Occurrence of immunoreactive enkephalins in a neurohemal organ and other nervous structures in the eyestalk of the shore crab,Carcinus maenas L. (Crustacea,Decapoda)

Summary Light-microscopical observations with immunofluorescence and peroxidase staining procedures revealed leu-enkephalin-like immunoreactivity in axon profiles of the sinus gland (SG) and in single small neurons in the optic ganglia of the eyestalk of Carcinus maenas. Electron microscopy of the SG showed reactivity to be associated with neurosecretory granules 82±23 nm in diameter. High performance liquid chromatography of SG-extracts revealed radioimmunoreactive substances with the retention times of synthetic met- and leu-enkephalin and met-enkephalin-Arg⁶-Phe⁷, respectively.     

Relationship between Rous sarcoma virus production in golden hamster cells and the cell cycle]

The axon terminals of neurosecretory cells, containing elementary granules of neurosecretory materials, have been described on the basis of light and electron microscopy. No allochthonous neurosecretory elements were found in the sinus gland. The discharge of the granules from some terminals of the sinus gland occur with the changed salinity of the environment. There is a great difference in the structure of the sinus gland when salinity is changed. The structure of the sinus gland and its modification under experimental conditions indicate a possibility of neurohormonal regulation of the hydromineral balance.     

Ultrastructure of Merkel corpuscles in the tongue of the finch,Lonchura striata

Summary Merkel corpuscles in the lingual mucosa of the finch, Lonchura striata, were examined by means of the argyrophilic reaction and electron microscopy. These corpuscles are composed of 12 to 20 flattened Merkel cells and enclosed nerve terminals. The present study demonstrated for the first time argyrophilia in avian subepithelial Merkel cells with the use of Grimelius silver stain. Electron-microscopically, the Merkel cell was characterized by the presence of numerous densecore granules, approximately 80 to 140 nm in diameter, as well as specialized contacts with nerve terminals. The granules showed a tendency to accumulate in the cytoplasm in close association with both nerve terminals and basal lamina. This study also provided unequivocal evidence for exocytotic discharge of Merkel-cell granules at the plasma membrane facing not only the nerve terminals but also the basal lamina. The exocytotic figures toward the nerve terminals can be regarded as synaptic discharge of Merkel-cell granules, but the possibility also exists that the Merkel-cell granules may exert a trophic effect on the nerve terminals. The exocytotic release of Merkel-cell granules toward the basal lamina with no relation to nerve terminals may suggest an endocrine (paracrine) function for the Merkel cell. The avian subepithelial Merkel cells qualify as paraneurons, but their exact nature and function remain enigmatic as is the case of intraepithelial Merkel cells in other vertebrates.     

Autoradiographic evidence that transport of newly synthesized neuropeptides is directed to release sites in the X-organsinus gland of Cardisoma carnifex

Summary Sections of isolated X-organ — sinus gland neurosecretory systems of the crab, Cardisoma carnifex, were studied by light-and electron microscopy with conventional and autoradiographic procedures. The somata only were exposed to a pulse of ³H-leucine (5 min-5 h) and the entire system perfused with chase medium for various times (1–72 h) before fixation. Within 1 h, radiolabel is concentrated in Golgi complexes and nascent granules of both large and small somata. Label is undetectable in the terminal region following a 10 h chase. It is found in the nerve tract near terminals at 14 h, while after a 19 h chase, label is concentrated in terminal profiles abutting blood sinuses of the neurohemal organ (sinus gland). Following a 72 h chase, label is distributed throughout the terminal region. Each of the six morphologically distinguishable terminal types shows labelling. These observations show that the vast majority of newly formed granules are initially transported to release sites of the perisinus terminals. They thus provide an explanation for previous analyses indicating that newly synthesized peptides are preferentially secreted.     

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.     

Ultrastructure and distribution of identified neurosecretory terminals in the sinus gland of the terrestrial isopod Oniscus asellus

An ultrastructural study of the sinus gland of the terrestrial isopod, Oniscus asellus, reveals that this structure consists of two regions: the bulb, which is attached by a narrow stalk to the optic lobe, and the lateral extension, which extends from the bulb along the optic tract to the compound eye. The bulb has a distal region containing only neurosecretory terminals, and a proximal region containing terminals, glial cells, and axons that give rise to the distally located terminals. In total, the sinus gland contains five types of terminals which can be distinguished by their location and the appearance of their neurosecretory granules. Three terminal types are located in the bulb and two in the lateral extension. The size of the terminals in the bulb varies among the three types, but the number of terminals is approximately the same for each type. Conversely, the two terminal types in the lateral extension are similar in size, but differ in number. Axons of two terminal types in the bulb can be traced to the central region of the protocerebrum; axons of one terminal type in the bulb and of terminals in the lateral extension can be traced to the optic lobe.     

The ultrastructure of the sinus gland of the crayfish Astacus leptodactylus (Nordmann)

Summary An ultrastructural study of the sinus gland of the crayfish Astacus leptodactylus demonstrates that this gland is mainly composed of glial cells, axons and axon terminals. On the basis of the size, shape and electron density of the neurosecretory granules, we could distinguish five different types of axon terminals.     

The presence of opioidergic pinealocytes in the pineal gland of the European hamster (Cricetus cricetus): an immunocytochemical study

By use of antibodies raised against leu-enkephalin and met-enkephalin immunoreactive, opioidergic bi- and multipolar cells were demonstrated in the pineal gland of the European hamster. Ultrastructural analysis of these opioidergic cells revealed them to be pinealocytes. Processes emerged from the cell bodies and terminated in club-shaped swellings containing many small clear and some larger granular vesicles. Some of the terminals made synapse-like contacts with non-immunoreactive pinealocytes. The presence of the opioidergic pinealocytes strongly indicates that the pineal gland of the European hamster, in addition to its pinealopetal nervous regulation, is regulated by intrapineal peptidergic pinealocytes via a synaptic mechanism. A possible paracrine role of the opioidergic cells must also be considered.     

Ultrastructural changes in isolated peptidergic nerve terminals induced by digitonin permeabilization and K+ stimulation in the sinus gland of the crab,Cardisoma carnifex

Summary The preparation of isolated peptidergic nerve terminals from the sinus gland (a neurohemal organ) of the crab (Cardisoma carnifex) is described. In this species the nerve endings can have diameters up to 30 m. They release neurosecretory material as judged by the decrease in the volumetric density of granules upon depolarization with potassium. Similar results were obtained after permeabilization of the nerve terminals with digitonin, but only in the presence of micromolar concentrations of calcium. This preparation should prove useful in correlating electrical events with other cellular processes involved in stimulus-secretion coupling.     

Species variability in the expression of met- and leu-enkephalin-like immunoreactivity in mammalian Merkel cell dense-core granules

Summary Immunogold staining failed to show met-enkephalin immunoreactivity in the Merkel cell dense-core granules of rats when examined by electron microscopy, but showed gold particle staining in the Merkel cell dense-core granules of mice and nude mice. Merkel cells of hamster, guinea pig, rabbit, cat and dog were also examined using a similar method, and different antisera dilutions. Immunogold particles were consistently found in the dense-core granules of mice and nude mice at all antisera dilutions, but not in the other species, except in the dog, where a very low labelling response was encountered. Merkel cells from skin touch domes or sinus hair follicles, did not exhibit any difference in peptide expression as far as met-enkephalin immunoreactivity was concerned. In addition, all species studied, including mice and nude mice, did not show leu-enkephalin immunoreactivity in their Merkel cell dense-core granules. It is concluded that species variability in peptide expression occurs in the Merkel cell dense-core granules, and may be closely related to the different methodologies used.     

Ontogenetic appearance of somatostatin-containing nerve terminals in the median eminence of rats

Summary The development of immunoreactive (ir) somatostatin-containing nerve terminals in the rat median eminence (ME) has been examined electron-microscopically. Nerve fibers containing ir particles scattered throughout the axoplasm are first seen in the external layer of the ME on day 18.5 of gestation, and, on day 21.5 appear to terminate on the basement membrane of the perivascular space of the portal vessels. After birth, the fiber terminals contain several membrane-limited granules, which are labeled with ir PAP particles. Ultrathin, Epon-embedded sections of ME, treated by the protein A gold-labeling method for somatostatin, demonstrate positively labeled granules in the nerve fibers in the postnatal ME, but in the prenatal tissue, no specific gold-labeling is found. These findings show that, in the external layer of the ME, somatostatin storing occurs in the granules in the axonal terminals after birth.     

Ultrastructural study of the neurosecretory granules in the sinus gland of the blue crab,Callinectes sapidus

Summary Seven morphologically different types of neurosecretory granules have been found in the axon terminals of the sinus gland of the blue crab, Callinectes sapidus. They differ from each other in size, shape, staining characteristics, solubility characteristics, core matrix characteristics, axon terminal matrix characteristics, presence or absence of space between the granule membrane and granule core matrix, and frequency of occurrence. Five of the types are segregated in different axon terminals and are believed to represent different hormone-protein complexes. Two of the types, which have lost part or all of their granular contents, are thought to be variants of the other five types. The differences in granular morphology are better revealed by some fixation procedures than others. Palade's acetate-veronal buffered osmium tetroxide, in particular, reveals striking differences. The following observations suggest that different hormone-protein complexes are segregated in different axon terminals and that these complexes may be morphologically distinguished at the level of the electron microscope.Supported by USPHS-NIH Training Grant GM-00669 and Grant GB-7595X from the National Science Foundation.     

Intragranular colocalization of arginine vasopressin and methionine-enkephalin-octapeptide in CRF-axons in the rat median eminence

Summary Ultrastructural appearances of axonal terminals containing corticoliberin (CRF) were examined in the rat median eminence prepared by a freeze-drying procedure. Immunolabeling was performed by using 5-, 8-, or 15-nm gold-antibody complexes for CRF, arginine vasopressin (VP) and methionine-enkephalin-octapeptide (Enk-8), singly or in combination. In intact animals, the CRF-containing secretory granules were only slightly labeled with goldanti-VP or -Enk-8. In adrenalectomized rats, granules within single axons appeared to be labeled with all the immunogold complexes. This intragranular colocalization of the three antigens was confirmed by using three neighboring sections of the same axon terminals which were stained separately with each one of the antibodies and visualized with the avidin-biotin-peroxidase complex method. The granules labeled for CRF had decreased 9 days after adrenalectomy but had increased again by day 21, while those labeled for VP steadily increased after adrenalectomy. However, this did not correspond with the appearances of cell bodies in the paraventricular nucleus; the cell bodies labeled for both CRF and VP steadily increased in number and in stainability. By contrast, Enk-8 immunoreactivity in the axonal terminals and cell bodies was not affected by adrenalectomy. These findings suggest that although the three peptides could be released simultaneously from the axonal terminals, VP may play some special role in the expression of CRF activity.     

‘Neurosecretion’ by a classic cholinergic innervation apparatus

Summary Nerve terminals forming typical synapses with adrenal chromaffin tissues have been examined in the goldfish, frog (Rana pipiens), hamster and rat. Presumptive secretory inclusions present in the terminals are of two distinct types. Electron-lucent synaptic vesicles 30–50 nm in diameter are densely clustered adjacent to membrane thickenings and presumably discharge their contents into the synaptic clefts. Secretory granules (i.e. large dense-cored vesicles) 60–100 nm in diameter are more abundant in other parts of the terminals. Sites of granule exocytosis have been observed in each of the animals investigated. They are usually encountered within apparently undifferentiated areas of plasmalemma and only rarely occur within synaptic thickenings. Granule exocytosis from within synaptic terminals and chromaffin gland cells is most readily observed in specimens exposed, prior to fixation, to saline solutions containing both tannic acid, and 4-aminopyridine and/or elevated levels of K⁺. These findings show that the pattern of secretory discharge, involving both synaptic and non-synaptic release, which is widespread in invertebrate central nervous systems, is also characteristic of vertebrate, peripheral cholinergic terminals.     

Fine structure of pars neuro-intermedia of the loach,Misgurnus fossilis L.

Summary Two types of glandular cells are present in the pars intermedia of the loach, Misgurnus fossilis. Basophils are characterized by the presence in their cytoplasm of numerous secretory granules containing electron-dense and homogeneous material and by scarce endoplasmic reticulum. Weak acidophils contain in their cytoplasm abundant endoplasmic reticulum and numerous granules of different electron densities.The distal part of the neurohypophysis is composed of several types of neurosecretory axons, strongly branched pituicytes, numerous capillaries, and connective tissue elements. The axon terminals form the neuroglandular, neurovascular and neurointerstitial contacts. Some axon terminals are closely apposed to the basement membrane separating neurohypophysis from meta-adenohypophysis. At points of absence of continuity of this membrane, some neurosecretory axons become directly contiguous with cytoplasmic membranes of the intermedia cells.The investigation was partly supported by a research grant from the Zoological Committee of the Polish Academy of Sciences.     

Tachykinin-like immunoreactivity in the sinus venosus of the dogfish (Scyliorhinus canicula)

The sinus venosus of the elasmobranch heart is characterized by the presence of large bundles of unmyelinated nerve fibres that bulge into the cardiac lumen, below the endocardium. In the dogfish (Scyliorhinus canicula), these fibres contain numerous dense-core membrane-bounded granules of about 200 nm in diameter. Most intramural ganglion cells of the sinus venosus also show densely packed granules similar to those found in the subendocardial fibres. We have observed strong substance-P-like immunoreactivity in the large fibre bundles and in the perikarya of the ganglion cells. Preabsorption of the antisera with fragment 7–11 of substance P has shown that the antisera recognize the tachykinin canonic sequence. Our findings suggest that an undetermined tachykinin is secreted in the elasmobranch heart, and that it is probably released into the blood stream in the context of a little-known neuroendocrine system.     

An ultrastructural study of relationships between the ovarian haemal system,follicle cells,and primary oocytes in the sea star,Asterias rubens

Summary The genital haemal sinus, present throughout the gonad wall of sea stars, is supposed to be the site of ultimate accumulation of nutrients for the germinal epithelium. Early vitellogenic pear-shaped oocytes are attached to this sinus by stalk-like processes. The ultrastructure of this association and of the oocyte-follicle cell complex is described with emphasis on mechanisms involved in oocyte nutrition.The genital haemal sinus, and sometimes portions of the surrounding genital coelomic sinus, contain a fine granular ground substance and amoeboid cells. Material similar to the haemal ground substance also fills vacuities in the inner basal laminae of the haemal sinus and intervenes between this layer and adjacent germinal and follicle cells in the ovarian lumen.Vitellogenesis is first detectable as numerous vacuoles accumulate within the oocyte-stalk near the haemal sinus; they contain flocculent material and often fuse with adjacent lysosome-like vacuoles. As vitellogenesis proceeds, oocytes develop complex and tenuous connections with the haemal sinus. These consist of a network of pseudopodia that interdigitate with thin sheet-like extensions of follicle cells. These cells are attached to the oolemma by microfilamentous processes and contain regularly arranged concentrations of glycogen granules and well developed rough endoplasmic reticulum.It is concluded, (1) that follicle cells provide each oocyte with a compartmentalized microenvironment within the ovarian lumen, (2) that such compartments are intimately associated with the nutrient laden haemal sinus, and (3) that nutritive and vitellogenic substances, derived extragonadally and stored temporarily in the ovarian wall, can pass through the oocyte-stalk.     

中华绒螯蟹窦腺神经末梢及X-器官神经分泌细胞的类型

在电子显微镜下观察了性未成熟的中华绒螯蟹黄蟹的窦腺及X－器官的超微结构。X－器官位于眼柄神经节终髓的腹外侧,与窦腺位置斜相对,窦腺主要由神经分泌细胞的末梢和胶质细胞组成。神经末梢含有大量的膜结构包围的颗粒、线粒体、粗面内质网和许多电子透明的小泡,末梢外周有时可见指状突起。依据颗粒的大小、形状、电子致密度以及胞质特征,可区分出6种类型的窦腺神经末梢及7种X－器官神经分泌细胞。观察了末梢中神经分泌颗粒的胞吐作用方式的释放过程,并且尝试对窦腺不同末梢中的颗粒及X－器官神经分泌细胞中的颗粒作了比较,发现二者之间具有较好的对应性,即电子致密度无大的变化,形态特征相似,只是大小稍有增加。     

Ultrastructural appearance of neurosecretory granules in the sinus gland of the crab after different fixation procedures

Summary The appearance of neurosecretory granules in the crab sinus gland was studied after fixation at different pHs. Whereas at pH 7.0 the neurosecretory granules were pleomorphic with respect to electron density, at pH 5.0 or 6.0 all the granules remained electron dense. The possible role of maturation as an explanation of this observation is discussed.ERA 493 CNRS     

Ultrastructural,fluorescence microscopic and microfluorimetric study of the innervation of the axial complex in the sea urchin,Sphaerechinus granularis (Lam.)

Summary In juxtaposition with the contractile epithelia of the axial complex of the sea urchin, Sphaerechinus granularis, several types of nerve fibers with different vesicle populations were determined. Nerve terminals, filled with clear vesicles and dense core vesicles, form synaptoid neuromuscular junctions. Close to the somatocoelic epithelia of the axial and terminal sinus septa, numerous axon profiles form a nerve plexus. Among the epithelial cells covering the plexus, two types of nerve cells can be distinguished which presumably produce neurosecretory and aminergic granules, respectively. Monoamine fluorescence (formaldehyde-induced fluorescence, Falck-Hillarp technique) was analyzed microspectrofluorimetrically. The emission spectrum of the fluorophores occurring in the present material shows a maximum at 475 nm and is characteristic of catecholamines; the excitation maximum at 380 nm after formaldehyde treatment is typical of catecholamines at low pH only. Since the peak ratio (370:320 nm) does not change after HCl-vapor treatment, the fluorophores are likely to be indicative of dopamine.