells in the frontal ganglion of the tobacco hornworm, Manduca sexta

The frontal ganglion of the tobacco hornworm, Manduca sexta (L.), was found to contain two neurosecretory (NS) cells (max dia = 40–45 μm). The cytoplasmic inclusions of the NS cells were stained purple with paraldehyde fuchsin, and marked fluctuations in amounts of NS material in the perikarya were observed, depending upon the developmental status of the insect. The perikarya of NS cells in the frontal ganglia of starved larvae and diapause pupae contained large accumulations of NS material, whereas feeding larvae and developing pharate adults showed relatively low amounts of neurosecretion. Electron microscopy revealed large accumulations of NS granules (dia = 80–240 nm) in the frontal ganglia of diapause pupae, but only slight accumulations of granules were seen in the NS cells of developing larvae and pharate adults.It was concluded that axonal transport and release but not synthesis is shut down during starvation and diapause, leading to accumulation of NS material in the perikarya. It is also suggested that the failure of many investigators to differentiate NS cells in the frontal ganglion of various insects may have been due to the selection of very active stages when the amount of available NS material was too low to be visualized by conventional staining techniques.     

Electron microscopic demonstration of acid phosphatase activity in the developing and mature heterophils of the chicken

Summary As shown by electron microscopic histochemistry using a modified Gomori lead salt technique, acid phosphatase is present in large dense granules and the Golgi apparatus —but not the light granules—in both immature and mature heterophils in the chicken. The large dense granules appear to form by budding from the Golgi cisternae while the light granules appear to be unassociated with the Golgi apparatus. The findings indicate that the large, dense granules are the lysosomes of the heterophils in the chicken.     

Ultrastructure of neurosecretory cells in the pars intercerebralis of Rhodnius prolixus (Hemiptera).

Six neuron types are distinguished in the pars intercerebralis of the starved fifth instar of Rhodnius prolixus. All neuron types contain electron dense secretory granules derived from Golgi complexes which are of characteristic size and morphology in each type. The neuron types are not thought to represent stages in a secretory cycle. The variety of neuron types described is related to that revealed by staining sections of the same cells with paraldehyde fuchsin. Active synthesis of neurosecretory granules continues throughout starvation and the lysosomal system appears to be involved in the continual degradation of secretory granules. Some of the variations in granule morphology observed may be a consequence of granule fusion and the importance of cytoplasmic events in the development of neurosecretory granules is discussed.     

Fine structure of nerve cells in a planarian

The fine structure of the nerve cell types in the white planarian Procotyla fluviatilis were described. Ganglion cells comprise the major portion of the brain. These cells are irregular in shape with several cytoplasmic processes and contain ribosomes, a sparse endoplasmic reticulum, microtubules, lysosomes, and a Golgi apparatus with numerous small vesicles. Granule-containing cells are situated in the peripheral regions of the brain and along the nerve cords. These cells contain ribosomes, rough-surfaced endoplasmic reticulum and a Golgi apparatus with associated dense granules. The granules occupy most of the cytoplasm and are ～ 750A in diameter with moderately dense contents, ～ 750A with opaque contents, and ～ 1000A with contents of medium density. These granules are similar to those in the nervous systems of higher animals that contain epinephrine, norepinephrine, and neurosecretory substance, respectively. Each cell contains predominantly one type of granule although there is some intermixing of granules and intermediate types between the three most abundant granules. Small clear vesicles, resembling cholinergic synaptic vesicles, and all types of dense granules occur in the neuropil and within nerve endings.     

The effect of fixation conditions on the ultrastructural appearance of gastrin cell granules in the rat gastric pyloric antrum

The ultrastructural appearance of gastrin cell (G cell) granules was studied after different fixation procedures. When the pH of prefixation was varied there was greater preservation of the electron density of granule cores after acidic (pH 5.0 and 6.0) than after neutral or alkaline (pH 7.0 and 8.0) prefixation. Increasing duration of prefixation at pH 7.3 resulted in progressive loss of electron density of the granule core with swelling and occasional rupture of the limiting membrane. In tissues where most granules had been rendered electron lucent by fixation, those granules remaining dense cored were preferentially located close to the Golgi zone. These findings indicate that the electron density of G cell granules is profoundly affected by conditions of fixation, and that immature granules are more resistant to loss of core density than mature granules. They also suggest that the gastrin granule in vivo, like other polypeptide granules, may have a "solid", osmotically inactive core.     

CYTOPLASMIC FINE STRUCTURE OF SCIARA SALIVARY GLANDS : I. Secretion

Cells from the anterior segment of the salivary glands of Sciara coprophila were found to synthesize and secrete into the gland lumen three morphologically distinct types of granule: 1) A large, electron-lucid granule, up to 1 µ in diameter, staining only faintly with pH 2 fast green and the PAS reaction; 2) an ellipsoid granule of moderate density, strongly fast green and PAS positive; and 3) a small spherical granule of high electron density. The cells contained numerous Golgi areas, up to an estimated 8,000 per cell. Evidence is presented for the transfer of material from the endoplasmic reticulum to the Golgi areas via small vesicles. Three types of Golgi areas were distinguishable, each containing intercisternal material resembling one of the three types of secretion granule. Patterns of secretion granule synthesis varied with the developmental stage of the larva as determined by counts of eye spots in the eye anlage. Lucid granules were most abundant in the youngest larvae, and decreased in abundance as larvae grew older, becoming virtually absent in prepupae. The small, dense granules were present in all gland cells, but became more prevalent in older larvae and prepupae. Ellipsoid granules were only occasionally present, and were independent of larval stage. It is suggested that lucid granules are digestive in function, since their abundance correlates with feeding patterns. Other granules may produce the external slime coating of the larvae, and also the mucoprotein component of the pupal cocoon.     

Light and electron microscopic observations of fabrication, release, and fate of biphasic secretion granules produced by epididymal epithelial principal cells of the fan-throated lizard Sitana ponticeriana cuvier

The epididymis of the fan-throated lizard Sitana ponticeriana was examined with light and transmission electron microscopy to understand the cellular mechanisms of fabrication of secretion granules in epithelial principal cells, granule release into the lumen, and the fate of the dense structured granules after reaching the lumen. Principal cells of the ductus epididymis, except at the cauda, secrete electron-dense biphasic granules copiously, which decrease in abundance from the initial segment to corpus. The principal cell possesses a prominent Golgi apparatus and all versions of endoplasmic reticulum (ER), rough, smooth, and sparsely granulated. The material of the dense portion of the secretion granules, after processing at the Golgi apparatus, appears to accumulate in large ER cisternae in the supranuclear cytoplasm. It undergoes condensation when the cisternae become condensing vacuoles. Mitochondria appear to play a role in dense granule formation. The condensing vacuoles are displaced toward the apical cytoplasm when the material of the less dense portion is added to the condensing vacuoles at the Golgi area. Thus, the less dense and dense portions of the secretion granules are secreted and added to the condensing vacuoles separately. The composite granules are released into the lumen by exocytosis when the less dense portion merges with the luminal content, whereas the dense portion maintains its structured identity. The latter, initially measuring 1-2 microm in diameter, increases in size several times. It is inferred that these granules release their content gradually, resulting in the appearance of vacuoles, and suggesting that the granules have an insoluble matrix in which there is a sparingly soluble material. The substance leaching out of the granules appears to contribute to keeping the sperm quiescent and alive during storage in the male reproductive tract.     

Precursors of monoamine-storage organelles in developing megakaryocytes of the rat

Summary Identification and distribution of the precursors of aminestorage organelles in rat megakaryocytes during cell maturation were studied, using the uranaffin reaction for adenine nucleotide. The precursors of the amine-storage organelles appeared as 200–300 nm vesicles having an uranaffin electron dense granule, whereas they appeared as empty vesicles by conventional glutaraldehyde-OsO₄ fixation. X-ray probe microanalysis confirmed the existence of U and P in the uranaffin reaction positive vesicles. The precursors appeared in the immature megakaryocytes, especially at the trans(mature) face of the Golgi apparatus, and rapidly increased in number in the maturing cells. The size of the uranaffin granules in the precursory organelles increased gradually during cell maturation and became almost equivalent to the dense body of blood platelets in the final stage of cell maturation.     

Electron microscope study of haemolymph cells of Decticus verrucivorus (Orthoptera, Tettigoniidae) in larva and imago stages

The haemolymph of larvae and imago stages of Decticus verrucirus was studied with electron and light microscope. PAS-positive and PAS-negative granules were detected in haemocytes. On the electronograms, granulocytes were recognized as the only type of haemocytes. In the cytoplasm of granulocytes, granules of two types were found: those of mitochondrial origin, and originating from the Golgi apparatus, respectively. The discharge of a secret is realized by the merocrine way. Four stages of granulocyte development have been distinguished: 1) granule formation and organelle development, 2) granule formation and accumulation, 3) active secretion, and 4) cell destruction.     

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.     

Ultrastructure of the neurosecretory cells in the brain of diapausing pupae of the tobacco hornworm, Manduca sexta (l)

The ultrastructure of seven different types of neurosecretory cells (NSC) found in the medial and lateral areas of the brain of diapausing Manduca sexta is described. The five different types of NSC in the medial area have characteristic differences in their shape, size, neurosecretory granules (NSG), and the morphology of their organelles. The cell types of the medial area accumulated the NSG, but did not appear to be synthesizing and packaging new NSG, whereas the NSC in the lateral region were synthesizing and packaging NSG during diapause. The possible significance of the relationship between the lateral and medial cells is discussed.     

The secretory cycle of supraoptic neurons in the rat

Summary After perfusion with formaldehyde and glutaraldehyde the supraoptic nucleus and infundibular process of the neurohypophysis of the rat were dissected and prepared for electronmicroscope observation. This study was carried out in specimens under normal water balance, in others fed on dry food and in rats submitted to forced hydration.Two extreme types of neurons with intermediary stages were recognized in the normal supraoptic nucleus. The main difference between them is in the content of ribosomes, development and dilatation of the vacuolar system and in the number of elementary neurosecretory granules. In both types lysosome-like particles are observed. The volume of the elementary granules increases 1.7 times along the hypothalamic-hypophyseal tract while the increase in the dense core of the granule is of the order of 4.3 times.After forty-eight hours on dry food there is a general depletion of secretory granules from the perikaryon and nearby axons, the ribosomes are numerous and the endoplasmic reticulum is dilated in all cells and contains a macromolecular filamentous material. With more prolonged dehydration the neurosecretory granules reappear in relation to the Golgi complex and the vacuolar system becomes progressively flattened. With forced hydration the number of granules in the perikaryon increases considerably.These observations are interpreted as indicative that the early stages of synthesis take place at the level of the ribosomes. The product, in a dilute macromolecular form, is transferred into the cisternae of the endoplasmic reticulum and then condensed into granules within the Golgi complex. The increase in size of the granules along the axon is discussed in relation to the progressive increase in hormone content.Supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas and by the Air Force Office of Scientific Research No 963-65.Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas.     

Ultrastructure of the corpus cardiacum and corpus allatum of the house cricket Acheta domesticus

Summary The ultrastructure of the corpus cardiacum (CC) and corpus allatum (CA) of the house cricket, Acheta domesticus, is described. Axon profiles within the CC contain neurosecretory granules 160–350 nm in diameter which are indistinguishable from those found in type I neurosecretory cells of the pars intercerebralis and in the nervus corporis cardiaci I. The CC itself contains two cell types: intrinsic neurosecretory cells and glial cells. Intrinsic NSC cytoplasm contains Golgi bodies and electron dense neurosecretory granules 160–350 nm in diameter. Synaptoid configurations with 20–50 nm diameter electron lucent vesicles were observed within axon profiles of the CC. The structure of the CA is relatively uniform with one cell type predominating. Typical CA cells possess large nucleoli, active Golgi complexes, numerous mitochondria, and occassional microtubules. Groups of dark staining cells scattered throughout the CA of some animals were interpreted as evidence of cellular death.This work was done while JTB was supported by USPHS Training Grant HD-0266 from NICHDI wish to express my thanks to Dr. Richard A. Cloney for sharing his expertise in electron microscopy     

Core formation and the acquisition of fusion competence are linked during secretory granule maturation in Tetrahymena

The formation of dense core secretory granules is a multistage process beginning in the trans Golgi network and continuing during a period of granule maturation. Direct interactions between proteins in the membrane and those in the forming dense core may be important for sorting during this process, as well as for organizing membrane proteins in mature granules. We have isolated two mutants in dense core granule formation in the ciliate Tetrahymena thermophila, an organism in which this pathway is genetically accessible. The mutants lie in two distinct genes but have similar phenotypes, marked by accumulation of a set of granule cargo markers in intracellular vesicles resembling immature secretory granules. Sorting to these vesicles appears specific, since they do not contain detectable levels of an extraneous secretory marker. The mutants were initially identified on the basis of aberrant proprotein processing, but also showed defects in the docking of the immature granules. These defects, in core assembly and docking, were similarly conditional with respect to growth conditions, and therefore are likely to be tightly linked. In starved cells, the processing defect was less severe, and the immature granules could dock but still did not undergo stimulated exocytosis. We identified a lumenal protein that localizes to the docking-competent end of wildtype granules, but which is delocalized in the mutants. Our results suggest that dense cores have functionally distinct domains that may be important for organizing membrane proteins involved in docking and fusion.     

Morphometric and electron microscopic studies of the secretory granules of neurosecretory cells of the supraoptic and paraventricular nuclei of white rats and mice]

The morphometrical and electron microscopic analysis of secretory granules in the perikaryons of neurosecretory cells of the supraoptic and paraventricular nuclei in male rats and mice has shown than in the cells of these nuclei in both species of animals there occur secretory granules of the same kind and size. Therefore this method fails to determine which of them contain oxytocin and which of them contain vasorpressin. The neurosecretory granules located in the Golgi apparatus zone are of a less size and have more osmiophilic cnetral material than the granules localized on the periphery which mainly have granular central material and are of a greater size. The distinctions in the size and type of secretory granules are associated with certain stages of their "maturation". Granular particles appear to be "swallen", more active forms of storing neurohormones. The presence of larger granular particles in the supraoptic nucleus of mice allows to suggest greater reactivity of this nucleus than in rats which is likely to be associated with a higher ability of mice, as compared with rats, to adaptate to disturbances in water-salt metabolism.     

MATURATION OF RAT MAST CELLS : An Electron Microscope Study

Electron microscope study of rat mast cell maturation corroborates certain interpretations of features of mast cell differentiation based on light microscope studies. In addition, the ultrastructural variation observed in the granules of differentiating mast cells suggests that granule formation begins with the elaboration of dense granules about 70 mµ in diameter inside Golgi vacuoles. These progranules appear to aggregate inside a membrane and fuse to form dense cords 70 to 100 mµ in diameter. These dense cords are embedded in a finely granular material possibly added to the developing granule by direct continuity between perigranular membranes and cisternae of rough endoplasmic reticulum. The dense cords and finely granular material then appear to be replaced by a mass of strands about 30 mµ in diameter, thought to be a reorganization product of the two formerly separate components. A process interpreted as compaction of the strands completes the formation of the dense, homogeneous granules observed in mature rat mast cells. The similarity between mast cell granule formation and the elaboration of other granules is considered, with special reference to rabbit polymorphonuclear leukocyte azurophil granules. The relationships between the ultrastructural, histochemical, and radioautographic characteristics of mast cell granule formation are considered, and the significance of the perigranular membrane is discussed.     

In vitro differentiation of hypothalamic magnocellular neurons of guinea pigs

Summary The development of neurophysin-oxytocin and neurophysinvasopressin containing neurons of the guinea pig was studied in vitro. Supraoptic (SO) and paraventricular (PV) nuclei were explanted from guinea pig foetuses at the 40th day of gestation and cultured in Maximov slides for nearly fifty days. The cultures were observed daily under a phase-contrast microscope. Explants were fixed every five days for observation with the electron microscope. At the time of explantation, magnocellular neurons were still immature. They acquired the morphologic characteristics of mature neurons, with axosomatic synapses, after about 10 days in vitro. After 15–20 days in vitro, they contained in addition neurosecretory granules (NSG), first in the Golgi region, then also dispersed in the cytoplasm. In the oldest culture (45–50 days), signs of granulolysis were regularly found. It appears that magnocellular neurosecretory neurons are able to differentiate in vitro from a primitive state in the absence of specific stimulation.The authors are indebted to M.J. Drian for help with tissue culture, and to D. Le Cren for photographic work