Localization of acetylcholinesterase in granule-containing cells of glomus-like bodies in pre- and postnatal rabbits by electron microscopy

The distribution of acetylcholinesterase (ACHE) was studied in the granule-containing cells which constitute the glomus-like bodies found near the origin of the great vessels in pre- and postnatal rabbits. Karnovsky's method for localization of ACHE at the electron-microscope level was used and suitable controls were carried out. In the granule-containing cells, ACHE reaction product was evident in the perinuclear cisternae and cisternae of the rough endoplasmic reticulum as well as at the cell membrane. ACHE activity was also localized at the axolemma of unmyelinated axons found near the granule-containing cells and around afferent synaptic terminals to these cells. Possible functions of ACHE associated with the monoamine-storing granule-containing cells are presented.     

Changes occurring in the epithelium covering the bronchus-associated lymphoid tissue of rats after intratracheal challenge with horseradish peroxidase

Summary Changes occurring in the epithelium covering bronchus-associated lymphoid tissue (BALT) in the rat after several intratracheal administrations of horseradish peroxidase (HRP) were studied using morphological and ultrastructural methods. The epithelium is invaded by W3/ 25-positive (T-helper) lymphocytes, the BALT epithelial cells become Ia-positive and develop microvilli; there is an apparent loss of cilia. The number of non-ciliated cells in stimulated BALT increases. The non-ciliated cells can be subdivided into two cell types, one with electron-dense cytoplasm and cytoplasmic granules and the other without granules. The electron-density of the latter cell type is intermediate between that of the ciliated cells and that of the granulecontaining non-ciliated cells. The granule-containing cell types may be responsible for the uptake of antigens, while the other non-ciliated cell may be involved in the production of the secretory component and the passage of secretory IgA.Supported by a research grant from the Nederlands Astma Fonds     

The ultrastructure of paraganglia associated with the inferior mesenteric ganglia in the guinea-pig

Summary The paraganglia of the inferior mesenteric ganglia in the guinea-pig are composed of small chromaffin cells containing an abundance of granule-containing vesicles. The chromaffin cells are almost completely surrounded by satellite cells. In areas in which satellite cell processes do not intervene, the membranes of adjacent chromaffin cells are closely apposed and often form specialized attachment zones. The paraganglia contain a dense capillary network, the endothelial cells of which are often extremely attenuated and show areas of fenestration. The processes of chromaffin cells approach close to the capillary walls and are often bare of satellite cells covering on the side facing the capillary. Evidence has been obtained for the exocytotic release of the contents of chromaffin cell vesicles into pericapillary spaces. Synapses of cholinergic and noradrenergic axons are seen on the chromaffin cells. The cholinergic axons degenerate when the praganglia are decentralized, but the noradrenergic axons, which appear to arise from the local inferior mesenteric ganglia, remain intact. The results suggest that the paraganglia have an endocrine function.     

Differentiation of embryonic chick sympathetic neurons in vivo: ultrastructure,and quantitative determinations of catecholamines and somatostatin

Summary The ultrastructural and transmitter development of lumbar sympathetic ganglia was studied in embryonic day-6 through-18 chick embryos. At embryonic day 6, ganglia are populated by two morphologically distinct types of neuronal cells and Schwann cell precursors. The neuronal populations basically comprise a granule-containing cell and a developing principal neuron. Granule-containing cells have, an irregularly shaped or oval nucleus with small clumps of chromatin attached to the inner nuclear membrane and numerous large (up to 300 nm) membrane-limited granules. Developing principal neurons display a more rounded vesicular nucleus with evenly distributed chromatin, prominent nucleoli, more developed areas of Golgi complexes, and rough endoplasmic reticulum and large dense-core vesicles up to 120 nm in diameter. There are granule-containing cells with fewer and smaller granules which still display the nucleus typical for granule-containing cells. These granule-containing cells may develop toward developing principal neurons or the resting state of granule-containing cells found in older ganglia. Both granule-containing cells and developing principal neurons proliferate and can undergo degeneration. At embryonic day 9 there are far more developing principal neurons than granule-containing cells. Most granule-containing cells have very few granules. Mitotic figures and signs of cell degeneration are still apparent. Synapse-like terminals are found on both developing principal neurons and granule-containing cells. Ganglionic development from embryonic day 11 through 18 comprises extensive maturation of developing principal neurons and a numerical decline of granule-containing cells. Some granule-containing cells with very few and small granules still persist at embryonic day 18. The mean catecholamine content per neuron increases from 0.044 femtomol at embryonic day 7 to 0.22 femtomol at embryonic day 15. Concomitantly, there is a more than 6-fold increase in tyrosine hydroxylase activity. Adrenaline has a 14% share in total catecholamines at embryonic day 15. Somatostatin levels are relatively high at embryonic day 7 (1.82 attomol per neuron) and are 10-fold reduced by embryonic day 15. Our results suggest the presence of two morphologically distinct sympathetic neuronal precursors at embryonic day 6: one with a binary choice to become a principal neuron or to die, the other one, a granule-containing cell, which alternatively may develop into a principal neuron, acquire a resting state or die.     

Differentiation of oxyntic cells during early ontogenesis in the bovine

The origin and the differentiation of oxyntic cells in fetal bovine abomasum were investigated using transmission electron and light microscopic methods. In the oxyntic gland region oxyntic cell precursors and immature oxyntic cells appear as early as at the end of the first trimester of gestation--much earlier than described in any other mammalian animal species. Immature oxyntic cells are characterized by long apical microvilli, by their triangular-shaped light cytoplasm rich in large and numerous mitochondria, by the existence of vesicular profiles and by the incipient invagination of the apical plasma membrane forming a primitive intracellular canaliculus expanding into central areas of the cell. The oxyntic cell represents the first exocrine cell type developing from secretory granule-containing cells in the base of the primitive gastric glands.     

Urethral chromaffin cells

Summary The urethral mucosa of the rat, rabbit and guinea-pig was examined with both fluorescence and electron microscopy. Employing the former technique, numerous brightly fluorescing flask-shaped cells were observed amongst the basal cells of the urethral epithelium in all three species. In the electron microscope cells with a similar shape and distribution are distinguished by their content of membrane-limited dense granules, extensive Golgi membranes and bundles of filaments. In favourable planes of section short microvilli extend from the apical region of these cells which are joined to neighbouring urethral epithelial cells by zonulae occludentes. These fluorescent, granule-containing cells are classified as urethral chromaffin cells.Fluorescent nerves were not observed in relation to the urethral epithelium although the electron microscope revealed axons lying singly or in groups both beneath and between the urethral epithelial cells. Many of these axons appear varicose and contain small, agranular vesicles, a few large granulated vesicles and numerous mitochondria. Occasionally a vesicle-containing axon lay adjacent to a urethral chromaffin cell. While a direct autonomic innervation of these cells could not be discounted it is concluded that the majority of nerves probably perform a sensory function.     

Catecholamine-storing cells in the adrenal medulla of the pre- and postnatal rat

Summary The development of the rat adrenal medulla was studied at the ultrastructural level with particular emphasis placed on early discrimination of different catecholamine-storing cells. The first granule-containing cells, phaeochromoblasts, were seen at day 15 of gestation migrating into the anlage of the cortex. These cells were characterized by a few small granules (80–120 nm in diameter) and a high nuclear to cytoplasmic ratio. Presumably due to differentiation into chromaffin cells, they were no longer present after the eighth postnatal day. Maturation of phaeochromoblasts was indicated by an increase in number and size of their storage granules and a decrease in the nuclear to cytoplasmic ratio. Noradrenaline and adrenaline cell types were first clearly discernible at day 21 of gestation. Another cell type, a giant cell, was also recognized at this stage. In the adult animal, noradrenaline, two morphologically different types of adrenaline, and small granule-containing cells were observed.By applying acetylcholinesterase histochemistry, it was found that at day 17 of gestation a small population of granule-storing cells showed strong positive staining in the endoplasmic reticulum. In the adult animal this cell type was further characterized by small-storage granules. Other chromaffin cells began to show weak staining within the endoplasmic reticulum at day 19 of gestation. This staining appeared more frequently within adrenaline than noradrenaline cells. However, even in the adult animal many cells of both types were completely negative.It is concluded that acetylcholinesterase histochemistry is a useful method for early discrimination of small granule-containing cells in the developing rat adrenal medulla.Supported by grants from the Deutsche Forschungsgemeinschaft     

Identification of a secretomotor centre in the brain of Locusta migratoria,controlling the secretory activity of the adipokinetic hormone producing cells of the corpus cardiacum

Summary After retrograde filling of axons terminating in the glandular lobe of the corpus cardiacum (CC) of Locusta migratoria with cobalt chloride, a paired group of about 15 cobalt containing cells was demonstrated in the lateral area of the protocerebrum. The axons of these cells run via the NCC II into the glandular lobe of the CC. These small neurons have the characteristics of secretory cells; they contain secretory granules of about 1000 Å in diameter. The axon terminals in the glandular lobe, making synaptic contacts with the glandular cells, contain secretory granules of the same size. It is therefore concluded that the cell groups in the protocerebrum control the activity of the glandular cells which produce an adipokinetic hormone. Arborizations of fibers of the lateral secretomotor cells are present in the dorsal neuropile of the protocerebrum, ventral of the mushroom bodies and along the tracts of the NCC I within the brain. It is proposed that these arborizations are sites of synaptic input. It is discussed that the axons of these cells might receive additional synaptic input in the storage lobe of the CC.The localization of cell bodies, the axons of which enter the storage part of the CC is described. The course of the axon tracts of the various cell groups in the protocerebrum and their connections with the NCC I and NCC II are demonstrated.Supported by the Foundation for Fundamental Biological Research (BION) which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO). The electron microscopical investigations were performed at the EM-unit of the Faculty of Biology, State University of Utrecht (Director: Prof. Dr. J.C. van de Kamer)The author is greatly indebted to Dr. A.M.Th. Beenakkers and Dr. H.H. Boer for their active interest and helpful advise. Thanks are also due to Mr. H. van Kooten and his staff for making the macro- and microphotographs, to Mr. L.W. van Veenendaal for preparing the electron micrographs and final assistance in the preparation of the photo pages and to Mr. D. Smit, who made the drawings     

Ultrastructure of the salivary glands of the planthopper,peregrinus maidis (ashmead) (Homoptera : Delphacidae)

The principal salivary gland of the planthopper, Peregrinus maidis (Ashmead) (Homoptera : Delphacidae), comprises 8 acini of only 6 ultrastructurally different acinar types. In these acini, secretory cells contain elongated vacuoles partly lined by microvilli and by microtubule bundles. These vacuoles are apparently connected with extracellular canaliculi deeply invaginated into secretory cells. Canaliculi of each acinus lead to a ductule lumen, which is lined with spiral cuticular intima, surrounded by duct cells. Striated muscle fibers, supplied with small nerve axons and tracheoles, are found in various acini of the principal gland, usually around secretory and duct cells.In the accessory salivary gland, the 2 large secretory cells contain no elongated vacuoles or canaliculi invaginations. However, in their central region, apically, these cells border a large microvilli-lined canal with its own canal cells. This canal is apparently connected with the cuticle-lined accessory duct, formed by duct cells. Nerve axons, but no muscle fibers, are found in the accessory gland and its duct. It is suggested that the system for transporting secretory material within acini of the principal gland, is basically different from that within the accessory gland.     

Granule containing cells and fibres in the sinus venosus of elasmobranchs

The concentrations of catecholamines in the heart chambers of elasmobranchs were measured by the fluorimetric method of Bertler et al. (1958). Noradrenaline (NA) can be detected in all the chambers, but the sinus venosus is by far the richest in NA. This can either be due to the presence of storage sites for this amine in the sinus wall, or to a transport of amine to the sinus venosus from the anterior chromaffin bodies. The sinus wall contains large numbers of "granule containing cells" and axon-like processes, both with numerous dense-core vesicles of about 1800 A diameter. The dense-core vesicles contain a uranophilic matrix indicating the presence of protein, phospholipids and/or nucleic acid. The reactions failed to demonstrate amine, which may be due to a loss of amine by diffusion, to a relatively low intravesicular amine concentration, or, to the absence of amines in these granule-containing cells and processes. Heavy accumulations of granule-containing processes occur in the subendothelial area. The endothelium contains fenestrae and pores through which granule-containing fibres protrude into the venous cavity. Granule-containing cells are innervated by presumed cholinergic nerve endings. It is suggested that the granule-containing cells and fibres belong to the neurosecretory system with a cholinergic input, releasing the contents of the dense-core vesicles into the blood stream at the level of the venous cavity.     

A light and fluorescence cytochemical and electron microscopic study of granule-containing cells in the intrapulmonary ganglia of Pseudemys scripta elegans

In the lung of the red-eared turtle, large numbers of intramural ganglia located in the intraparenchymal connective tissue are demonstrated. Numerous cells in close proximity to the principal ganglionic neurons displayed a bright blue-white formaldehyde-induced fluorescence. Microspectrofluorometric analysis revealed the presence of dopamine (DA) in all cells measured. Subsequent light histochemical staining of the fluorescent sections showed the DA-containing cells to display argentaffinity. Electron microscopy of serial sections revealed cells characterized by dense-cored vesicles corresponding to the intensely formaldehyde-induced fluorescent cells. The argentaffin technique performed directly on ultrathin sections selectively stained the dense-cored vesicles. After fixation with glutaraldehyde followed by dichromate, x-ray microanalysis showed the chromium to be incorporated into the dense granules. Cholinergic-type nerve endings formed axosomatic synaptic contacts with the DA-containing cells, which can therefore be considered as intrinsic postganglionic elements. No efferent synapses from the granule-containing cells to the principal ganglionic neurons could be observed. The granule-containing cells occurred solitarily and in clusters, partially invested with satellite cells, and usually located near fenestrated capillaries; they displayed cytoplasmic processes and indicated emiocytotic granule release. Adjacent granule-containing cells were separated by spaces about 20 nm wide, gradually widening to form intercellular channels with apically projecting microvilli and primary cilia. It is concluded that the intrapulmonary granule-containing cells of the red-eared turtle belong to the APUD system. Furthermore, morphologically these cells appeared to possess a special sensory apparatus which designates them as paraneurons. The possible physiological significance of these intrapulmonary granule-containing cells is discussed.     

The morphology of the Limulus visual system

Summary The lateral optic nerve of Limulus polyphemus, the horseshoe crab, contains 4 types of axons, which originate from eccentric cells, retinula cells, rudimentary eye cells, and from unidentified cells in the brain that give rise to the efferent fibers. Though small in diameter in a young animal, the eccentric cell axons in the adult grow to the same size as the rudimentary eye axons, which are originally the largest fibers in the nerve of the small Limulus. Cytoplasmic content, particularly the orderly distribution of microtubules, is identical in the three types of visual fibers. The segregation of rudimentary eye axons into a separate grouping within the optic nerve in small animals gives way to a homogeneous distribution in the adult. Interrupting the optic nerve leads to a proximal pile-up of secretory granules in a few fibers. The identity of these granules with those in the synaptoid terminations of photoreceptors establishes these fibers as efferent. The same operation leads to a conspicuous hypertrophy of subsurface cisternae within retinula cell axons.This study constitutes Publication No. 483 from the Oregon Regional Primate Research Center, supported by Grants FR00163 and EY 00392 from the National Institutes of Health and by a Bob Hope Grant-in-Aid by Fight-for-Sight, Inc., New York City.The author wishes to thank Mrs. Audrey Griffin for patient and excellent technical assistance.     

A pair of rosette glands in the embryo and zoeal larva of an estuarine crab Sesarma haematocheir, and classification of the tegumental glands in the embryos of other crabs

A pair of rosette glands (one of the tegumental glands in crustaceans) is present at the root of the dorsal spine of the thorax in mature embryos of the estuarine crab Sesarma haematocheir. Each rosette gland is spherical, 45-50 microm in diameter. This gland consists of three types of cells: 18-20 secretory cells, one central cell, and one canal cell. The secretory cells are further classified into two types on the basis of the morphology of secretory granules. There are 17-19 a cells, and only one b cell per rosette gland. An a cell contains spherical secretory granules of 2-3 microm in diameter. The granules are filled with highly electron-dense materials near the nucleus but have lower electron-density near the central cell. The secretory granules contained in the b cell have an irregular shape and are 1-1.5 microm in diameter. The density of the materials in the granules is uniform throughout the cytoplasm. The secretory granules contained in both the a and b cells are produced by the rough endoplasmic reticulum. Materials in the granules are exocytotically discharged into the secretory apparatus inside the secretory cell, sent to the extracellular channels in the central cell, and secreted through the canal cell. The rosette gland can be distinguished from the epidermal cells 2 weeks after egg-laying and the gland matures just before hatching. Materials produced by this gland are secreted after hatching and secretion continues through five stages of zoeal larvae. These rosette glands were never found in the megalopal larva. Rosette glands are found in the embryos of Sesarma spp. and Uca spp. In other crabs, tegumental glands are also found at the same position as in the embryo of S. haematocheir, but the fine structure of their glands is largely different from that of the rosette gland. On the basis of the morphology of secretory cells (a-g cell types), the tegumental glands of a variety of crab embryos can be classified into four types, including rosette glands (type I-IV). The function of these tegumental glands is not yet known, but different types of the gland seem to reflect the phylogeny of the crabs rather than differences of habitat.     

Ultrastructural studies on the secretory cycle of the neurosecretory cells and the formation of herring bodies in the paraventricular nucleus of the rat

Summary The ultrastructure of the neurosecretory cells in the paraventricular nucleus of the normal male rat was studied by electron microscopy during various functional states. Four morphologically distinct types of neurosecretory cells were observed. It appears that they do not represent different classes of cells but different phases of secretory activity of a single cell type. The perikarya of the neurosecretory cells show a definite cycle of formation and transportation of secretory granules. We have designated the phases of this cycle as: (1) phase of synthesis, (2) phase of granule production, (3) phase of granule storage and (4) phase of granule transport. The neurosecretory granules appear to be moved in bulk into the axons, forming a large axonal swelling filled with granules as a result of one cycle in the neurosecretory process. Thus it may be postulated that a secretory cycle in the perikaryon of the neurosecretory cell seems to result in the formation of a Herring body in its axon, and that its content is then conveyed to the posterior pituitary.     

The mouse vomeronasal glands: a light and electron microscopical study

The glands of adult mouse vomeronasal organ (VNO) were studiedwith light- and electro-microscopical techniques. The vomeronasalglands (VN-Gs) consist of several individual glandular complexesdistributed along the long axis of the VNO. The secretory productsreleased from VN-G cells enter into the lumen of the VNO inthe region of transition between the neuroepithelium and thereceptor-free epithelium. The acini show the typical morphologicalfeatures of serous glands. The secretory cells of these aciniare characterized by a round to oval nucleus and a well-developed,rough endoplasmic reticulum, both preferentially located inthe basal part of the cell. The supranuclear region is occupiedby the Golgi apparatus and secretory granules varying in sizeand electron density. They accumulate towards the apical partof the cell. Secretory cells are connected by tight junctions,desmosomes and membrane interdigitations, moreover, they arealso coupled by gap junctions. Axonal terminals containing clearvesicles and dense-cored vesicles are frequently seen betweenthe secretory cells. Secretory cells are directly related tothe thin basal lamina of the acinus; myoepithelial cells arenot present. In the lamina propria, numerous smooth muscle cells,blood vessels and nerve bundles containing both myelinated andunmyelinated axons can be observed. An automatic regulationof the activity of the VN-Gs is discussed in relation to thevomeronasal pump.     

PC1/3, PC2 and PC5/6A are targeted to dense core secretory granules by a common mechanism

There are seven members of the proprotein convertase (PC) family of secreted serine proteases that cleave their substrates at basic amino acids, thereby activating a variety of hormones, growth factors, and viruses. PC1/3, PC2 and PC5/6A are the only members of the PC family that are targeted to dense core secretory granules, where they carry out the processing of proteins that are secreted from the cell in a regulated manner. Previous studies have identified alpha-helices in the C-termini of the PC1/3 and PC2 proteases that are required for this subcellular targeting. In the current study, we demonstrate that a predicted alpha-helix in the C-terminus of PC5/6A is also critical for the ability of this domain to target a heterologous protein to the regulated secretory pathway of mouse endocrine AtT-20 cells. Analysis of the subcellular distribution of fusion proteins containing the C-terminal domains of PC1/3, PC2 and PC5/6A confirmed that all three domains have the capacity to redirect a constitutively secreted protein to the granule-containing cytoplasmic extensions. Analysis of the predicted structures formed by these three granule-sorting helices shows a correlation between their granule-sorting efficiency and the clustering of hydrophobic amino acids in their granule-targeting helices.     

Horizontal cells of the carp retina: Golgi impregnation and Procion-Yellow injection

Summary Application of two methods for the selective staining of neurons, Golgi impregnation and intracellular marking with Procion Yellow, has revealed the anatomical arrangements of the horizontal cells in the carp retina. There are two basic horizontal-cell types, those with axons and these without. The former can be subdivided into three groups on the basis of the pattern of branching of the dendrites. These three structural groups are also functionally distinct, as shown by the electrical recordings made during Procion-Yellow injection. The axons of these three types of cell project into the proximal part of the inner nuclear layer, where they expand to form morphologically indistinguishable terminals. Fine horizontal processes leave the surfaces of these axon terminals. The functional behavior of a terminal resembles that of the associated soma. The horizontal cells lacking axons vary in morphological appearance, but they are similar functionally.This work was supported by the Deutsche Forschungsgemeinschaft     

The Pituitary Gland of the Roach Leuciscus rutilus

Abstract Two endocrine cell types, PbH+ and PAS+ cells, were recognized in the pars intermedia (PI) of the roach, Leuciscus rutilus, by light and electron microscopy. They can be distinguished by the appearance of their secretory granules. The PAS+ cells stain with toluidine blue in semithin sections as opposed to the PbH+ cells. After 5 days' treatment with metopirone the PbH+ cells are highly activated whereas the PAS+ cells show no response. The PbH+ cells are assumed to produce MSH. In roaches adapted to diluted sea water (10%) the transfer to fresh water for 1—1 1/2 hours or reserpine injections for the same period had no notable effect on neither cell type. Three kinds of axons innervate the PI. Two of these, which have granules averaging 111 nm and 151 nm in diameter, originate in the nucleus preopticus (NPO). The third type of axons has granules of an average size of 74 nm and is probably aminergic. The glia cells in this part of the neurohypophysis (NH) are granular pituicytes, which are innervated by all three axon types. The border between the NH and the PI is composed of a double basement membrane from which a network extends into the NH. This network, the basement membrane and the capillaries are the main sites for the terminations of the axons although nerve fibers also invade the endocrine tissue. Only axon type 3 was observed to synapse frequently with the PbH+ cells.